LINC02595 promotes tumor progression in colorectal cancer by inhibiting miR‐203b‐3p activity and facilitating BCL2L1 expression

Abstract Colorectal cancer (CRC) is one of the most prevalent tumors worldwide. Recently, long noncoding RNAs (lncRNAs) have been recognized as key regulators in postgenomic biology. Numerous lncRNAs have been identified as diagnostic biomarkers and therapeutic targets. However, the molecular mechanisms underlying the role of lncRNAs in CRC progression are not fully understood. Differentially expressed lncRNAs and messenger RNAs were investigated using a microarray approach in five paired primary CRC tumor tissues and the corresponding nontumor tissues and confirmed in an additional 116 paired tissues and 21 inflammatory bowel disease tissues and 15 adjacent normal tissues by a quantitative real‐time polymerase chain reaction. We also performed comprehensive transcriptome profiling analysis on Gene Expression Omnibus and The Cancer Genome Atlas datasets. We identified LINC02595 and evaluated its clinical significance as a plasma biomarker. The function of LINC02595 was evaluated using a panel of in vivo and vitro assays, including cell counting kit‐8, colony formation, cell cycle, apoptosis, RNA fluorescence in situ hybridization, luciferase reporter, immunohistochemistry, and CRC xenografts. We found that LINC02595 is upregulated in tumor tissues and blood samples of patients with CRC and CRC cell lines. Functional research found that LINC02595 promotes CRC cell growth, influences the cell cycle, and reduces apoptosis in vitro and vivo. Mechanistically, LINC02595 promoted BCL2‐like 1 (BCL2L1) expression through miR‐203b‐3p sponging. Our research demonstrated that LINC02595 is an oncogene in CRC and established the presence of a LINC02595‐miR‐203b‐BCL2L1 axis in CRC, which might provide a new diagnostic biomarker and therapeutic targets for the treatment of this disease.

therapeutic efficacy of surgery. Therefore, discovering highly effective diagnostic biomarkers and understanding the molecular mechanisms underlying CRC tumorigenesis are needed to significantly increase the survival rate of patients with CRC.
In the present study, our objective was to identify novel lncRNAs in CRC through a comprehensive analysis of microarray, The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) datasets. We determined the lncRNA expression profile in primary tumor tissue and paired adjacent normal tissue using the lncRNA and messenger RNA (mRNA) microarray. Through a detailed analysis of the expression of lncRNAs in various tissues by quantitative real-time polymerase chain reaction (qRT-PCR) and verification in TCGA and GEO datasets, we found that LINC02595 was significantly upregulated in CRC tissues. We further evaluated the function of LINC02595 through bioinformatics and experimentally in CRC cells.
Moreover, we established LINC02595 as a novel biomarker and oncogene that functions as a ceRNA of miR-203b-3p to regulate the expression of BCL2-like 1 (BCL2L1) in CRC. This study identified a potential new biomarker and therapeutic target for CRC.

| Microarray and computational analysis
The samples (five colorectal primer tumor tissues and their adjacent nontumor tissues) were used to purify RNA and synthesize doublestranded complementary DNA (cDNA), which was hybridized to the Human LncRNA Gene Expression Microarray V4 (CapitalBio Corp, Beijing, China). The lncRNA and mRNA array data were subjected to data summarization, normalization, and quality control using GeneSpring software V13.0 (Agilent). A fold-change (FC) of >2 and a threshold of p < .05 were used to select differentially expressed lncRNAs.

| Expression profile analysis from GEO and TCGA
Three CRC raw microarray datasets (GSE110715, GSE109454, and GSE70880) were downloaded from the GEO database. Raw data for 506 patients with CRC were downloaded from TCGA data portal.
Perl and R package were used to analyze all the datasets with FC > 2 and p < .05 as a threshold.

| Gene set enrichment analysis
Gene set enrichment analysis v3.0 (GSEA v3.0) was carried out using the JAVA program, and enrichment analysis were performed on normalized mRNA expression profiles. Random sample permutations (n = 1,000) were used to calculate the p value. A false discovery rate (FDR) <0.01 and a nominal p < .05 were considered as significant.

