Regulatory roles of non-coding and exosomal RNAs in colorectal cancer: spotlight on angiogenesis.

Introduction
CRC is one of the primary contributors to cancer-related mortality globally, characterized as a prevalent and aggressive malignancy [1, 2]. Even though systemic treatment for metastatic CRC has advanced significantly, patient mortality remains high [3, 4]. To identify novel treatment strategies, it is essential to study the mechanisms underlying CRC progression [5]. Chemotherapy and optimal surgical resection currently constitute the fundamental treatments for early-stage CRC [6, 7]. Nonetheless, patients with advanced-stage CRC continue to experience significant challenges, including tumor heterogeneity, metastasis, and drug resistance following combined radiation and chemotherapy, all of which diminish treatment efficacy [8–11]. Comprehending the molecular mechanisms underlying cancer advancement may lead to novel targeted therapies for highly malignant cancers [12]. CRC develops through a number of genetic changes, transforming healthy colonic epithelium into an adenoma and then into an adenocarcinoma. CRC is caused by genetic mutations and environmental changes that target DNA repair genes, tumor suppressor genes, oncogenes, or ncRNAs, such as miRNA or lncRNA [13, 14]. One new area regarding non-invasive cancer diagnostic applications is the measurement of circulating levels of ncRNAs in serum or plasma [15, 16]. Particularly, ncRNAs with stable expression have recently attracted a lot of attention due to their potential predictive value as biomarkers [17–20]. A novel delivery vehicle for nucleic acids and proteins, including ncRNA, has been identified: exosomes, which are vesicles measuring 30 to 150 nm in diameter [21]. According to recent research, exosomes may constitute a key mode of intercellular communication during tumor metastasis and TME remodeling [22, 23]. Furthermore, tumor metastasis and angiogenesis are significantly influenced by ncRNA that is present in exosomes from various cell types, including cancer cells and M2 macrophages [24–26] Angiogenesis is a complex process that is controlled both time-dependently and regionally by a fine balance between the quantity and quality of anti- and pro-angiogenic molecule expression [27]. Recent research has combined discoveries on how miRNAs interact with lncRNAs, circRNAs, and exosomal ncRNAs in CRC angiogenesis, highlighting their potential as diagnostic tools and treatment targets. Understanding these molecular regulators may lead to novel, targeted treatments that improve CRC patient outcomes by controlling angiogenesis and metastatic spread. (Fig. 1).

Angiogenesis and colorectal cancer
Tumors require the rapid formation of new vascular networks to grow. Tumors develop new blood vessels from the capillary network through angiogenesis [28]. According to pertinent research, solid tumors are unable to enlarge past 2–3 mm without triggering their own blood flow. According to this theory, tumor development and angiogenesis are related [29]. Consequently, angiogenesis is necessary for tumor initiation and development. To develop novel treatments that prevent tumor angiogenesis, it is essential to comprehend the molecular and cellular processes that control this process. The degradation of the basement membrane, along with the proliferation and migration of ECs, contributes to tumor angiogenesis through different processes. The mechanism of tumor angiogenesis is influenced by VEGF and its receptors, which serve as significant pro-angiogenic factors. Through complete engagement with three major receptors for tyrosine kinase activity (VEGFR3, VEGFR2, and VEGFR1), components of the VEGF family have the capacity to activate intracellular signaling pathways. These pathways then have an impact on endothelial cells, causing formation of capillaries and cell mitosis [30]. A number of members of the VEGF family may dramatically boost the proliferation and differentiation of vascular ECs, induce angiogenesis, and improve capillary permeability when combined with VEGFR2 [31]. Through the PLC-γ-MEK-MAPK pathway, VEGFR2 controls the expression of associated genes, resulting in EC proliferation [32]. Via the PI3K pathway, VEGFR-2 may control cell migration. Angiogenesis, endothelial cell survival, and inhibition of endothelial cell apoptosis may all result from, may all result from PI3K/Akt pathway activation by the tumor microenvironment. Through activating the HDM2 and kinases p70S6K1 in tumor tissue, the PI3K/Akt pathway has been shown to regulate the expression of VEGF and HIF-1 [33, 34]. Furthermore, PTEN can promote the PI3K/Akt/VEGF/eNOS signaling pathway, which in turn inhibits tumor angiogenesis [35]. The intricate process of tumor angiogenesis relies on the cooperative action of several regulatory elements [36]. FGF-2, SDF-1α/β, VEGF-A, and VEGF-2 may all be released in large quantities by HIF-1 activating cells within a tumor’s internal environment. These elements promote the development and remodeling of blood vessels [37]. MMPs from pre-stimulated angiogenic cells break the basement membrane and accelerate vasculogenic mimicry (VM). Stable vascular networks affect tumor growth. Platelets release TGF-α, ANG-1, and PDGF-BB to stabilize and mature the complex vascular network. Figure 2: Tumor angiogenesis signaling pathways [38].

MicroRNA biogenesis
In animals, canonical miRNAs are encoded either as single genes or they exist as clusters of miRNAs ranging in number from a couple of hundred to several hundreds. Polycistronic transcripts, containing clustered miRNAs, undergo processing to produce mature miRNAs. The introns of many genes that code for proteins contain miRNAs [39–41]. Nevertheless, it is unusual for miRNAs to have functional connections with host genes. In most cases, RNA Pol II transcribes miRNA genes to produce primary miRNAs (pri-miRNAs), which are capped and polyadenylated [42]. The mature miRNA contains 20–25 nucleotides that are lodged in the stem of a hairpin seen on the precursor miRNA (Fig. 2). When it comes to the first step of the miRNA synthesis process, a nuclear protein complex known as the microprocessor is responsible for converting pri-miRNAs into single hairpins known as pre-miRNAs [43]. Less well understood supplementary factors include the DDX17 and DDX5, the dsRBP DGCR8; in flies, Pasha, and the RNase III [44–47]. Exp5 is the export receptor that directly interacts with pre-miRNAs, facilitating their transportation to the cytoplasm [48–50]. A dsRNA measuring 20–25 nucleotides in length, originating from the pre-miRNA stem, is enzymatically processed in the cytoplasm by the RNase III-type enzyme Dicer [51, 52]. Dicer and the dsRBP TRBP; or TARBP2 work together in humans, while the miRNA-Dcr1 and the dsRBP Loquacious interact jointly in Drosophila melanogaster. There are four Dicer-like enzymes in Arabidopsis thaliana (DICER-LIKE 1 (DCL1)-DCL4) but lack the Drosha enzyme. DCL1, which is one of these enzymes, is responsible for the processing of pre-miRNAs in conjunction with the dsRBP HYPONASTIC LEAVES 1, which is essential for the accurate cleavage of miRNA. This suggests that pre-miRNAs adhere to broadly mechanistic capabilities shared by species that are very distant in evolutionary terms [53, 54]. Finally, a protein from the Argonaute family is recruited during RISC loading to assume control of the ds-miRNA generated by Dicer. This protein chooses one strand to convert into the mature miRNA, which is referred to as the guide strand, while discarding the other strand, which is known as miRNA* [55]. RISC is formed when AGO proteins that have been encoded with miRNAs separate from Dicer. Box 1 summarizes the mechanisms underlying miRNA-mediated gene silencing. Recent evidence indicates that Exp5, Dicer, and Drosha are all integral to the miRNA biogenesis process. Based on recent studies [56], separate reductions of the three proteins have been carried out in a cell line, and subsequent sequencing of miRNAs was performed. Drosha silencing stopped the transcription of canonical miRNAs, but several remained detectable in Dicer-knockout cells, suggesting that alternative pathways can still synthesize them. Also, miRNA biogenesis was barely affected by Exp5 deletion, suggesting that other export routes are in play [56]. These findings suggest that these apparently widely recognized mechanisms might be considerably more complicated than earlier believed, and that additional study is necessary to explain miRNA biogenesis [57].

