Long noncoding RNA LOC441461 drives cancer growth and is associated with poor clinical outcomes.

INTRODUCTION
Lung cancer is primarily driven by genetics and environmental exposure, whether through pollution, radiation, or tobacco consumption [1]. It remains the leading cause of cancer-related deaths worldwide, with over 40% of cases of nonsmall-cell lung cancer (NSCLC) already metastatic at the time of diagnosis [2]. Most patients with NSCLC receive a diagnosis at an advanced stage, resulting in a low clinical cure rate of approximately 15%. The two major histological subtypes of NSCLC are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Lung cancer cells are frequently involved in key cancer-associated pathways related to cell proliferation, programmed cell death, angiogenesis, invasion, and metastasis [3]. The transformation of normal lung epithelial cells into malignant cells is thought to occur over multiple steps involving both genetic and epigenetic alterations, leading to clonal expansion and invasive disease [4].
Evidence suggests that noncoding RNAs play a critical role in cancer biology, including the development, progression, metastasis, and drug resistance of tumors [5]. Long noncoding RNAs (lncRNAs), which are RNA transcripts longer than 200 nucleotides, have been shown to participate in several regulatory mechanisms; they (1) modulate protein-coding gene expression, (2) alter epigenetic regulation, (3) influence alternative splicing, and (4) act as decoys for microRNAs (miRNAs) [5]. The human genome is estimated to encode approximately 15,000–17,000 lncRNAs, although the functions of most of these lncRNAs remain unclear. Recent studies have highlighted the involvement of deregulated lncRNAs in lung cancer initiation, progression, and therapy resistance [6]. Numerous dysregulated lncRNAs have been implicated in lung cancer, such as HOTAIR, H19, and MALAT1 [78], although the roles of many lncRNAs in oncogenesis remain unknown.
LOC441461 is a 563-base pair lncRNA that shares a bidirectional promoter with the STX17 gene on human chromosome 9 (coordinates: 99,886,317–99,906,601). According to Wang et al., LOC441461 is upregulated in colorectal cancer tissues, and its high expression correlates with poor patient survival [9]. Interestingly, LOC441461 expression was found to be lower in primary colon tumors and liver metastases relative to normal mucosa, and LOC441461 knockdown reduced colon cancer cell proliferation by arresting the cancer cell cycle and inducing apoptosis [9]. By contrast, Lee et al. found that LOC441461 was downregulated in human gastric cancer and that its knockdown promoted cancer cell proliferation and metastasis [10]. Their findings suggest that LOC441461 may regulate transcriptional activity through interactions with several critical transcription factors. These contrasting findings indicate that LOC441461 may function differently depending on the cancer type. However, its role in lung cancer remains largely unexplored [10]. In this study, we investigated the clinical relevance of LOC441461 in lung cancer using public databases and explored its biological function through in vitro proliferation and cell cycle assays.

MATERIALS AND METHODS

Cell culture
The lung cancer cell lines A549, H1299, H1355, and CL1-5 and the human embryonic lung cell line HEL-299 were obtained from the American Type Culture Collection. All cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

LOC441461 expression according to the Cancer Genome Atlas data
The Cancer Genome Atlas (TCGA) project collects RNA expression data from both tumor and matched normal tissues obtained from hundreds of patients with lung cancer. In this study, we downloaded all level-3 RNA sequencing data for lung cancer from the TCGA Data Portal (https://tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm). These level-3 datasets contain processed expression values for each lncRNA, which have been derived through Illumina HiSeq sequencing. Specifically, we obtained expression profiles and corresponding clinical data for LUAD and LUSC. In addition, the expression levels of LOC441461 in human cancer were obtained and analyzed from the Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/index.html).

Extraction of RNA
Total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s protocol. Briefly, tissues were homogenized in 1 mL of TRIzol reagent, after which 0.2 mL of chloroform was added to separate the phases. RNA was then precipitated from the aqueous phase using 0.5 mL of isopropanol. The concentration, purity, and quantity of the extracted RNA were assessed using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies Inc., USA).