| RNA extraction and qRT-PCR analysis
Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad) following the manufacturer's instructions. For plasma samples, total RNA was isolated from cell-free plasma (200 µl) using a miRNeasy Serum/Plasma Kit (Qiagen, Germany) following the manufacturer's instructions. cDNA was synthesized using a Transcriptor First-Strand cDNA Synthesis Kit (Roche) following the manufacturer's instructions.
qRT-PCR was performed using a FastStart Universal SYBR Green Master (ROX; Roche) and a LightCycler 480. Relative expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 expression. The primers used for target amplification are presented in Table 1. All qRT-PCR results were analyzed using HT29, and RKO cells were maintained in Roswell Park Memorial Institute 1640 (HyClone, Logan, UT), Caco-2 and 293T were maintained in Dulbecco's modified Eagle's medium containing high glucose (HyClone, Logan, UT). All media were supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY). The cells were grown in a humidified 5% CO 2 incubator at 37°C.

| Subcellular fractionation
The cytoplasm and nuclear fractions of cells were separated using the PARIS Kit (Invitrogen) following the manufacturer's instructions.
qRT-PCR was used to analyze the ratios of expression, and the results were calculated using the −ΔΔ 2 Ct values. U6 and GAPDH served as the nuclear and cytoplasmic markers, respectively. sh-LINC02595 or sh-empty vector in the presence of 5 μg/ml polybrene for 24 hr followed by puromycin (4 μg/ml) selection for 5 days.

| RNA fluorescence in situ hybridization
LINC02595 expression was confirmed by qRT-PCR.

| Mouse xenografts
The mouse experiments were approved by the Institutional Ethics Committee of China Medical University. Ten BALB/c immunodeficient mice 4-6 weeks of age) were inoculated subcutaneously with stable HT29-sh-empty vector or HT29-sh-LINC02595 cells (4 × 10 6 in 0.2 ml) into the mouse abdomen. Tumor volumes were measured as (length × width 2 )/2 every 5 days. All the mice were euthanized 20 days postinoculation.

| Cell proliferation
CRC cell proliferation was measured using the cell counting kit-8 assay (Dojindo Lab, Japan) following the manufacturer's protocol.
Cell proliferation was also evaluated using the colony formation assay. CRC cells (1 × 10 3 /well) were seeded into six-well plates. After incubation for 10 days, the cells were fixed with formaldehyde, stained with 0.5% crystal violet, rinsed with H 2 O, and air-dried.
Colonies were counted using ImageJ software. 2.14 | Dual-luciferase reporter assay

| RESULTS
3.1 | Comprehensive analysis of lncRNAs expression profiles in CRC using lncRNA microarray, TCGA transcriptome profiling, and GEO microarray datasets The initial lncRNA and mRNA microarray analysis to identify lncRNAs and mRNAs that are dysregulated in CRC was performed using five pairs of matched CRC and adjacent non-cancer tissue samples. We identified 236 lncRNAs and 1,500 mRNAs that were upregulated, and 620 lncRNAs and 895 mRNAs that were downregulated in the CRC tissues compared with the adjacent normal tissue (FC > 2; p < .05; Figure 1a,b We also used lncRNA PVT1, which was previously identified in CRC, as a control. The results from this additional analysis were consistent with microarray results and identified ENST00000456532.1 (LINC02595) as the most significantly overexpressed lncRNA in CRC tissues (p < .05; Figure 1c).
To determine if LINC02595 plays a pivotal role in the pathogenesis of CRC, we analyzed the transcriptome profiles of CRC in the TCGA dataset. A total of 506 patients with CRC were enrolled in the study, which contained 534 tumors and 41 adjacent noncancer tissues. Using a cutoff threshold, we identified 869 upregulated and 756 downregulated mRNAs. In addition, 823 lncRNAs were differentially expressed (logFC > 2 and FDR < 0.01), including 622 upregulated and 201 downregulated lncRNAs ( Figure S1a,b).
We found that LINC02595 was also significantly upregulated in 534 unpaired and 41 paired patients with CRC samples (p < .05; Figure 1d,e). Correlation analysis between the LINC02595 expression levels and patient clinical features did not identify any association between LINC02595 expression and gender, age, tumor location, tumor invasion depth, or distant metastasis in CRC (Table   S3). Furthermore, the OS rate of patients with underexpressed LINC02595 was better than that of patients with overexpressed LINC02595. However, this difference was not statistically significant (p = .36; Figure S1c). Finally, a comprehensive transcriptional analysis of three independent microarray datasets (GSE110715, GSE109454, and GSE70880) from the GEO dataset confirmed that LINC02595 was upregulated in CRC tissues (p < .05; Figure 1f,g). Taken together, these results revealed that LINC02595 upregulation might have a critical role in the progression of CRC. association was observed with other parameters (e.g., gender, age, and cancer subtype; Table 2).