MicroRNAs and angiogenesis in CRC
Some cancers, including CRC, may have hypoxia as a trigger in their development. Hypoxia may inhibit cell death, which results in cancer proliferation, resistance to apoptosis, and decreased probability of survival [58]. Li et al. proposed in an article that miR-148a suppresses the expansion, cell death, spread, and invasion of malignancies through specific inhibition of ROCK1 and BCL-2 [59, 60]. another study indicated that miR-148a restricts the EMT of hepatic carcinoma through the selective inhibition of c-Met [61]. Recent studies indicate that miR-148a restricts tumor development and decreases the possibility of initial tumor regrowth [62], attenuates the resistance to chemoradiation, and promotes cell death by interacting with c-Met in CRC sufferers [63]. Additionally, it implicitly prevents VEGF release by selective suppression of HIF-1α [64], and demonstrates an apoptotic influence through modifications in Mcl-1 transcription level in CRC [65]. According to Xu et al. (2018), the Met/ERK/HIF-1α/VEGF-A system is inhibited when chemokine CC ligand 19 (CCL19) promotes miR-206, which in turn suppresses CRC Neovascularization [66]. Researchers demonstrated that Met modulates HIF-1α levels through a protein synthesis pathway [67]. A prior investigation revealed Rho/ROCK cascade is critical for HIF-1α transcription within ovarian cancer cellular models and serves as a primary mediator influencing angiogenesis in ovarian malignancies [68]. By specifically targeting the promoter area of Mcl-1, Wu et al. (2020) suggest HIF-1α may improve Mcl-1 transcription, allowing it to function as a gene regulatory element [69].
Tsai et al. found that miR-148a reduces Mcl-1 and VEGF secretion by modulating ROCK1/c-Met and downregulating HIF-1α in hypoxic conditions [70]. MiR-148a significantly decreased ROCK1 and Met/HIF-1α/Mcl-1 axis synthesis in HT29 and HCT116 CRC cell lines. The angiogenic tube formation assay showed that miR-148a inhibited neovascularization and improved bevacizumab’s efficacy. The cell viability analysis revealed the inhibitory effect of miR-148a on the HT29 and HCT116 cell lines. miR-148a and bevacizumab demonstrated collaborative cancer-inhibiting properties in-vivo. The levels of blood miR-148a in mCRC individuals displaying limited cure were elevated compared to that in mCRC individuals experiencing advancement of the disease. The findings indicated that miR-148a diminished vascularization and induced apoptosis in CRC cells by downregulating Mcl-1 and HIF-1α/VEGF through selective inhibition of ROCK1/c-Met. Additionally, blood miR-148a levels possess prognostic and predictive significance for individuals with metastatic CRC undergoing bevacizumab treatment [70].
An oncomiR, miR-1290, is is expressed at elevated levels in CRC in comparison to healthy individuals, which promotes tumors proliferation [71]. MiR-1290 was identified as an oncogene in various malignancies, including CRC, pancreatic, and a variety of gastrointestinal and solid tumors [72, 73]. Soheilifar et al. found that CRC tissues have higher miR-1290 than adjacent normal margins [74]. When comparing cancers with efficient mismatch repair (MMR) to those without, individuals with stages 2 or 3 develop Drug resistance due to the upregulation of miR-1290 through hMSH2 selective suppressing [75]. Wu et al. explored how miR-1290 induces inflammatory mediator prevention and nuclear polyploidization at CRC by targeting kinesin family member 13B (KIF13B) [76]. Furthermore, miR-1290 has been demonstrated to facilitate the restructuring of malignant cells through engagement of the Wnt cascade mechanism, leading to elevated production of c-Myc and Nanog. Additionally, the transcription of Nanog, Oct4, and Sox2 may be elevated in hypoxic circumstances, that is critical for vascularization of tumors [77]. Higher c-Myc levels in CRC tumors result in elevated transcription of HIF-1α and VEGF, consequently facilitating the development of vessels [78]. miR-1290’s function in controlling THBS1, SCAI, and DKK3 is highlighted in the study of Soheilifar et al. THBS1, an extracellular matrix-embedded glycoprotein, is produced by multiple cell types, such as endothelial cells (EC). Its reduction is significantly linked to enhanced growth and neovascularization in murine models of CRC [79, 80]. SCAI limits the growth of several malignancies, including CRC, by preventing the Metastasis of malignant cells [81]. DKK3 acts as a tumor suppressor and functions as a diagnostic tool for CRC [82], Its restriction enhances growth and vascular development of HUVECs [83]. Possibilities for miR-1290 to promote cancer and angiogenesis in CRC were studied by Soheilifar et al. Lenti-miR-1290 was introduced across HCT116, SW480, and HUVECs [84]. Through bioinformatics evaluation, THBS1 is determined as an innovative estimated target for miR-1290. Western blotting, RT-qPCR, and luciferase reporter assays have been employed for confirming the regulation of miR-1290-modulated genes, such as THBS1, DKK3, and SCAI within HCT116 CRC cells and HUVEC endothelial cells. Cell viability, wound healing, tube formation assays, and flow cytometry have been used to evaluate cell progression, division, metastasis, and tube development. DKK3, SCAI, and THBS1 transcription was significantly reduced by MiR-1290. They found miR-1290 enhances division, metastasis, and neovascularization, primarily by inhibiting DKK3, SCAI, and THBS1. The results suggest a possible novel function for miR-1290 in tumorigenesis and angiogenesis in CRC [84].
Stem-cell-like glioma cells found in CNS malignancies have been shown to enhance neovascularization and cancer development through elevated production of VEGF [85, 86]. VM could act as a significant alternative route for circulation in instances where vascular development declines [87]. Recurrent acid-Schiff-positive and CD31-negative cancer cells form VM systems in vivo and tube-like structures and structured connections in three-dimensional in vitro systems [88]. VM was found in many malignant cancers, including CRC, and induces EMT to develop CSC features [89]. Dang and Ramos identified malignant cells exhibiting cancer stem cell characteristics capable of forming Vessel-like formations in oral squamous cell carcinoma [90]. Vascularization and VM frequently reside together in malignant cancers, neither of which are pertinent for the development of cancer stem cell features.
Regulating CSC features require complicated systems comprising multiple gene expression regulators, including Nanog, Oct4, SOX2, and different miRNAs [91, 92]. SOX2 is pivotal in developmental pathways and is essential for the maintenance of cancer stem cell (CSC) features [93, 94], contributing to malignant cancer development, metastasis, and opposition to typical therapies across different kinds of cancer [95, 96]. Numerous miRNAs are being identified as the preserver of stemness. Variables including miR-371–5p, miR-200c, and miR-638 were known as regulators of SOX2-triggered cancer stem cell features in CRC [97, 98]. Chen et al. examined the significance of SOX2 in modulating CSC features, blood vessel development, and VM in CRC, while also investigating its fundamental processes using animal models and cell culture studies. CRC stemness, vascularization, and VM are linked to the miR-450a-5p-SOX2 axis, suggesting a treatment target.
