SCARNA20 influences the occurrence and development of lung cancer by inhibiting tumor cell proliferation, migration, and invasion.

Introduction
Lung cancer is a malignant tumor with some of the highest global incidence and mortality rates. According to the American Cancer Society’s 2024 survey, lung cancer has the second-highest incidence rate among all cancers, while its mortality rate ranks first, nearly 2.5 times higher than colorectal cancer, which ranks second (1). Although treatment methods for lung cancer are continuously evolving, its 5-year survival rate remains low, as approximately two-thirds of patients are diagnosed at an advanced stage (2).
Currently, with advances in sequencing technologies and the exploration of the lung cancer genome, various mechanisms of tumorigenesis and progression, including signaling pathways and immune checkpoints, are being extensively studied. Targeted therapies developed for specific signaling pathways and treatments targeting immune checkpoints such as programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have provided new treatment options for lung cancer patients and achieved significant therapeutic outcomes (3). However, despite targeted therapy offering new directions for lung cancer patients, tumors inevitably develop resistance due to gene mutations, amplification, and other reasons. Furthermore, due to the complexity of the tumor immune microenvironment, the clinical benefits of immune checkpoint inhibitors are limited, with the overall response rate (ORR) of single-agent immunotherapy being only about 10–20% (4). Therefore, it is necessary to continue in-depth exploration of the molecular mechanisms underlying lung cancer occurrence and development to identify new diagnostic and therapeutic targets for NSCLC patients.
In recent years, a growing body of research has shown that various small non-coding RNAs (sncRNAs), including small nucleolar RNAs (snoRNAs), play important roles in many biological processes, and their dysregulation is closely related to cancer progression (5,6). With the development of whole-genome expression analysis and RNA-Seq technologies, an increasing number of snoRNAs have been confirmed to be associated with tumorigenesis (7). In lung cancer, SNORA42 is predominantly overexpressed in NSCLC, and its expression level is negatively correlated with prognosis. Increasing SNORA42 levels enhances cell proliferation and clonogenic ability, whereas downregulating its expression inhibits NSCLC cell proliferation by inducing p53-dependent apoptosis (8). SNORD88C can serve as a non-invasive diagnostic biomarker for NSCLC, promoting cell proliferation and metastasis in both in vitro and in vivo experiments. It enhances the translation activity of its downstream gene SCD1 through 2-O-methylation, which subsequently inhibits autophagy and promotes invasion and migration (7). However, reports on snoRNAs in the field of non-small cell lung cancer (NSCLC) are currently limited, and their specific mechanisms of action are not yet fully understood, requiring further research for clarification.
In our previous studies, we screened SCARNA20 through high-throughput snoRNA sequencing and bioinformatics analysis of clinical specimens and found it to be significantly down-regulated in NSCLC patient samples, suggesting it may act as a novel tumor suppressor gene playing an important role in tumor cell development. Literature review revealed that its downregulation might be associated with Epstein-Barr virus (EBV) lymphoma development (7), but research in the field of NSCLC is lacking. Therefore, this paper focuses on the biological function of SCARNA20, validating its role through in vitro and in vivo experiments, and preliminarily exploring its potential mechanism of action via transcriptome sequencing. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-2010/rc).

Methods

Cell lines
Human NSCLC cell lines A549, NCI-H1299, and NCI-H1437 were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). All cell lines were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, USA) and maintained at 37 ℃ in a humidified incubator with 5% CO2. Cells were passaged every 2–3 days at a split ratio of 1:3 to 1:4 using a digestion solution containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) (1:1 mixture, Gibco) at 37 ℃ for 2–3 minutes. For long-term storage, cells were frozen in complete medium containing 10% dimethyl sulfoxide (DMSO) using a programmed freezer and stored in liquid nitrogen. All experiments were performed using cells in the logarithmic growth phase (passages 5–20). Cell morphology was regularly monitored under an inverted phase-contrast microscope to confirm the absence of microbial contamination and stable phenotypic characteristics.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells or tissue samples using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer. For reverse transcription, 1 µg of total RNA was used with the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) in a 20 µL reaction volume. Quantitative PCR was performed using TransStart Tip Green qPCR SuperMix (TransGen, Beijing, China) on a 7500Fast DX Real-Time PCR System (Applied Biosystems, Foster City, USA). The 20 µL reaction mixture contained: 2 µL complementary DNA (cDNA), 0.4 µL each of forward and reverse primers (final concentration 200 nM), 10 µL of SYBR Green Mix, and nuclease-free water to volume. The thermal cycling conditions were: 95 ℃ for 180 s; followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 30 sec then 72 ℃ for 30 s; finally, a melting curve analysis was performed. Each sample was assayed in triplicate.