Clinical samples
Thirty lung cancer tissues and corresponding adjacent normal samples were obtained from the Biobank of Taipei Tzu Chi Hospital, Taiwan. Informed consent was obtained from all patients by the Biobank of Taipei Tzu Chi Hospital. This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Taipei Tzu Chi Hospital (14-IRB-042).

Real-time polymerase chain reaction
Complementary DNA was used for quantitative real-time polymerase chain reaction (PCR) analysis with LOC441461-specific primers. Gene expression was quantified using Fast SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). The expression level of LOC441461 was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ΔCt method (ΔCt = Ct_LOC441461 − Ct_GAPDH). The specific primers used in this study are listed in Supplementary Table 1.

siRNA transfection
In this study, LOC441461 expression was knocked down using siRNA oligonucleotide transfection. The detailed sequences are provided in Supplementary Table 1. A scrambled oligonucleotide was used as a negative control (NC), and all siRNAs were purchased from GenDiscovery Biotechnology (Taipei, Taiwan). Briefly, lung cancer cells were transfected with 10 nM siLOC441461 (siLOC441461#1, siLOC441461#2, or a pooled combination of siLOC441461#1 and siLOC441461#2) or scrambled control using Lipofectamine RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific). Knockdown efficiency was assessed by real-time PCR.

Proliferation
A total of 2500 lung cancer cells were seeded into 96-well plates and transfected with siLOC441461 or a scrambled NC. Cell viability was measured at 0, 1, 2, and 3 days using the CellTiter-Glo One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA).

Colony formation assay
A total of 4000 cells were seeded into each well of a six-well plate and transfected with either si-LOC441461 or a scrambled NC using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific). After 3 days of incubation at 37°C, the culture medium was replaced with fresh medium, and the cells were further incubated for 10 days to allow colony formation. At the end of the incubation period, colonies were fixed with 4% formaldehyde for 2 min and stained with crystal violet solution (0.5% crystal violet, 5% formaldehyde, 50% ethanol, and 0.85% sodium chloride) for 2 h. After being air-dried, the wells were rinsed with distilled water. To quantify colony formation, the bound crystal violet dye was solubilized with 1 mL of 10% acetic acid in each well, and the absorbance was measured at 595 nm by using a spectrophotometer.

Cell cycle analysis
Lung cancer cells were transfected with siLOC441461 (a combination of siLOC441461#1 and siLOC441461#2) or a scrambled NC using Lipofectamine RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific). After 48 h, 1 × 106 cells were harvested and fixed in 70% ethanol at −20°C overnight. The cells were then stained with 4′,6-diamidino-2-phenylindole (ChemoMetec, Gydevang, Lillerød, Denmark) and analyzed with the NucleoView NC-3000 software (ChemoMetec).

Western blotting
Cells were harvested 48 h after transient transfection, washed with phosphate-buffered saline, and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) at 4°C for 30 min. Protein samples (40 μg per lane) were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis; 10% or 12% polyacrylamide gel, then was transferred to membranes. The membranes were then blocked for 1 h at room temperature with blocking buffer (50 mM Tris–HCl at pH 7.6, 150 mM NaCl, 0.1% Tween-20, 5% nonfat dry milk, and 0.05% sodium azide) and incubated overnight at 4°C with the primary antibodies. After being washed, the membranes were incubated for 1 h at room temperature with the following HRP-conjugated secondary antibodies. After the membranes were washed three times with Tris-buffered saline containing 0.1% Tween-20, immunoreactive bands were visualized using the WesternBright ECL detection system (Advansta, Menlo Park, CA, USA). The antibodies used in this study are listed in Supplementary Table 2.