SOX2 is a gene expression regulator critical for preserving the unique features of CSCs. The study designed to examine the function of SOX2 in regulating CSC features, vascularization, and VM in CRC, along with possible biological mechanisms. SOX2 and miR-450a-5p levels in CRC tumor specimens were evaluated by using immunohistochemistry. An elevated level of SOX2 is linked to poor outcome, but it also facilitates vascular development and VM. The ablation of SOX2 led to the termination of stemness traits, angiogenesis, and vasculogenic mimicry, alongside a decline in the expression of CD31, VE-cadherin, and CD133, as evidenced by in vitro assays. The diminished expression of SOX2 was shown to hinder tumor development in vivo. Moreover, miR-450a-5p targeted SOX2’s 3’UTR, limiting its effects on cancer stem cells, angiogenesis, and VM. Additionally, elevated levels of SOX2 maintained the regulatory blockade exerted by miR-450a-5p on CRC characteristics, blood vessel development, and VM. Clinical evidence demonstrated an opposite relationship among SOX2 and miR-450a-5p. Individuals exhibiting elevated SOX2 levels and reduced miR-450a-5p production demonstrated a less favorable outcome compared to those with the opposite production patterns. In conclusion, they clarified an individual function of the miR-450a-5p-SOX2 axis in regulating cellular self-renewal capacity, angiogenesis, and VM, that could be useful as a potential target for more efficient treatment in CRC [99].
Growth, metastasis, and specialization of endothelial cells are critical processes in vascular development. VEGF, a critical vascularization inducer, facilitates endothelial cell expansion and metastasis. Targeting VEGF, for example through the administration of the commonly used medication bevacizumab, is now identified as a successful innovative method for addressing CRC. Research suggests that SRC, an Intracellular tyrosine kinase, controls vascular development through the SRC-STAT3-VEGF signaling. SRC kinase signaling inhibitor 1 (SRCIN1), an anti-oncogene associated with SRC, plays an essential part in inhibiting cancer development and proliferation. SRCIN1 additionally controls the FAK-mediated signaling [100], the EGFR signaling, and Ras/ERK signaling cascade through the inactivation of SRC [101]. Thus, SRCIN1 is likely implicated in the regulation of CRC vascularization. Sun et al. proved that miR-181a augmented vascularization in CRC, while SRCIN1 suppressed it. MiR-181a targets SRCIN1 in CRC cells. SRCIN1 inhibition by miR-181a activates SRC, increasing VEGF release and vascularization. These results highlighted the significance of SRCIN1 and miR-181a in controlling vascularization and identified miR-181a to be a possible drug target for CRC [102].
Sun et al. investigated the role of miR-181a in promoting CRC angiogenesis [102]. Their findings demonstrated that miR-181a enhances angiogenesis both in vitro, as shown by capillary tube formation assays, and in vivo, confirmed by Matrigel plug assays. Mechanistically, bioinformatics identified the anti-oncogene SRCIN1 as a direct target of miR-181a, a finding the authors validated in CRC cell lines.
To map the downstream pathway, the researchers used RT-qPCR, ELISA, and western blotting. They further confirmed the clinical relevance using patient samples, where fluorescence in-situ hybridization (FISH) and immunofluorescence showed an inverse correlation between miR-181a and SRCIN1 levels. The authors concluded that miR-181a-mediated suppression of SRCIN1 activates the SRC kinase. This activation, in turn, upregulates VEGF production, ultimately driving vascularization. This study thus identifies the dysregulation of miR-181a as a key factor in malignant angiogenesis, suggesting its suppression as a novel anti-angiogenic therapeutic strategy for CRC [102].
It has been determined that the ILF3 genome is the source of transcription for the NF90 group [103]. The two different primary peptide variants have been named NF90 (also referred to as DRBP76 and NFAR1) or NF110 (which is in addition referred to as ILF3, NFAR2, and TCP110), with estimated weights of 90 and 110 kDa, respectively [103]. NF90 and NF110 exhibit homology within their N-terminal and central areas; however, their C-terminal areas are entirely distinct [103]. NF90 has been initially isolated as a Transcription factor that regulates the IL-2 transcription [104]. Nonetheless, its role extends beyond T cells, as NF90 has been shown to influence various other mRNAs. Furthermore, it plays a role in controlling protein synthesis, genomic stability maintenance, RNA modification, host immunity against viruses, and mitosis [105]. Recent findings indicate that NF90 influences vascular development in breast cancer, while the NF90/NF45 structure facilitates E6 carcinogen upregulation in cervical carcinoma cells altered from human papilloma virus [106]. The regulation of miR-590-5p is implicated in tumor formation, with evidence indicating that this gene may function as an oncogene in cervical cancer and as a suppressor of cancer growth in renal carcinoma [107, 108].
Zhou et al. examined the effects of differing levels of miR-590-5p in cancerous and healthy cells [109]. The outcomes indicated that miR-590-5p concentrations are reduced in human CRC tumors compared to adjacent healthy tissue. Likewise, they found that miR-590-5p suppression accelerated CRC growth using their xenograft mice model. Conversely, the overexpression of miR-590-5p resulted in a reduction of angiogenesis, tumor progression, and metastatic spread of malignancy to the lungs in mice. NF90 functions as an upregulator of VEGF mRNA stability and peptide synthesis, and has been recognized as the principal target of miR-590-5p. Increased NF90 levels restored VEGFA production and restored tumor vascularization after miR-590-5p decreased it. NF90-shRNA diminished the tumorigenicity induced by the miR-590-5p inhibitor. MiR-590-5p exhibited an inverse correlation with NF90 and VEGFA in CRC cells. A suppressive feedback mechanism was suggested by NF90 deficiency’s decrease in pri-miR-590 and increase in mature miR-590-5. Because it blocks CRC neovascularization and spread via a unique NF90/VEGFA route, the results imply that miR-590-5p could be a therapeutic choice for human CRC [109].
Oligosaccharides, glycoprotein or glycolipid sugar chains, or GDP-fucose can be transferred to oligosaccharides by members of the FUT family of fucosylation synthases [110, 111]. The FUT gene presents an intriguing therapeutic possibility due to its role in modulating glycan structures (fucosylation) on the cell surface [112]. A group consisting of three genes (FUT3, FUT6, and FUT5) is located in 1 cM in the human chromosomal region 19p13.3 [113, 114], and exhibits over 90% sequence identity [115, 116]. Due to the above physiological features, these genes exhibit similar physiological roles [117]. FUT3, FUT5, and FUT6 are linked with the incidence and spread of GI cancer, specifically through variations in the levels of α−2,3-sialyltransferases and α−1,3/4 [118, 119]. Recent investigations indicate that elevated FUT3 levels in CRC facilitates tumors spread [112]. According to Liang et al., FUT5 and FUT6 could encourage CRC metastasis and development. Furthermore, earlier investigation indicates that FUT might be controlled by miRNA in cancer of the breast [120]. The study examined the regulation of FUT6 and FUT5 through miRNAs in CRC.
Liang et al. investigated the expression levels of FUT and miR-125a-3p among CRC cell populations and a pair of human CRC cell lines using q-PCR [121]. The findings indicate that, FUT6, FUT5, and miR-125a-3p exhibit variation in expression in typical versus tumor cells. FUT regulation by miRNA has emerged to affect the progression and spread of breast and hepatocellular tumor cells, as indicated by earlier studies. The authors hypothesized that FUT6 and FUT5 could be controlled through miR-125a-3p. Luciferase reporter assays have been utilized for discovering possible regulated genes of miR-125a-3p. Further research demonstrated that an elevated level of miR-125a-3p restricts the development, metastasis, spread, and vascular development of CRC cells by suppressing FUT5 and FUT6. FUT6, miR-125a-3p, and FUT5 levels significantly altered the PI3K/Akt signaling process, but transfection with miR-125a-3p-mimics reversed their effects. FUT5 and FUT6 cannot accelerate CRC development via the miR-125a-3p-regulated PI3K/Akt signaling pathway. MiR-125a-3p may be a biomarker for CRC treatment [121]. Table 1 presents a compilation of miRNAs implicated in the vascular development of CRC cells.