Cell proliferation assay [Cell Counting Kit-8 (CCK-8)]
The CCK-8 kit (Beyotime Biotechnology, Shanghai, China) was used according to the manufacturer’s protocol. Cells in the exponential growth phase were digested with 0.25% trypsin-EDTA, centrifuged, the supernatant was discarded, and the pellet was resuspended in culture medium and counted. Cell suspensions (100 µL/well, 1,000 cells/well) were seeded into 96-well plates and incubated (37 ℃, 5% CO2) for 24 hours. Then, 10 µL of CCK-8 solution was added directly to the surface of the medium in each well. After shaking and pre-mixing for 30 seconds using a microplate reader (Detie, Nanjing, China), the plates were incubated for 2 hours, and the absorbance at 450 nm was measured and recorded using the microplate reader.

Cell cycle analysis
Approximately 1×106 cells were collected and digested with 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, USA), fixed overnight at 4 ℃ with 70% cold ethanol, and washed with PBS. Fixed cells were washed twice with PBS and resuspended in 500 µL of propidium iodide (PI) staining solution (containing 50 µg/mL PI and 100 µg/mL RNase A, Yeasen, Shanghai, China). 0.5 mL of the prepared propidium iodide (PI) staining solution was added to each cell sample, and cells were gently resuspended after incubation at 37 ℃ in the dark for 15 minutes. Cell cycle distribution was analyzed using a flow cytometer (Beckman CytoFLEX, Brea, USA) with an excitation wavelength of 488 nm, collecting fluorescence in the FL2 channel. The percentages of cells in G0/G1, S, and G2/M phases were determined using FlowJo software. Experiments were independently repeated three times.

Cell apoptosis detection
Lung cancer cell lines were resuspended in 1X binding buffer and approximately 5×105 cells were collected, washed with PBS, and resuspended in 100 µL of 1× binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of PI were added sequentially, stained at room temperature in the dark for 15 minutes using the Annexin V-FITC/PI Apoptosis Kit (Invitrogen, Thermo Fisher Scientific, Waltham, USA). Cell apoptosis was detected using a flow cytometer (Beckman, Brea, USA), and results were analyzed with the specialized software. Each experiment was repeated three times.

Cell colony formation assay
Cells in the logarithmic growth phase were digested and seeded into 6-well plates at a density of 500 cells per well in 2 mL of complete medium. The medium was refreshed every 3 days. After culturing for 14 days under standard conditions (37 ℃, 5% CO2), cells were washed with PBS, fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), stained with 0.5% crystal violet aqueous solution for 5 minutes, washed with PBS, air-dried, and photographed. Colonies containing >50 cells were counted, and clone counts were statistically analyzed.

Transwell assay
Cells in the logarithmic growth phase were digested and resuspended in serum-free medium, adjusted to a preset cell density. Cell suspension was added to the upper chamber of the Transwell insert, while the lower chamber contained complete medium in a 24-well plate. After incubation for 24 hours, non-migrated cells on the upper surface of the membrane were removed. Cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.5% crystal violet for 20 minutes. The upper surface of the membrane was wiped clean with a cotton swab. Cells that penetrated the membrane were counted under a microscope in five randomly selected fields per membrane. The experiment was divided into Transwell migration assay and Transwell invasion assay based on whether the upper chamber membrane was pre-coated with Matrigel (Chemicon, Billerica, USA; #ECM550) before cell seeding.

Wound healing assay
According to the experimental design, approximately 1×106 cells were seeded per well in a 6-well plate. After overnight incubation, the medium was replaced with 2% serum medium. A straight scratch was made on the bottom of the well using a 200 µL pipette tip. After washing twice with PBS, 2% serum medium was added again, and an image was taken (recorded as 0 hour). The plates were incubated under standard conditions (37 ℃, 5% CO2), and images of the same scratch location were captured at 0, 6, 12, and 24 hours using an inverted microscope. The scratch width was measured using ImageJ software. The migration rate (%) was calculated as [(width at 0 h − width at t h) / width at 0 h] × 100%.