| LINC02595 is upregulated in CRC tissues
There have been many studies that have demonstrated that patients with inflammatory bowel disease (IBD) have a high risk of developing CRC. Therefore, we hypothesized that LINC02595 expression might also be elevated in patients with IBD. To evaluate this hypothesis, LINC02595 expression was measured in 21 IBD and 15 adjacent normal tissues. We found that LINC02595 was overexpressed in the IBD tissues but to a lesser extent than in patients with CRC (p = .004; Figure 2b). These data suggest that LINC02595 could be an important marker for the early diagnosis of CRC.

| LINC02595 is a potential diagnostic biomarker for CRC
Many studies have demonstrated that lncRNAs could represent a potential new class of diagnostic biomarkers. As LINC02595 was upregulated in CRC tumor tissues, we investigated whether LINC02595 expression was increased in peripheral blood plasma, which would make it a potential biomarker for CRC diagnosis. To this end, qRT-PCR was performed to detect LINC02595 expression in plasma. We found that LINC02595 expression was increased in the plasma from CRC patients compared with control samples (p = 3.856e−04; Figure 2c). Based on the receiver operator curve (ROC) analysis between patients with CRC and controls, the cut-off value for plasma LINC02595 was 4.29 with an area under the curve (AUC) of 0.781. The sensitivity and specificity for LINC02595 expression in the plasma were 0.667 and 0.84 (Figure 2d).

| LINC02595 knockdown inhibits CRC cell proliferation
To investigate the biological function of LINC02595 in CRC,

| LINC02595 inhibits apoptosis
To identify whether CRC cell proliferation was influenced by apoptosis, we assessed the effects of LINC02595 on apoptosis. We found that HT29 and RKO cells transfected with LINC02595 smart silencer exhibited a markedly increased rate of apoptosis compared with control cells (p < .05; Figure 3a,b). In contrast, LINC02595 overexpression significantly inhibited apoptosis in HCT116 cells (p < .05; Figure 3c). Furthermore, LINC02595 knockdown caused the downregulation of Bcl2, Survivin protein levels and upregulation of the BAX protein level of these key cell apoptosis regulators (Figure 3d).
3.6 | LINC02595 knockdown induces G1/G0 cell cycle arrest in CRC cells We next evaluated the effect of LINC02595 knockdown on cell cycle progression. HT29 and RKO cells transfected with LINC02595 smart silencer had increased percentages of cells in the G1/G0 phase of the cell cycle (p < .05; Figure 4a,b). LINC02595 overexpression reduced the percentages of cells in this phase (p < .05; Figure 4c). Furthermore, LINC02595 knockdown caused the downregulation of CDK4, CDK6 protein levels of these key cell cycle regulators (Figure 4d).

| LINC02595 knockdown inhibits the growth of CRC xenografts
To determine whether LINC02595 affected tumorigenesis in vivo, the effect of LINC02595 knockdown on the growth of CRC xenografts was evaluated. We found that sh-LINC02595 HT29 cells formed smaller tumors in immunodeficient mice compared with the HT29-sh-empty control cells (p < .05; Figure 5a-c). As expected, these tumors expressed significantly lower levels of LINC02595 than the sh-empty control tumors (p < .05; Figure 5d). Furthermore, the sh-LINC02595 tumors had lower Ki67-positive rates than the control tumors (P < 0.05; Figure 5e). Taken together, these data suggested that LINC02595 promotes tumor growth in vivo.