Interaction between MiRNAs and LncRNAs in CRC: focus on angiogenesis
LncRNAs primarily exert their physiological functions by effectively sequestering microRNAs. One way to silence target genes’ activity is through the RISC, that consists of sequences that are compatible between microRNAs and their target RNAs [41]. LncRNAs and miRNAs additionally have a dynamic binding interaction. LncRNA PVT1 modulates the subsequent gene of interest RUNX2 via both the miR-30d-5p/RUNX2/PVT1 axis [157] and the miR-455/RUNX2/PVT1 axis [158, 159]. N6-methylation, the predominant epigenetic modification, is present in approximately 90% of RNAs. It serves as a key regulator for regulating mRNA expression, preparation, degradation, and protein synthesis, as well as influencing various ncRNAs. Changes in m6A are pivotal regulators in the formation and development of multiple illnesses, like glioblastoma, CVD, and CRC [160–164]. M6A epigenetic alteration is an active and reversible procedure primarily caused by methyltransferases (“writers”), demethylases (“erasers”), or m6A-binding proteins (“readers”) recognition enzymes, which serve to promote, exclude, and detect the m6A methylation site, accordingly. The topic of lncRNA methylation regulation is gaining more and more attention in the scientific literature. LncRNA FAM225A exhibited upregulation in nasopharyngeal carcinoma (NPC) Malignant tissues, and higher transcript rate of FAM225A showed a connection with unfavorable a medical outcome. MeRIP investigations demonstrated that knockdown of METTL3 leads to a reduction of m6A levels in FAM225A, which was accompanied by less stable synthesis and transcription of FAM225A [165]. Research has highlighted the important function of HNF1A-AS1 in gastric cancer, NSCLC, and HCC [166–169]. In CRC, research indicates that HNF1A-AS1 primarily exerts physiological functions by Cross-regulating interactions with miRNAs, including miR-34a and miR-124 [170, 171]. Their study thoroughly examined the processes that explain how HNF1A-AS1 regulates the cell cycle in CRC, a topic that had never been earlier investigated. Functional tests demonstrated that HNF1A-AS1 can enhance development, metastasis, and vascular development in CRC. Mechanistically, by building the AGO2/HNF1A-AS1/miR-93-5p framework, HNF1A-AS1 was found to function as a sponge for miR-93-5p, therefore causing the following upregulation of CCND1. Additionally, it can maintain CCND1 mRNA structure through METTL3-triggered m6A modulations. HNF1A-AS1 might inhibit PDCD4, thereby regulating CCND1 through the PI3K/AKT signaling cascade activation. So, evidence revealed that HNF1A-AS1 could function as a diagnostic marker and an innovative prognostic indicator in CRC.
Bian et al. conducted research analyzing HNF1A-AS1 in CRC patients and cell lines through RT-qPCR [172]. HNF1A-AS1’s role in CRC progression has been studied using tube forming assays, transwell assays, CCK8 assays, flow cytometry, colony formation assays, and animal models. Luciferase report assays, bioinformatic assessment, RIP assays, and RNA pull-down assays have been used to identify the HNF1A-AS1 target peptide and possible downstream targets. The findings suggest that HNF1A-AS1 had been elevated in CRC and correlated with a poor outcome. HNF1A-AS1 enhanced growth, metastasis, and vascularization, expedited cellular period, and decreased cell death in CRC. Bioinformatics estimates and additional research demonstrated that HNF1A-AS1 can enhance CCND1 levels through inhibiting PDCD4 or by absorbing miR-93-5p in a competitive manner. METTL3 facilitated the changes in m6A of HNF1A-AS1, thereby influencing its RNA durability. CCND1, HNF1A-AS1, IGF2BP2 and may function as an integrated system for controling the durability of CCND1. The findings indicate an innovative process whereby the m6A-associated IGF2BP2/CCND1/HNF1A-AS1 Signaling facilitates CRC cellular development. This transpires via the sequestration of miR-93-5p, resulting in the upregulation of CCND1. This article discusses HNF1A-AS1’s role in cellular progression and its potential as a CRC prognostic indicator [172].
The CTCF functions as a DNA-attachment protein that regulates both three-dimensional genome structures and critical elements of DNA transcription [173]. CTCF exhibited upregulation in CRC samples relative to near healthy CRC tissues. Its elevated levels enhanced the growing capacity of CRC cells and tumor expansion, indicating that CTCF may serve as a possible indicator and treatment option for CRC [174]. Given the established roles of CTCF and OGFRP1 in CRC cells, as well as the bioinformatics-based estimation which says miR-423-5p targets CTCF directly, this study aims to confirm the interplay between OGFRP1, CTCF, and miR-423-5p during EMT, vascular development and cellular metastasis [175].
The levels of CTCF, OGFRP1 and miR-423-5p have been assessed in NCM460, a common CRC cell lines [175]. Flow cytometry, Colony formation assays, CCK-8, Scratch assay and Western blotting have been utilized to examine the growth, cell death, EMT, spread, and metastasis of HCT116 and SW620 cells following gain- and loss-of-function studies. The modified cells were co-cultured with HUVECs to test tube formation for vascular development. ELISA detected VEGF in HCT116 and SW620 cell conditioned medium. A dual-luciferase reporter assay confirmed CTCF, miR-423-5p, and OGFRP1 associations. CRC tissues exhibited higher transcript rates of CTCF and OGFRP1, alongside a reduced production amount of miR-423-5p, in comparison to NCM460 cell type. In HCT116 and SW620 cells, inhibiting OGFRP1 or CTCF and overproducing miR-423-5p decreased growth, EMT, spread, and metastasis and increased cell death. Vascular development was reduced in HUVECs grown with miR-423-5p mimic, si-CTCF, or si-OGFRP1. Inhibiting miR-423-5p may help CRC cells’ OGFRP1 levels. OGFRP1 and CTCF bind miR-423-5p. OGFRP1 promotes CRC growth, angiogenesis, and EMT via miR-423-5p/CTCF [175].
LINC00467 was identified as a promoter of malignancy formation through the adsorption of miRNA. In HNSCC, LINC00467 acts as a ceRNA by sequestering miR-1285-3p, which controls the synthesis of TFAP2A. This mechanism promotes metastatic characteristics in the malignancy and suppresses cell death in the tumor cells [176]. Furthermore, LINC00467 may facilitate the advancement of prostate carcinoma through the miR-494-3p/STAT3 signaling cascade [177]. Furthermore, research indicates that miR-128-3p functions as a cancer inhibitor for multiple genes in CRC [178, 179].
According to the theory put up by Chang et al., LINC00467 regulates miR-128-3p/VEGFC, which in turn has an effect on CRC. The study effort was to examine the function and process of LINC00467 in CRC and to present an empirical foundation for understanding the mechanisms behind the development of such serious illness.
RT-qPCR indicated that LINC00467 synthesis amount within CRC tissue samples and in vitro models were considerably enhanced, correlating tightly with the clinical characteristics of CRC [180]. Reducing LINC00467 expression in CRC cells inhibited growth, angiogenesis, and metastasis in vitro and in vivo. The expression of LINC00467 increased CRC tumorigenesis. Bioinformatics modeling and luciferase reporter assays showed LINC00467’s miR-128-3p interaction. Rescue experiments showed that lowering miR-128-3p levels restored CRC cell function after LINC00467 was cut off. VEGFC is miR-128-3p’s target, which may help overcome cell development suppression. The results obtained indicated that LINC00467 functions in CRC development and vascular development through the miR-128-3p/VEGFC pathway. Their results enhance the comprehension of the biological processes involved in CRC and propose possible targets for medical approaches addressing CRC [180].
Several studies have elucidated the functions of lncRNAs in cancer growth and tumorigenesis. UCA1 is one of the most thoroughly investigated lncRNAs in relation to cancer. Initially determined as an cancer-promoting lncRNA in Urothelial cancer [181], the function of UCA1 is documented through investigations related to multiple cancers, like HCC [182], breast cancer [183, 184], NSCLC [185], RCC [186], gastric cancer [187] and CRC [188, 189]. These investigations demonstrated that UCA1 enhances cell growth, facilitates cell spread and metastasis, and inhibits cell death. The biological processes and related mechanisms associated with UCA1-associated cancer advancement, specifically in CRC, remain inadequately understood. a significant process of lncRNA is their potential role as biological sponges for microRNAs. As informed by LncCeRBase [190], over 300 papers have documented over 430 connections among lncRNAs and microRNAs. These associations result in a reduction of microRNAs, restore the expression of the protein-coding targets of microRNAs, and subsequently influence various physiological functions. New investigations have shown that UCA1 interacts with miR-495. UCA1 may facilitate cell growth [186, 191], increase metastasis [191] and inhibit cell death [192]. MiR-495 is already widely documented to perform inhibitory functions and influence multiple facets of cancer, including cell death [193], resistance to drugs [194], metastasis [193, 195, 196], metabolic processes [197] and development [195]. Consequently, miR-495 might be an indirect objective of UCA1, which promotes cancer growth through UCA1. Dysregulation of SP1 and SP3 is observed in several types of cancer [198]. SP1 facilitated the advancement of CRC cellular growth via the regulation of lipid synthesis [199]. Numerous pharmacologic substances demonstrated anti-CRC-like effects via SP1 inhibition [200, 201]. SP1 has the potential to change the organization of chromatin and influence gene activity by attaching to GC boxes via its Cys2/His2 zinc fingers [202]. SP3 and SP1 share a highly preserved DNA attachment region and biological function. MiR-495 binds to the SP1 3′ UTR, restricting protein synthesis [203]. Furthermore, UCA1 may function as a sponge for miR-495. Consequently, it is suggested that UCA1 may function as a ceRNA to control SP1/SP3 associated processes involved in tumor development in CRC. Using samples of tissue and cell lines from CRC, the current study investigates the UCA1-miR-495-SP1/SP3 cascade and its effects on EMT and DNMT-related tumoral characteristics.
In the research by Liu et al., shRNAs were used to reduce some genes [204]. MicroRNA mimic and pcDNA-UCA1 plasmids have been employed for the increased levels of miR-495 and UCA1, accordingly. MTT has been used to assess cell survival and responsiveness to FU. Transwell assays were performed to evaluate cell dissemination and metastasis. Neovascularization has been evaluated via tube formation analysis. Protein identification was performed using Western blotting, while quantitative PCR was employed for mRNA monitoring. Dual-luciferase assays examined miR-495, UCA1, and SP1/SP3 interactions. RNA pulldown was used to study miR-495-UCA1 interaction. UCA1 expression increased significantly in CRC tissues. In CRC cell lines, UCA1 increased angiogenesis, dissemination, metastasis, cell proliferation, EMT, and 5-fluorouracil resistance. UCA1 levels inversely correlated with MiR-495. The findings showed that UCA1 sequestered miR-495, inhibiting SP1/SP3 synthesis. SP1/SP3 induced DNA methyltransferases, which helped form UCA1-associated tumors. For the purpose of examining the interactions that take place between MiR-495, UCA1, and SP1/SP3, the dual-luciferase assay employed was utilized. The utilization of RNA pulldown was employed in order to conduct an investigation into the interaction that exists between miR-495 and UCA1. CRC tissues showed a significant increase in UCA1 expression [204].
A prior investigation indicated that LINC01232 may in competition attach to miR-654-3p, leading to a reduction in its production in ESCC cells, which in turn promotes the synthesis of HDGF [205]. The control of miRNA because of LINC01232 in CRC remains unclear. Yuan et al. conducted a bioinformatics evaluation revealing the fact that LINC01232 has the capacity to attach to miR-181a-5p. The role of miR-181a-5p in the development of COAD was recently established [206, 207]. They suggested that miR-181a-5p regulates LINC01232’s effect on COAD progression. This study examined miR-181a-5p and LINC01232 expression in CRC adenocarcinoma and normal cells. MiR-181a-5p and LINC01232 roles in COAD development were then assessed by altering their gene activity. Their research may reveal a key COAD drug target.
The research conducted by Yuan et al. aimed to investigate the biological processes that involve LINC01232 to the development of COAD [208]. The findings indicate that LINC01232 has significant levels in COAD cells and promotes development, vascular development, spread, and metastasis of these cells. Downregulation of LINC01232 enhanced the synthesis of p53 and p16, whereas suppressed the biosynthesis of c-myc, cyclin D1, and Bcl-2in COAD tissues. Conversely, elevated levels of LINC01232 produced different outcomes. LINC01232 exhibited an opposite relationship with miR-181a-5p, same as the downregulation of miR-181a-5p had the ability to counteract the impact of siLINC01232 on cell growth, vascular development, spread, and metastasis. A miR-181a-5p mimic can also counteract LINC01232’s increased expression. SiLINC01232 increased Bax and E-cadherin and decreased vimentin, VEGF, SDAD1, and N-cadherin, with limited attenuation by miR-181a-5p blocker. LINC01232 suppresses miR-181a-5p synthesis to improve COAD cell growth, spread, metastasis, and vascularization [208]. Table 2 presents a compilation of lncRNA implicated in the vascular development of CRC cells.