Subcutaneous tumor Xenograft model
Six-week-old BALB/c nude mice (Vital River, Beijing, China) were randomly divided into two groups for the experiment. A549 cells overexpressing SCARNA20 (1×107 cells per mouse) were injected subcutaneously to establish the animal model. Starting on day 7 post-inoculation, tumor length (L) and width (W) were measured every 5 days using a caliper, and tumor volume was calculated using the formula V = 0.5 × L × W2. After 32 days, mice were sacrificed, tumors were collected, and the weight of each tumor was recorded. The average tumor size and weight for each group were calculated and compared between groups. Tumor tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned for hematoxylin and eosin (H&E) staining and immunohistochemical staining for Ki-67 and Caspase-3. Pathological analysis and scoring were performed by at least two independent pathologists. During evaluation, the entire section was first scanned at low magnification (40×) to assess heterogeneity. Ki-67 expression was determined by the percentage of positive cells (positive cell number/total cell number × 100%). Caspase-3 staining was considered positive if brown-yellow or brown reaction products were seen in the cytoplasm. Semi-quantitative grading for Caspase-3 expression was determined based on staining intensity and the percentage of positive cells: (I) staining intensity: colorless (0 points), light yellow (1 point), brown-yellow (2 points), brown (3 points). (II) Percentage of positive cells: scored on a scale of 1–100. The final score was the product of the two scores, with a total score ranging from 0 to 300. Animal experiments were performed under a project license (No. ZRDWLL260007) granted by institutional ethics committee of China-Japan Friendship Hospital, in compliance with China national guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Statistical analysis
Statistical analyses were conducted using R software (version 4.3.1, sourced from https://www.r-project.org), gene set enrichment analysis (GSEA) software (version 4.3.2, sourced from https://www.gsea-msigdb.org) and GraphPad Prism software (version 10.6.0, sourced https://www.graphpad.com/). Depending on the distribution of the metric data, values were expressed as mean ± standard deviation for continuous variables and as frequencies (percentages) for categorical data. Wilcoxon rank-sum test and one-way analysis of variance (ANOVA) were used to compare differences between groups. All tests were two-sided, and P<0.05 was considered statistically significant.

Results

Efficient overexpression of SCARNA20 in lung cancer cell lines
To verify the expression level of SCARNA20 in lung cancer cell lines, we performed PCR experiments in multiple lung cancer cell lines to determine its basal expression level and ultimately selected A549, H1299, and H1437 for subsequent cellular experiments. We first verified the overexpression efficiency of SCARNA20 in these cell lines. As shown in Figure 1A, transfection resulted in good overexpression efficiency. In contrast, knockdown of SCARNA20 in the same cell lines did not yield consistent results, and the knockdown efficiency was less than 60% in all cases (Figure 1, Figure S1).

SCARNA20 inhibits lung cancer cell proliferation in vitro
To investigate the biological function of SCARNA20, we conducted a series of in vitro cell experiments. CCK-8 cell proliferation assay results showed that lung cancer cell lines overexpressing SCARNA20 had significantly reduced absorbance compared to the control group, indicating an inhibitory effect on tumor proliferation (Figure 1B-1D). Colony formation assays showed similar results, with the overexpression group significantly inhibiting colony formation compared to the control group (Figure 1E-1H, Figure S2). This indicates that SCARNA20 can significantly inhibit the in vitro proliferation of lung cancer cells.

SCARNA20 may exert its tumor-suppressive effect by promoting apoptosis and altering the cell cycle
To explore the reason for the inhibition of cell proliferation, we used flow cytometry to detect the apoptosis rate and cell cycle changes, investigating the relationship between proliferation inhibition and these factors (Figure 2A, Figure S3). As shown, the apoptosis rate was significantly increased in the overexpression group compared to the control group (Figure 2B-2D). Regarding the cell cycle, in A549, H1299, and H1437 cell lines, the proportion of cells in the G0/G1 phase was significantly higher in the overexpression group than in the control group. In H1299 and H1437 cell lines, no significant difference was observed in the proportion of cells in S phase or G2/M phase between the Ctrl and OE groups. However, in the A549 cell line, the OE group had more cells in S phase and fewer cells in G2/M phase than the Ctrl group (Figure 2E-2G). These experiments suggest that overexpression of SCARNA20 can promote apoptosis of lung cancer cells in vitro and may exert its tumor-suppressive effect by altering the cell cycle.