Statistical analysis
Data from real-time PCR or the TCGA database were analyzed to examine the relative expression levels of LOC441461 in lung cancer cells. For the expression levels of LOC441461 in five lung cancer cell lines, a one-way ANOVA was performed to assess overall differences among the groups, followed by one-way ANOVA post hoc tests with Bonferroni correction to compare pairwise differences between each cell line. A P < 0.05 was considered statistically significant. Cumulative survival curves were drawn using the Kaplan–Meier method, and survival curves were compared using the log–rank test. For the cell proliferation assay, repeated measures ANOVA was used to evaluate the interaction between group and time. Post hoc pairwise comparisons between the two groups (N.C. vs. siLOC441461) at each time point (days 0, 1, 2, and 3) were conducted using ANOVA with Bonferroni correction. Cell proliferation and colony formation experiments were performed in triplicate. Histograms are used to present the mean values, and error bars indicate standard deviations. The data were analyzed using Student’s t-test. Differences were considered significant if P < 0.05.

RESULTS

High LOC441461 expression in lung cancer is strongly associated with poor outcomes
In the present study, we examined LOC441461 expression in various human cancers. As shown in Figure 1, LOC441461 was underexpressed in brain tumors (P < 0.001), oral cancer (P < 0.001), esophageal cancer (P < 0.05), stomach cancer (P < 0.001), kidney cancer (P < 0.01), ovarian cancer (P < 0.001), bladder cancer (P < 0.001), and cervical cancer (P < 0.05). By contrast, LOC441461 was markedly overexpressed in head and neck cancer (P < 0.001), breast cancer (P < 0.001), lung cancer (P < 0.001), and prostate cancer (P < 0.05). These findings suggest that LOC441461 may play cancer-type-specific roles in tumorigenesis. We further investigated the clinical relevance of LOC441461 for lung cancer prognosis using another independent datasets. TCGA data indicated that compared with that in normal tissue, LOC441461 expression was significantly higher in LUAD (P < 0.001) but nonsignificantly different in LUSC [P = 0.76; Figure 2a]. Real-time PCR analysis revealed that LOC441461 was significantly upregulated in LUAD compared with the corresponding adjacent normal tissues [P < 0.001; Figure 2b]. To examine the clinical impact of LOC441461 expression in LUAD, we obtained detailed clinical information and expression data for LOC441461 from the TCGA database. Using these data, we evaluated the association of LOC441461 expression with overall survival (OS), progression-free survival (PFS), and disease-free survival (DFS) in these patients. A cutoff value for LOC441461 expression was determined through receiver operating characteristic curve analysis. On the basis of this cutoff, patients were divided into high and low LOC441461 expression groups. Kaplan–Meier survival analysis revealed that high LOC441461 expression had a significant association with poorer PFS (P = 0.018) and showed a trend toward associations with OS (P = 0.081) and DFS [P = 0.051; Figure 2c-e]. A univariate analysis indicated that high LOC441461 expression exhibited a similar trend toward poorer OS (hazard ratio [HR]: 1.31, 95% confidence interval [CI]: 0.97–1.76; P = 0.082) and DFS (HR: 1.53, 95% CI: 0.99–2.34; P = 0.053) and significant associations with shorter PFS [HR: 1.39, 95% CI: 1.06–1.84; P = 0.019; Table 1]. In multivariate analysis, high LOC441461 expression remained significantly associated with poorer PFS [adjusted HR: 1.45, 95% CI: 1.10–1.92; P = 0.009; Table 1]. A further analysis with data from an independent database validated these findings, indicating that high LOC441461 expression was significantly correlated with poor OS (HR: 1.34, P = 0.00019) and PFS (HR: 1.29, P = 0.026), as shown in Supplementary Figure 1a and b. Collectively, these findings suggest that LOC441461 is significantly overexpressed in lung cancer. Thus, high LOC441461 expression may serve as a prognostic biomarker for poor outcomes in LUAD.