Interaction between circular RNAs and MiRNAs in CRC: focus on angiogenesis
At first, Guo et al. looked at circ3823, a gene labeled as hsa_circ_0001821 in RNA-seq, to see whether it might be a diagnostic biomarker for CRC. Their research showed that circ3823 inhibited the expression of miR-30c-5p, which resulted in angiogenesis, proliferation, and metastasis of CRC. This was accomplished by increasing the expression of TCF7 and its targets, CCND1 and MYC. Furthermore, the m6A alteration was found in circ3823. The m6A recognition protein YTHDF3 and the demethylase ALKBH5 controlled circ3823 degradation. Their research suggests that circ3823 may be a CRC diagnostic or therapeutic target [213]. Differences in circRNA expression between CRC tumor and non-tumor tissues were examined using high-throughput sequencing in the research by Guo et al. [213]. In situ hybridization and qRT-PCR were used to measure circ3823 in CRC tissues and blood. We then used functional in vitro and in vivo assays to examine how circ3823 affects CRC tumorigenesis, metastasis, and angiogenesis. The ring structure of circ3823-2 was validated through Sanger sequencing, RNase R analysis, and the Actinomycin D assay. The mechanisms of circ3823 were validated using RNA pull-down studies, RIP, FISH, and dual luciferase reporter assays. Patients with CRC exhibiting elevated circ3823 expression experienced a poorer prognosis. According to ROCs, serum circ3823 expression had good specificity and sensitivity for identifying CRC, suggesting it could be used as a diagnostic biomarker. Functional studies show Circ3823 promotes CRC cell proliferation, angiogenesis, and metastasis in vivo and in vitro. Circ3823 competes with miR-30c-5p to reduce TCF7 repression. MYC and CCND1 upregulation accelerates CRC. Circ3823 had m6A modifications. The m6A modification regulates circ3823 degradation. The circ3823/miR-30c-5p/TCF7 axis promotes CRC development, angiogenesis, and metastasis, suggesting it may be a novel CRC marker or treatment target. Furthermore, the m6A modification regulates circ3823 degradation [213]. Chen et al. studied circ-ERBIN. It was made from ERBIN (ERBB2IP) exons 2–4. By interacting with miR-138-5p and miR-125a-5p, circ-ERBIN enhances CRC angiogenesis, metastasis, invasion, and proliferation. Combined upregulation of 4EBP-1 increases HIF-1α protein expression and activates the pathway. The study discovered that circ-ERBIN disrupts the HIF-1α pathway, revealing its role in CRC progression [214]. They employed qRT-PCR to identify circRNA and messenger RNA expression in CRC cells and tissues [214]. The location of circ-ERBIN was examined using FISH. In vivo and in vitro studies using circ-ERBIN knockdown and overexpression cell lines examined EdU assay, CCK8 colony formation, transwell, cancer progression, and metastatic models. We used IHC, western blots, and luciferase reporter assays to study mechanismsBoth in vivo and vitro studies proved that an overexpression of Circ-Erbin promoted the growth, movement, and metastasis of CRC cells. Studies done both in vitro and in vivo showed that an overexpression of Circ-Erbin promoted the spread, migration, and metastasis of CRC cells. CRC cells contain an excessive amount of Circ-Erbin. Overexpression of circ-Erbin in CRC results in an increase in the production of HIF-1α and a boost in angiogenesis. In CRC cells, Circ-Erbin functions as a sponge for miR-138-5p and miR-125a-5p, targeting 4EBP-1 and accelerating cap-independent HIF-1α protein translation. The research identified that the miR-125a-5p/miR-138-5p/4EBP-1 axis is crucial for circ-ERBIN-mediated activation of HIF-1α, indicating its potential as a target for CRC treatment [214]. Chen et al. studied circ_0000467’s role in CRC; miR-4766-5p inhibited tumor growth in both GC and CRC [215, 216]. Whether circ_0000467 can serve as a miR-4766-5pin CRC sponge is an open question, though. In CRC, KLF12 expression was elevated, and this upregulation aided cell proliferation [217]. Within the scope of this study, the hypothesis that miR-4766-5p targeted KLF12 was investigated. One of the most important functional mechanisms in CRC is the regulatory network of circRNA, miRNA, and mRNA [218]. They focused on circ_0000467’s connection axis with KLF12 and miR-4766-5p in addition to its biological function in CRC. The levels of circ_0000467, miR-4766-5p, and KLF12 were investigated in the study by Chen et al. utilizing q-RTPCR [219]. It was determined that the colony formation assay and the cell counting kit-8 were both effective methods for determining the rate of cell proliferation. Flow cytometry was utilized in order to measure apoptosis. The invasion and migration tests performed on transwell plates were used to evaluate cell metastasis. The test for tube formation revealed the presence of angiogenesis. For the purpose of determining the percentages of all protein expressions, the western blot was utilized. An investigation into intergenic binding was carried out using a dual-luciferase reporter assay. In order to conduct the in vivo experiment known as circ_0000467, xenograft models were constructed. It was discovered that CRC tissues and cells had a high level of Circ_0000467 expression. While circ 0000467 knockdown was successful in killing CRC cells, it was also successful in reducing cell growth, metastasis, and angiogenesis. Circ_0000467 reduced miR-4766-5p expression. Circ_0000467 reduced colorectal cancer by upregulating miR-4766-5p. Although miR-4766-5p inhibited KLF12 expression, overexpression of the target gene negated its effects on CRC cells. KLF12 gene expression increased with miR-4766-5p targeting by Circ_0000467. Circ_0000467 downregulation in vivo reduced the carcinogenesis of CRC through the use of KLF12 and miR-4766-5p. CRC was brought on by the regulation of miR-4766-5p KLF12 that was mediated by sponges. Therefore, circ_0000467 may be helpful in the diagnosis and treatment of CRC [219].
According to the GEO databases, Circ_0084615, which is generated from ASPH, is elevated in CRC patient samples. Nevertheless, exactly what circ_0084615 does in CRC is still mostly unknown.
Involved in tumor growth are miRNAs, a family of ncRNAs of 18–22 nucleotides [220]. Glioma, bladder urothelial carcinoma, gastric cancer, and anaplastic thyroid carcinoma have been suppressed by miR-599 [221, 222]. In addition, the miR-599/lncRNA MCM3AP-AS1/miR-599/ARPP19 axis was involved in the development of CRC cells [223]. However, the exact ways in which miR-599 mediates PC are still not well understood. One cut homeobox 2 (ONECUT2, or OC2) is linked with cancer cell growth, motility, and differentiation [224]. In CRC, ONECUT2 acted as a target of miR-429, which in turn affected growth, invasion, and EMT [225]. Bioinformatics research revealed that the miR-599 protein contains the circ_0084615 and ONECUT2 binding sites. The connections between circ_0084615, miR-599, and ONECUT2 are not well established. Jiang et al. identified the links between ONECUT2, miR-599, and circ_0084615 in CRC formation and found their expression patterns in CRC. Their research was conducted with the intention of examining the functions and procedures of circ_0084615 in CRC environments [226]. For the levels of miR-599, circ_0084615, EIF4A3, or ONECUT2, as well as for cell proliferation capacity, they used qRT-PCR, western blotting, and IHC assays. Cell invasion and migration were assessed using wound-healing and transwell tests. Tube formation assays assess angiogenesis. RIP, RNA pull-down, and dual-luciferase reporter assays examined EIF4A3, ONECUT2, miR-599, and circ_0084615. Circ_0084615 was tested on mice xenografts. Circ_0084615 was higher in CRC tissues and linked to lymph node survival, metastasis, differentiation, and TNM stages. Reduced Circ_0084615 expression decreased CRC cell migration, angiogenesis, invasion, and carcinogenesis in vitro and in vivo. Sponged circ_0084615 knockdown and MiR-599 inhibition reduced CRC cell angiogenesis, proliferation, and motility. It was found that miR-599 targets ONECUT2. Overexpressing ONECUT2 restored miR-599’s CRC malignancy effect. Importantly, EIF4A3 upregulated circ_0084615. Via miR-599/ONECUT2, EIF4A3-induced circ_0084615 promoted CRC [226].
Hsa_circ_0081069 is a recently discovered circulation that the majority of cancer researchers are not aware of. In a GEO database analysis of CRC ncRNA profiling, hsa_circ_0081069 exhibited elevated levels in CRC tissues compared to adjacent normal tissues (GSE197991). Backsplicing from COL1A2 pre-mRNA produces Hsa_circ_0081069. Supplementary Figure S1B, C shows that CRC tissues express COL1A2 extensively and CRC patients have poorer survival [227, 228]. Therefore, Xie et al. suggest that hsa_circ_0081069 may be crucial to CRC control.
CRC tissues and cell lines expressed more Hsa_circ 0081069. Reduced hsa_circ 0081069 expression reduced CRC cell invasion, migration, and angiogenic potential. They then found miR-665 and E2F3, downstream components that cause cancer in hsa_circ_0081069 CRCcells. Research suggests that targeting the hsa_circ_0081069 and miR-665/E2F3 axis may be a promising treatment for CRC. Xie and colleagues investigated whether hsa_hsa_circ_0081069 affects CRC progression [12]. Q-RTPCR was used to measure gene expression. The functional significance of the ShRNA-mediated silencing of hsa_circ_0081069 was evaluated through the utilization of the CCK-8 proliferation, Transwell migration and invasion, and tube formation assays. Xenograft mice were used to study CRC cell tumorigenicity and metastasis. Hsa\_circ\_0081069 was found to be more common in CRC tissues and cells. Hsa_circ_0081069 downregulation in CRC cells inhibited migration, invasion, angiogenesis, and proliferation. Silencing of Hsa_circ_0081069 reduced tumorigenesis of colorectal cancer cells in an alternative xenograft animal model. Hsa_circ_0081069 has also been demonstrated to adversely affect miR-665, which is an interacting partner of hsa_circ_0081069. MiR-665 suppressed E2F3 expression by binding to its messenger RNA. They showed that the miR-665/E2F3 axis was responsible for hsa_circ_0081069’s functional involvement in controlling CRC cells’ malignant phenotype. Taken as a whole, their findings provide more evidence that hsa_circ_0081069 may be useful as a marker for CRC prognosis. The miR-665/E2F3 and hsa_circ_0081069 axis may be targets for future CRC treatments [12].
Researchers recognize that miRNAs possess significant potential as therapeutic and diagnostic biomarkers for CRC and are integral to the disease’s progression [229, 230]. MiR-149-5p is shown to be downregulated in CRC cells, which results in the inhibition of the motility of CRC cells through the targeting of BGN, as demonstrated by a previous investigation [231]. On top of that, CRC cells expressed SLC38A1, and inhibiting its expression prevented the proliferation and migration of CRC cells [232]. There is currently no information available regarding the connections that exist between miR-149-5p, circ_0003602, and SLC38A1. Circ_0003602 was the subject of Wu et al.‘s investigation into its effectiveness in CRC. In addition to that, additional research was carried out to investigate the possible modulatory network of circ_0003602 in CRC. Consequently, this research has the potential to usher in a new era in the treatment of CRC. An investigation into Hsa_circ_0003602 will be carried out in order to ascertain the part that it contributes to the progression of CRC cell [5]. Q-PCR detected circ_0003602, miR-149-5p, and SLC38A1 expression. To characterize circ_0003602, RNase R tests were run. For invasion, cell viability, apoptosis, angiogenesis, and migration, CCK-8 tests, wound healing assays, tube formation assays, transwell invasion assays, flow cytometry analysis, and others were used. All protein levels were measured by western blotting and immunohistochemistry. The metabolism of glutamine was monitored using glutamate, α-ketoglutarate, and glutamine test kits. Dual luciferase reporter experiments confirmed miR-149-5p and circ_0003602 or SLC38A1 targeting. We used a xenograft tumor model to study circ_0003602’s role in CRC tumor development. Circ 00003602 was upregulated in CRC cells and tissues. Circ_0003602 silencing prevented cancer cell angiogenesis, invasion, migration, glutaminolysis, and cell death in labs and living organisms. By downregulating circ_0003602, miR-149-5p was directly increased, reducing CRC cell cancerous activity. SLC38A1’s 3’ UTR was directly degraded by MiR-149-5p. Overexpression of MiR-149-5p, which led to a reduction in SLC38A1, was found to reduce the risk of colorectal cancer cell cancer. Circ_0003602 induced an increase in the expression of SLC38A1 in CRC cells by sponging miR-149-5p. Through the miR-149-5p/SLC38A1 axis, Circ_0003602 knockdown inhibits colorectal cancer formation, providing new insights into treatment and a theoretical foundation [5]. Table 3 presents a compilation of circular RNAs implicated in the vascular progression of CRC cells.