SCARNA20 inhibits lung cancer cell invasion and migration ability
Next, we verified the effect of SCARNA20 on the invasiveness and migrative ability of lung cancer cells. Through Transwell assays with or without an extracellular matrix (ECM) layer, recording the number of cells penetrating the membrane, it was observed that overexpression of SCARNA20 significantly reduced the migration efficiency (Figure 3A,3B, Figure S4) and invasion ability (Figure 3C,3D, Figure S5) of lung cancer cells. Simultaneously, the wound healing assay showed that the migration efficiency of the overexpression group was significantly lower than that of the control group (Figure 3E,3F, Figure S6).

SCARNA20 inhibits lung cancer cell growth in vivo
To further verify the biological function of SCARNA20, we established a mouse subcutaneous xenograft model. Tumor diameter was measured regularly to calculate tumor volume, and pathological section observation and immunohistochemical staining were performed on tumors collected after sacrificing the mice. Mice were sacrificed 32 days after successful inoculation. Upon complete dissection of the tumors, it was evident that the tumor volume in the overexpression group was significantly smaller than that in the control group (925.26 vs. 463.53 mm3, P<0.001), and the tumor weight was also significantly lower than the control group (1.2747 vs. 0.5247 g, P<0.001), indicating that SCARNA20 overexpression can inhibit tumor growth in vivo (Figure 4A-4C). Pathological section observation of the tumors under H&E staining (Figure 4D) showed that the maximum cross-sectional area of the tumors in the overexpression group was significantly smaller than that in the control group (Figure 4E), while the necrotic area showed no significant difference (Figure 4F). Consequently, the proportion of necrotic area was significantly higher in the overexpression group (Figure 4G), suggesting that SCARNA20 overexpression inhibits tumor growth in vivo and promotes central necrosis. Regarding malignancy, no significant difference was observed in tumor heterogeneity, but the number of mitotic figures was significantly lower in the overexpression group than in the control group (Figure 4H). To further verify tumor cell proliferation activity and apoptosis, we performed Ki-67 and Caspase-3 immunohistochemical staining and pathological scoring. Staining results showed that Ki-67 was mainly expressed in the nuclei of tumor cells, predominantly distributed in the peripheral tumor tissue away from the central necrotic area. Compared to the control group, the overexpression group had a lower pathological score, suggesting less active proliferation (Figure 5A,5B). Caspase-3 was mainly expressed in the cytoplasm of individual cells at the junction between tumor tissue and the central necrotic area. Compared to the control group, the overexpression group had a higher pathological score at the junction, suggesting increased apoptosis (Figure 5C,5D).