| LINC02595 acts as a miR-203b-3p sponge
Previous studies showed that lncRNAs could function as ceRNAs to exert their effects on cells. To determine whether LINC02595 could function as a molecular sponge for microRNA (miRNA), the distribution of LINC02595 in RKO cells and HT29 cells were determined by qRT-PCR and FISH assay. This analysis demonstrated that LINC02595 localized to both the nucleus and cytoplasm (Figure 6a). Through FISH assay, we found that LINC02595 was mostly located in the cytoplasm ( Figure 6b). All these results suggested that LINC02595 could regulate gene expression at both the transcriptional and posttranscriptional levels. Bioinformatics analysis DIANA was used to predict the miRNAs that could bind to LINC02595. We found that miR-3942-3p, miR-4715-3p, and miR-203b-3p generated the highest scores for binding the LINC02595 sequence.
We found that only miR-203b-3p significantly decreased the luciferase activity of WT LINC02595 (Figure 6c). In addition, we constructed a reporter vector in which the potential miR-203b-3p binding site in the sequence of LINC02595 was mutated (MT-LINC02595), as expected, con-transfection of the MT-LINC02595 with miR-203b-3p mimic could not repress the luciferase activity (p < .05; Figure 6d). Furthermore, miR-203b-3p expression was downregulated in 116 CRC tumors tissues compared with matched control tissue (p = 1.254e−29; Figure 6e) and in
These data suggest that LINC20595 may regulate CRC proliferation, apoptosis, and the cell cycle by sponging miR-203b-3p. between these two groups showed enrichment for the nuclear factor-κB (NF-κB) signaling pathways (p < .05; Figure 8b). These findings suggested that LINC02595 regulates cell proliferation through NF-κB signaling to regulate downstream target genes. Of the 11 potential target genes, only BCL2L1 was related to NF-κB signaling. Aakko et al. (2019) previously demonstrated that BCL2L1 has a binding site for miR-203b-3p in breast cancer. Thus, BCL2L1 was chosen as a target for our further studies to understand whether BCL2L1 and miR-203b-3p have the same mechanism in CRC. A dual-luciferase assay was performed to confirm the bioinformatics results. HEK293T cells were cotransfected with WT-BCL2L1 and miR-203b-3p mimic or NC. We found that miR-203b-3p significantly decreased the luciferase activity of WT-BCL2L1, but not the MT-BCL2L1 (p < .05; Figure 8c). In addition, miR-203b-3p overexpression significantly reduced BCL2L1 mRNA and protein levels in HT29 cells (p < .05; Figure 8d,e). In contrast, miR-203b-3p knockdown promoted BCL2L1 mRNA and protein expression (p < .05; Figure 8d,e). As LINC02595 could function as a ceRNA for miR-203b-3p, we investigated whether LINC02595 could indirectly regulate the expression of BCL2L1. We found that LINC02595 knockdown reduced BCL2L1 mRNA and protein expression, whereas LINC02595 overexpression promoted BCL2L1 protein expression only in CRC cells (p < .05; Figure 8d,e). To further demonstrate that LINC02595 could regulate BCL2L1 expression through sponging miR-203b-3p, western blot analysis assays showed that transfection of the miR-203b-3p mimic abolished BCL2L1 increase in LINC02595 upregulated cells (p < .05; Figure 8f). Furthermore, the miR-203b-3p knockdown could rescue the BCL2L1 protein level downregulation induced by LINC02595 (p < .05; Figure 8f). Finally, we performed dual-luciferase reporter assays in HT29 cells, the results showed that the transfection of WT-LINC02595 plasmids but not the MT- whereas LINC02595 knockdown reduced Luc-BCL2L1 luciferase activity significantly, and transfection of the miR-203b-3p inhibitor antagonized this decrease (p < .05; Figure 8g). All these results showed that LINC02595 could regulate BCL2L1 expression by sponging miR-203b-3p in CRC.