Sorting microRNAs into exosomes: exosomal microRNAs and angiogenesis in CRC
The new microRNA N-72, which targets MMP2 in hAMSCs, is shown to regulate EGF-induced migration [240]. However, the connection between miRNA N-72 and cancer remains unclear. According to a prior study by Li et al., serum levels of miRNA N-72 were elevated in CRC patients compared to healthy individuals, indicating its potential impact on CRC progression.
The researchers found miRNA N-72 in CRC cell and serum. When present, miRNA N-72 increased EC migration, permeability, and tube-forming [241]. In vivo, miRNA N-72 was demonstrated to promote tumor development, angiogenesis, and metastasis of CRC using animal xenograft models. MiRNA N-72 was found to be transported into ECs through exosomes, blocking cell junctions by targeting CLDN18 expression, ultimately leading to tumor progression. This discovery suggests a novel CRC angiogenesis mechanism and the potential of secreted miRNA N-72 as a biomarker and therapeutic target. The research emphasizes the significance of comprehending the correlation between miRNA N-72 and cancer advancement [241]. MiR-21 is involved in various human malignancies, which include NSCLC, ovarian cancer, and CRC [242–244]. It targets tumor suppressor genes, facilitating the development of malignant biological behavior [242, 243]. Even though miR-21-3p and miR-21-5p share a common ancestor, miR-21-5p has received the lion’s share of attention in the literature regarding miR-21, perhaps due to the stability and utility it provides [242–246]. MiR-21 expression in CRC cells enhances proliferation, migration, and incursion [243, 244]. Latest research demonstrated that miR-21 expression is notably enhanced in exosomes from CRC patients’ plasma, which is related to liver metastases and TNM stage [247, 248]. Additionally, Recipient endothelial cells may have an enhanced ability to form tubes if exosomes generated by adipose-derived stem cells overexpressing miR-21 are any indication [249]. At this time, the effects of exosomal miR-21-5p on CRC vascular ECs are unknown.
He et al. found that vessels near CRC overexpressed miR-21-5p, a protein involved in vascular permeability and angiogenesis, and correlated positively with its expression in cancer epithelium. Exosomes carried MiR-21-5p from CRC cells to ECs, increasing its expression [250]. The microRNA MiR-21-5p inhibits KRIT1 in recipient HUVECs, activates β-catenin signaling, and increases Ccnd1 and VEGFa, promoting angiogenesis and vascular permeability in CRC. The vasculature near the CRC had a negative correlation between KRIT1 and miR-21-5p. Exosomes from CRC patients expressed more MiR-21-5p than those from healthy donors. Exosomal miR-21-5p affects vascular permeability and angiogenesis in CRC and may be a therapeutic target [250].
A recent study found that GC upregulates circCOL1A1, which promotes metastasis and growth via the miR-145/RABL3 axis [251]. Cancer cell exosomes promote endothelial cell angiogenesis, and Gene Ontology analysis links circCOL1A1 to angiogenesis. It promotes CRC progression and is highly expressed in CRC tissues [252]. Cancer cell-derived exosomes additionally promote angiogenesis in ECs [253]. The biological function and underlying mechanism of circCOL1A1, as well as the pro-angiogenic role of exosomal circCOL1A1 in CRC, remain unclear. Bioinformatics analysis indicated a direct relationship between circCOL1A1 and EIF4A3. EIF4A3 is associated with lncRNAs or circRNAs in multiple cancers [254–258]. lncRNA H19 promotes cCRC growth through its interaction with EIF4A3, whereas Circ_cse1l acts to inhibit CRC growth [257, 258]. LINC00667 promotes angiogenesis in NSCLC by means of EIF4A3-mediated stabilization of VEGFA [256]. Bioinformatic analyses indicate that members of the Smad family, namely Smad1, 2, 3, 4, 6, 7, and 9, are likely binding partners for EIF4A3 [259, 260]. The studies indicate that circCOL1A1 may enhance angiogenesis in CRC by recruiting EIF4A3, possibly via the activation of the Smad pathway.
Hu et al. proved that exosomal circCOL1A1 from CRC cells boosts HUVEC angiogenesis. The recruitment of EIF4A3, observed at elevated levels in CRC tissues, indicates that circCOL1A1 overexpression or exosomal circCOL1A1 contributes to the promotion of angiogenesis in HUVECs. Furthermore, by binding to and stabilizing Smad2/3 mRNA, EIF4A3 promotes angiogenesis. Exosomal circCOL1A1 activated Smad2/3, which helped to speed up the growth of tumors and the process of angiogenesis both in vitro and in vivo. This article provides an explanation of the anti-angiogenic targeted therapy for colorectal cancer. We were successful in identifying the exosomes that are present in colorectal cancer cells by employing techniques such as western blot, electron microscopy, and nanoparticle tracking. Assessments of HUVECs’ migratory and angiogenic capabilities were carried out through the use of wound healing assays as well as tube formation assays. Utilizing bioinformatics, RNA pull-down, RNA immunoprecipitation, and FISH tests, it was found that circCOL1A1, EIF4A3, and Smad2/3 mRNA interactions were identified. This was accomplished through the utilization of these techniques. It was determined that the xenograft experiment yielded the desired results [261].
A study reveals that hsa-miR-183-5p, a microRNA associated with CRC, is up-regulated in the cancerous cell [262]. This miRNA has been linked to other cancers, including RCC and lung adenocarcinoma [263]. Lung adenocarcinoma is oncogenic due to its interactions with multiple target genes [264]. MiR-183 targets FOXO1, an insulin signaling target that regulates metabolic homeostasis and organismal survival, to increase NSCLC cell survival [265]. This is linked to angiogenesis and tumor development. A study also investigated the functions of microRNA-183-5p in exosomes secreted from CRC cells [266].
In another study, the effects of microRNA-183-5p were investigated in exosomes that were secreted from CRC cells. In order to determine which microRNAs were expressed in a different manner, an analysis of microarrays was carried out. In a study, HMEC-1 cells were co-cultured with CRC cell exosomes to determine their role in angiogenesis. The use of nude mice allowed for the evaluation of the preventative effect of exosomal miR-183-5p in vivo. This evaluation was brought about by the utilization of nude mice. The research findings indicate that exosomes from CRC cells, which overexpress miR-183-5p, facilitate the progression of symptoms related to CRC, with FOXO identified as a regulatory factor. One potential therapeutic biomarker for colorectal cancer is exosomes that have a higher expression of miR-183-5p [267].
Advanced-stage cancers exhibiting gene mutations are associated with poorer outcomes primarily due to their more aggressive biological characteristics [268]. Previous research has shown that B-cell receptor-associated protein 31, or BAP31, is an integral ER membrane protein that regulates the fate of many molecules and is involved in immunomodulation, neuroinflammation, tumor and disease development, and more [269–272]. BAP31 has recently been identified as a novel CTA and proved to be associated with cervical cancer metastasis and growth [273, 274]. In advanced clinical stages II and III, BAP31 expression increased most [275]. In liver metastatic colorectal cancer tissues, the expression of BAP31 was significantly higher than in primary CRC tissues [275]. It is shown that BAP31 controls invasion and migration in lung, cervical, and ovarian malignancies [276]. Cancer metastasis occurs via multistep pathways, with angiogenesis identified as a rate-limiting factor in tumor metastasis [277, 278]. The aberrant expression of BAP31 in CRC is shown to have a significant impact on adjacent tissues within the TME.
San et al. looked into how fibroblast transition involves exosomes from CRCs and the abnormal expression of BAP31. Exosome miRNA expression profiles from BAP31-overexpressing colorectal cancers were analyzed in a methodical manner in order to determine the impact that BAP31 expression had on the levels of exosomal miRNA in colorectal cancers. They also showed that BAP31 expression affects tumor microenvironment angiogenesis via the BAP31/miR-181a-5p/RECK axis. Their research looked at how BAP31 controls the tumor microenvironment and its effect on CRC angiogenesis. Exosomes released by BAP31-regulated CRCs first influenced the in vivo and in vitro conversion of non-cancerous fibroblasts to CAFs [267]. Exosomes released by CRC that overexpress BAP31 had their microRNA expression profiles examined. They discovered that exosomal microRNAs, namely miR-181a-5p, underwent a dramatic shift when BAP31 was expressed in CRC. Endothelial cells were able to differentiate into angiogenesis with the help of fibroblasts overexpressing miR-181a-5p. In order to drive fibroblast transformation into proangiogenic CAFs, the researchers found that miR-181a-5p directly targeted the 3’-untranslated region of RECK. This offers additional evidence that the miR-181a-5p/RECK axis may serve as a mechanism through which exosomes originating from CRC influence fibroblast differentiation into proangiogenic CAFs [267].
Human cancer endothelial cells, including those from renal cell carcinoma that has metastasized and hepatocellular carcinoma, express endogenous miR-221-3p, which promotes angiogenesis; Embryogenesis is another area where this miRNA is involved [279–281]. One example of a microRNA that can increase tumor angiogenesis is exosomal miR-221-3p, which is expressed in cervical squamous cell carcinoma. This miRNA can do this by reducing the expression of thrombospondin-2 in vascular ECs [].
Dokhanchi et al. conducted a study on patients who were diagnosed with colorectal cancer. The researchers looked into the expression of four microRNAs: miR-203-3p, miR-19a-3p, let-7f-5p, and miR-221-3p. A further investigation was conducted to determine whether or not the expression of these miRNAs was associated with the lymph node metastatic status of patients who were diagnosed with chemotherapy-resistant CRC. In the process of modulating the SOCS3/STAT3/VEGFR-2 expression pathway, they made the discovery that the expression of miR-221-3p, which is released by patients with CRC, stimulates the angiogenesis of tumors. This was accomplished by observing the effects of the pathway. A CRC signature consisting of four serum-derived microRNAs was investigated by Dokhanchi et al. for its potential therapeutic application [282]. In addition, miR-221-3p-containing EVs increased endothelial cell angiogenesis. They used RT-qPCR to examine the relationships between lymph node metastases and four serum-derived miRs: 19a-3p, 221-3p, 203-3p, and let-7f-5p. Included were CRC patients. Receiver operating characteristic curve analysis evaluated these devices’ diagnostics. Extracted EVs from CRC-conditioned medium were tested for purity. MiR-221-3p was administered to ECs through CRC-derived extracellular vesicles to examine its pro-angiogenic effects on cellular proliferation, tube formation, and transwell migration. An in-silico simulation showed miR-221-3p regulates SOCS3. The functions were verified by Western blots and luciferase assays. Serum-derived miR-203-3p, miR-19a-3p let-7f-5p, and miR-221-3p levels differed significantly between colorectal cancer patients and controls. This study found a positive correlation between lymph node metastasis and miR-19a-3p, miR-203-3p, and miR-221-3p expression. In addition, miR-221-3p appears to directly target SOCS3. CRC-EV-released miR-221-3p targeted SOCS3 in ECs to modulate STAT3/VEGFR-2 signaling. EC migration and vessel formation increase with CRC-EVs. Overexpression of miR-221-3p allowed CRC-EVs to replicate their proangiogenic effect. This highlights the role of EV-derived miR-221-3p in EC differentiation. Four circulating microRNAs—miR-19a-3p, miR-203-3p, miR-221-3p, and let-7f-5p—were used to create a CRC biomarker panel. Furthermore, miR-221-3p targets SOCS3, causing in vitro EC angiogenesis [285]. CRC angiogenesis involves several exosomal ncRNAs, as shown in Table 4.