Discussion
snoRNAs are a class of sncRNAs located in the nucleolus of eukaryotic cells, with lengths ranging from 60 to 300 nucleotides (9). Most snoRNAs are encoded within introns (10) and can be classified into two types based on their structure: C/D box (SNORDs) and H/ACA box (SNORAs) (11). There is also a classification that considers small Cajal body-specific RNAs (scaRNAs), which possess both C/D and H/ACA sequences, as a separate category (12), such as SCARNA20 studied in this paper, whose unique structure is the most different compared to other snoRNAs, making it possible to have more complex biological functions simultaneously. Regarding their physiological functions, C/D box snoRNAs can guide site-specific 2’-O-methylation of target nucleotides, increasing hydrophobicity and potentially protecting RNA from hydrolysis (13). H/ACA box snoRNAs primarily guide the isomerization of uridine to pseudouridine, thereby breaking the original N-glycosidic bond and forming a C-C glycosidic bond for modification (12). As early as the 1960s and 1970s, researchers discovered snoRNAs in mammalian cells and demonstrated their involvement in ribosomal RNA modification (14), such as methylation and pseudouridylation (15). Although the role of snoRNAs was discovered early, they were largely overlooked by cancer researchers for a long time until recent genomic sequencing revealed that the number of snoRNAs is much higher than previously anticipated; a recent study annotated at least 2,000 snoRNAs in the human genome, bringing snoRNAs back into the spotlight for researchers (16).
In this study, we found that overexpression of SCARNA20 was efficient in various lung cancer cell lines, consistent with our preliminary results. In in vitro cell experiments, SCARNA20 overexpression exhibited significant tumor-suppressive effects, not only inhibiting cell proliferation and promoting apoptosis but also suppressing cell migration and invasion abilities. The results from animal experiments validated these conclusions. Immunohistochemical analysis of dissected mouse tumors showed significantly weakened cell proliferation and increased apoptosis in the overexpression group. These results indicate that SCARNA20 can significantly inhibit the migration efficiency and invasion ability of lung cancer cells, thereby delaying the occurrence and development of lung cancer. Through cell cycle analysis, we found that in the three cell lines A549, H1299, and H1437, the proportion of cells arrested in the G0/G1 phase was significantly higher in the overexpression group compared to the control group, suggesting that SCARNA20 may exert its tumor-suppressive effect by influencing the cell cycle. Literature review revealed that recent studies have found that snoRNAs can indirectly affect the cell cycle and promote apoptosis by activating the p53 pathway (17). Researchers found that SNORA13 is essential for cellular senescence, but not through its canonical pseudouridylation role in translation modification. Instead, it directly binds to ribosomal protein RPL23, delaying the assembly of the 60S large subunit, leading to the accumulation of free ribosomal proteins (such as RPL5, RPL11, RPL23). These free ribosomal proteins bind to and inhibit MDM2 (a negative regulator of p53), thereby activating the p53 pathway and ultimately causing irreversible cell cycle arrest in G1 phase (17). These studies indicate that snoRNAs can also influence tumorigenesis and progression through non-canonical functions, which inspired our research. Regarding the potential mechanism of action of SCARNA20, we performed transcriptome next-generation sequencing on overexpression and control cells and conducted bioinformatics analysis. Differential analysis of the transcriptome sequencing results identified 2604 differentially expressed genes, of which 1,088 were upregulated and 1,516 were downregulated (Figure 6A,6B). Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that the differentially expressed genes were enriched in cytokine-cytokine receptor interaction and classical pathways related to tumorigenesis and progression, such as MAPK and TNF signaling pathways (Figure 6C,6D). GSEA pathway enrichment analysis confirmed this finding (Figure 6E). These bioinformatics analysis results provide direction for our future research.
Certain snoRNAs exhibit cell-type-specific expression patterns. For instance, SNORD29 is predominantly expressed in neuronal cells, while SNORD46 and SNORD42A are enriched in mammary glands and lymphocytes (18). This makes snoRNAs promising biomarkers for cancer diagnosis and therapeutic targeting. As potential diagnostic biomarkers, the expression levels of SNORA42 and RNU2-1f fragments are notably higher in lung cancer patients compared to healthy controls, with serum RUN2-1f levels paralleling the overall tumor mass (8,19), showing diagnostic value. Additionally, Liao et al. (20) found that the levels of snoRNAs in peripheral blood also hold diagnostic value for tumors. They analyzed the stability of snoRNAs in plasma using qRT-PCR and found that they were resistant to enzymatic degradation. Even after storing the plasma at −80 ℃ for a month, the snoRNAs remained stable. The tissue-specific expression and stability of snoRNAs make them excellent potential biomarkers for cancer diagnosis and prognosis. The review by Yan et al. (21) provides a detailed account of the current clinical potential of snoRNAs and the challenges in translational medicine. SnoRNA is localized in the nucleolus of the cell nucleus and requires the formation of a complex with proteins [small nucleolar ribonucleoprotein (snoRNP) complexes] to function and avoid degradation. Structurally, it lacks the characteristic “binding pockets” found in traditional small-molecule drugs, making it difficult to target directly due to its molecular properties. This also makes the development of small-molecule drugs targeting snoRNA extremely challenging. In recent years, in response to these challenges, antisense oligonucleotides (ASOs) have demonstrated broad prospects as a therapeutic approach for targeting non-coding RNAs (22). ASOs can specifically target non-coding RNAs via base-pair complementarity, thereby blocking their normal functions or inducing degradation, offering a more feasible translational pathway for developing snoRNA-targeted therapies. In our future research, we will explore translational medicine perspectives by integrating biomarkers with the development of ASOs targeting SCARNA20.

Conclusions
SCARNA20 acts as a tumor suppressor in NSCLC, inhibiting malignant phenotypes including cell proliferation, migration, and invasion, both in vitro and in vivo. Its mechanisms may involve promoting apoptosis, inducing cell cycle arrest, and modulating critical cancer-related pathways. SCARNA20 represents a potential novel therapeutic target for NSCLC and it is worth further in-depth study.

Supplementary
The article’s supplementary files as