| BCL2L1 is upregulated in CRC
To understand the function of BCL2L1 in CRC progression, we measured BCL2L1 expression levels in 116 paired CRC and normal tissues and CRC cell lines by qRT-PCR. We found that BCL2L1 expression levels were significantly upregulated in the CRC tumor tissues and cell lines compared with the adjacent nontumor tissues (p < .05; Figure 9a,b). These results were confirmed by TCGA transcriptome profiling analysis (p < .05; Figure 9c). Furthermore, IHC showed that BCL2L1 protein levels were also upregulated in the CRC tumors compared with the adjacent nontumor tissue (p < .05; Figure 9d). Correlation analysis revealed a positive correlation between the LINC02595 and BCL2L1 expression levels in the 116 CRC tissues (r = 0.769; p = 6.248e−24; Figure 9e) and the TCGA dataset (r = 0.231; p = 5.325e−08; Figure S1d). Together, these results identified an important LINC02595/miR-203b-3p/BCL2L1 regulatory axis in CRC (Figure 9f). In recent years, Xian et al. (Xian & Zhao, 2019) found that KCNQ1OT1 was upregulated in methotrexate-resistant cells and promoted the methotrexate resistance of CRC cells by regulating miR-760/PPP1R1B signaling. G. Li, Wang, Wang, Xu, and Zhang (2018) showed that LINC00312 was downregulated in CRC tissues   (Li, Chen, Zhao, Kong, & Zhang, 2011;T. Li, Gao, & Zhang, 2015).
In this study, we found that miR-203b-3p was downregulated in CRC cell lines and tissues compared with adjacent nontumor tissues, and overexpression of miR-203b-3p inhibited cell proliferation, increased the rate of apoptosis, and caused G1/G0 cell cycle arrest. Furthermore, we found that the inhibition of these effects by the LINC02595 smart silencer could be rescued by a miR-203b-3p inhibitor. We also found a negative correlation between the expression levels of LINC02595 and miR-203b-3p in 116 CRC samples. However, miR-203b-3p was upregulated in CRC samples from the TCGA dataset, which was in contrast with our experimental conclusion.
lncRNAs act as ceRNA through the inhibition of its miRNA target gene. In our study, we identified BCL2L1 as the target gene of miR-203b-3p using bioinformatic tools (miRDB, miRTarBase, TargetScan). GSEA analysis showed that high expression of LINC02595 was enriched for the NF-κB signaling pathway, and BCL2L1 is one of the most important components of NF-κB signaling. In 1986, NF-κB was found to interact with the 11-base pair sequence in the immunoglobulin light-chain enhancer of B cells.
Multiple papers demonstrated that it was a key factor involved in cell proliferation, inflammation, cell death, and cell survival. Two general NF-κB signaling pathways, which include the classical and alternative pathways were shown to correlate with tumor progression. The targets of NF-κB signaling involving the classical NF-κB signaling pathway include antiapoptotic proteins (e.g., BclxL, BCL2) and pro-proliferative proteins (e.g., cyclin D1, MYC; Karin, 2006). X.  found that the adaptively expressed endogenous epidermal growth factor could activate the cyclin D1/P53/PARP signaling pathway in pancreatic cancer cells. Aakko et al. (2019) also demonstrated that BCL2L1 has a miR-203b-3p binding site in breast cancer. BCL2L1 is a member of the Bcl2 protein family that regulates mitochondrial-mediated apoptosis by regulating the release of proapoptotic factors from the mitochondria (Chen et al., 2005). BCL2L1 has been identified as an important survival factor in many tumor types (Obasi et al., 2018;Zhang et al., 2015;P. F. Zhang et al., 2019). In this study, we demonstrated that BCL2L1 is overexpressed in CRC compared with adjacent nontumor tissue, and a positive correlation existed between the expression levels of LINC02595 and BCL2L1 in 116 CRC tissues and the TCGA dataset.
Our study systematically analyzed the correlation of LINC02595, miR-203b-3p, and BCL2L1 in the modulation of CRC progression. We also demonstrated through the analysis of a large number of samples that LINC02595 might be a potential target for diagnosis and treatment of CRC. However, there are still several limitations to our study. First, our cohort study lacks survival time information, which affected our survival analysis based on LINC02595 expression. Furthermore, the plasma sample size presented in our study was confined to one ethnicity. Therefore, the study results might not apply to the general population. Finally, we only analyzed the role of LINC02595 in the progression of CRC as a ceRNA for miR-203b-3p to regulate the expression of BCL2L1. Analysis of its role in CDK6, CDK4, BCL2, Survivin, and BAX protein expression is currently in progress. How LINC02595 exerts its effect on the NF-κB signaling pathway requires further study.