LOC441461 plays an oncogenic role in promoting the growth of lung cancer cells
The aforementioned findings indicate that LOC441461 is overexpressed in lung cancer and that high expression is correlated with poor survival and disease progression. However, the biological role of LOC441461 on lung cancer growth remains unclear. Thus, we examined the effects of LOC441461 knockdown on the growth and motility of lung cancer cells. We first determined the expression levels of LOC441461 in various lung cancer cells by using real-time PCR. According to the results, A549, H1299, and H1355 cells had higher LOC441461 expression compared with HEL299 cells [Figure 3a]. Furthermore, LOC441461 expression was predominantly localized in the cytoplasm of A549 and H1299 cells [Supplementary Figure 2a and b]. Because A549 and H1299 cells had high LOC441461 expression, we selected them for further study. We transfected these cells with small interfering RNA (siRNA; siLOC441461#1 and siLOC441461#2) for 24 h and observed significantly lower LOC441461 expression in these cells relative to the expression in a NC group [Figure 3b and c]. We then assessed the biological function of A549 and H1299 cells with LOC441461 knockdown. As illustrated in Figure 3d, f and g, the cell proliferation and colony formation ability of the A549 cells were significantly lower after LOC441461 knockdown. Similarly, we observed that LOC441461 knockdown significantly inhibited H1299 cell proliferation and colony formation ability in the control group comparisons [Figure 3e-g].

LOC441461 regulates lung cancer cell growth by impairing cell cycle progression
The aforementioned results indicate that LOC441461 knockdown suppresses lung cancer cell proliferation and colony formation ability, including in A549 and H1299 cells [Figure 3]. We further analyzed the apoptosis and cell cycle progression of lung cancer cells with LOC441461 knockdown. As shown in Figure 4a and b, LOC441461 knockdown induced a modest but statistically significant increase in apoptosis in both cell lines compared with their respective control groups. These results indicate that LOC441461 involves in the growth of LUAD cells, although the magnitude of apoptosis induction was limited. In addition, we conducted an image flow assay and found that in A549 cells, siLOC441461 pool transfection was associated with increased prevalence of the G0/G1 phase and significantly lower prevalence of the S and G2/M phases [Figure 4c]. However, in H1299 cells, siLOC441461 pool transfection was associated with decreased prevalence of the G0/G1 and S phases and increased prevalence of the G2/M phase [Figure 4d]. Thus, the effect of LOC441461 knockdown on cell cycle progression differed between A549 and H1299 cells. We further examined the expression levels of cell cycle-related genes in A549 and H1299 cells with LOC441461 knockdown. Our results indicated that in A549 cells, LOC441461 knockdown was associated with lower expression levels of CDK4 and cyclin A2, B1, and D1 and higher expression levels of p21 and p27 [Figure 4e]. However, in H1299 cells, only the expression levels of CDK4 and the cyclin D1 were lower after LOC441461 knockdown. Because A549 cells are p53 wild-type cells and H1299 cells are P53 null cells, this difference in the effect of LOC441461 knockdown between the two cell types might have stemmed from the p53 status of H1299 cells. However, the mechanism underlying this difference should be investigated in future studies.