Discussion and future perspectives
The role of non-coding RNAs in regulating colorectal cancer angiogenesis represents a dynamic and promising frontier in oncology. This review has outlined the complex, multi-layered network involving miRNAs, lncRNAs, circRNAs, and exosomal ncRNAs. It is now clear that specific miRNAs, such as miR-148a and miR-181a, have proven potential as therapeutic targets by modulating pro-angiogenic factors like VEGF and HIF-1α. Similarly, lncRNAs (e.g., HNF1A-AS1, UCA1) and circRNAs (e.g., circ-ERBIN, circ3823) function as critical regulators, often by “sponging” these miRNAs.

The integrated CeRNA network
A critical insight from this review is that these ncRNAs do not operate in isolation. Instead, they form an intricate regulatory web known as the competing endogenous RNA (ceRNA) network. In this network, lncRNAs and circRNAs act as “miRNA sponges,” competitively binding to and sequestering tumor-suppressive miRNAs [292]. This sequestration effectively “liberates” the target mRNAs of those miRNAs—often key angiogenic drivers like VEGF, HIF-1α, and TCF7—leading to their overexpression and promoting neovascularization. This interplay highlights a sophisticated mechanism of gene regulation where the dynamic balance between sponges and their miRNA targets dictates the angiogenic phenotype. Exosomes add another layer of complexity, allowing cancer cells to “export” these ncRNA sponges (like circCOL1A1) or pro-angiogenic miRNAs (like miR-21-5p) to remodel the TME and induce angiogenesis in recipient endothelial cells.