DISCUSSION
Although our findings indicate that LOC441461 functions as a potential oncogenic factor in lung cancer by promoting cell cycle progression and inhibiting apoptosis, its expression pattern appears to differ between lung cancer subtypes. Analysis of TCGA data revealed that LOC441461 is significantly overexpressed in LUAD but not in LUSC. These findings highlight the necessity of differentiating among cancer types when assessing LOC441461 expression and function in human malignancies. In colon cancer, LOC441461 was reported to be upregulated and associated with enhanced tumor cell proliferation and migration, indicating a tumor-promoting role [9]. This finding is consistent with our observations in LUAD, where LOC441461 knockdown suppressed proliferation and induced apoptosis. However, LOC441461 may play a tumor-suppressing role in gastric cancer. Low LOC441461 expression was found to be correlated with poor prognosis in gastric cancer, and LOC441461 knockdown increased the growth and motility of gastric cancer cells and exerted tumor-suppressing effects through the regulation of cell adhesion and genes related to the epithelial–mesenchymal transition [10].
In this study, we identified a novel oncogenic lncRNA, LOC441461, that is involved in the growth of LUAD cells. We also found that LOC441461 overexpression is associated with poor PFS in patients with LUAD. However, the exact mechanism through which LOC441461 promotes cell growth remains unclear. Our study showed that LOC441461 is primarily expressed in the cytoplasm, suggesting that it may regulate lung cancer cell proliferation by interacting with miRNAs to suppress the translation of tumor suppressor genes [9]. A previous study demonstrated that miR-335 suppresses the motility of metastatic breast cancer cells by regulating a set of genes, including SOX4 [1112]. Gene expression omnibus (GDS3138) analysis revealed that LOC441461 expression was downregulated in lung metastatic breast cancer cells with miR-335 overexpression. Studies have indicated that miR-335 expression acts as a tumor suppressor in the inhibition of lung cancer cell growth and metastasis [13141516]. These studies’ findings suggest that the miR-335-LOC441461 axis may regulate lung cancer cell growth by acting as a miR-335 sponge. However, further experiments are needed to fully elucidate the mechanism of LOC441461.
Interestingly, our findings demonstrate that the effects of LOC441461 knockdown on cell cycle progression and apoptosis differ for A549 (p53 wild-type) versus H1299 (p53-null) cells, suggesting the existence of some mechanisms that do not depend on p53 status and other mechanisms that do. Other research has revealed that p53 status directly leads to drug-treatment-induced cell arrest by regulating p21 expression [1718]. In A549 cells, LOC441461 knockdown significantly upregulated the expression of the cyclin-dependent kinase inhibitors p21 and p27 and reduced the levels of CDK4 and the cyclin D1, A2, and B1 [Figure 4]. These changes were associated with G0/G1 cell cycle arrest, increased apoptosis, and p21 expression [19]. Given that p21 is a direct transcriptional target of p53, our findings imply that LOC441461 may negatively regulate the p53 signaling pathway. Upon LOC441461 silencing, p53 activity is likely to be restored, leading to enhanced p21-mediated cell cycle inhibition and apoptotic induction. By contrast, in H1299 cells lacking functional p53, LOC441461 knockdown did not alter p21 or p27 expression but still reduced CDK4 and cyclin D1 levels. Interestingly, these cells were more likely to be in the G2/M phase and to undergo apoptosis, indicating a p53-independent mechanism of G2/M arrest. This suggests that LOC441461 may also regulate cell cycle progression through other pathways, potentially involving mitotic regulators such as PLK1, CDC25C, or aurora kinases. The observed apoptosis in the two cell lines following LOC441461 depletion underscores the potential oncogenic role of LOC441461 in LUAD.

CONCLUSION
Overall, our findings reveal that LOC441461 contributes to LUAD cell proliferation by modulating cell cycle regulators and apoptosis through p53-dependent and p53-independent pathways. Targeting LOC441461 may represent a promising therapeutic strategy for LUAD regardless of p53 status.

Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of interest
Dr. Ching-Feng Cheng, an editorial board member at Tzu Chi Medical Journal, had no role in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.

SUPPLEMENTARY MATERIAL
Supplementary Figure 1Clinical effects of LOC441461 expression in lung cancer according to GENT data. Correlations of LOC441461 with (a) overall survival and (b) progression-free survival. HR: Hazard ratio

Supplementary Figure 2Subcellular localization analysis of LOC441461 in lung cancer cell lines. After nuclear and cytoplasmic fractionation, total RNA from A549 (a) and H1299 (b) cells was extracted and subjected to reverse transcription followed by real-time polymerase chain reaction (PCR). GAPDH was used as a cytoplasmic marker, and U6 as a nuclear marker. The expression levels of LOC441461 in the nuclear and cytoplasmic fractions were quantified separately by real-time PCR