Therapeutic strategies and biomarker potential
The clinical potential of these findings is twofold. First, ncRNAs are highly promising diagnostic and prognostic biomarkers. Exosomal ncRNAs, in particular, are stable and readily detectable in liquid biopsies (e.g., serum or plasma). This opens the door for non-invasive screening, predicting metastatic risk, or monitoring patient response to anti-angiogenic therapies like bevacizumab.
Second, these networks present a rich source of novel therapeutic targets [293]. Several strategies are emerging:

miRNA Replacement Therapy: Using synthetic miRNA mimics to restore the levels of tumor-suppressive miRNAs (e.g., miR-148a, miR-590-5p) that are lost in CRC.

miRNA Inhibition: Employing antagomirs (antisense inhibitors) or small molecules to block oncogenic “oncomiRs” (e.g., miR-181a, miR-1290), thereby inhibiting their pro-angiogenic functions.

Targeting the Sponges: Using antisense oligonucleotides (ASOs) or siRNAs to degrade specific oncogenic lncRNAs and circRNAs. This would “release” the tumor-suppressive miRNAs they sponge, restoring their inhibitory function.

Exosome-Based Delivery: Engineering exosomes themselves as delivery vehicles to carry these therapeutic ncRNAs, or developing drugs that block the uptake of pro-angiogenic exosomes by endothelial cells.

Challenges and future directions
Despite this immense potential, significant translational hurdles remain. The primary challenge is the safe and effective in vivo delivery of RNA-based therapeutics. These molecules are notoriously unstable and require sophisticated delivery systems, such as lipid nanoparticles (LNPs) or polymer-based nanocarriers, to protect them from degradation and ensure specific uptake by tumor cells while sparing healthy tissues [294].
Furthermore, the ceRNA network is characterized by significant redundancy and complexity. Targeting a single lncRNA or circRNA may be insufficient if other ncRNAs compensate for its loss. The potential for off-target effects of miRNA mimics and inhibitors is also a major safety concern that must be thoroughly evaluated.
Future research must therefore focus not only on discovering new ncRNA interactions but also on developing robust, clinically viable delivery systems. Integrating multi-omics data with computational biology will be essential to map these complex networks and predict the most effective combination therapies. By addressing these challenges, targeting the ncRNA-driven angiogenic pathways may one day offer a powerful new strategy to reduce CRC metastasis, overcome therapeutic resistance, and ultimately improve patient outcomes.