Abstract
Background. Numerous studies have indicated the engagement of long non-coding RNA (lncRNA) in various cancer types, including colorectal cancer (CRC). However, the functional and mechanistic roles of lncRNAs in CRC remain largely elusive.
Objectives. The aim of this study was to explore the function and mechanism of lncRNA BVES-AS1 in CRC.
Materials and methods. The expression levels of BVES-AS1 were validated in CRC tissues and paired normal samples using quantitative real-time polymerase chain reaction (qPCR) Subsequently, the biological functions of BVES-AS1 in CRC cells were investigated both in vitro and in vivo. Various experimental techniques such as western blot, fluorescence in situ hybridization, RNA-sequencing (RNA-seq), biotin-labeled miRNA pulldown assay, dual-luciferase reporter gene assay, and RNA-protein immunoprecipitation (RIP) assay were employed to elucidate the potential mechanism of BVES-AS1.
Results. The findings of this study demonstrated that BVES-AS1 expression was downregulated in CRC tissues compared to normal tissues, and its expression level was associated with tumor infiltration and tumor-nodule-metastasis (TNM) stage. Furthermore, BVES-AS1 was found to suppress CRC cell proliferation, migration and metastasis both in vitro and in vivo. Mechanistically, BVES-AS1 acted as a sponge for miR-1269a and miR-1269b, thereby regulating SVEP1. Additionally, the silencing of SVEP1 activated the PI3K/AKT pathway.
Conclusions. These results suggest that BVES-AS1 plays a crucial role in the progression of CRC through the miR-1269a/b-SVEP1-PI3K/AKT axis, providing new insights into the therapeutic strategies for CRC.
Key words: miRNA, PI3K/AKT, colorectal cancer, BVES-AS1, SVEP1
Background
Colorectal cancer (CRC) is the 2nd leading cause of cancer-related deaths. The number of cancer-related deaths caused by CRC accounts for 9.4% of all cancers.1, 2 Local recurrence, metastasis and resistance to therapy are still the main reasons for cancer-related death in CRC.3, 4 At present, the mechanisms of tumorigenesis and metastasis in CRC remain unknown. Thus, clarifying the tumorigenesis, metastasis and therapeutic resistance potential molecular mechanisms of CRC is imperative.
Studies have indicated that lncRNAs can regulate CRC by serving as scaffolds, signals, decoys, or guide molecules to interact with mRNA, chromatin, miRNA, or protein.5, 6 It has been reported that lncRNAs participate in the proliferation, migration, invasion, and apoptosis processes of CRC. For example, DLGAP1-AS2 mediates the ubiquitination of Trim21 and the degradation of ELOA to promote CRC cell proliferation and metastasis.7 LncRNAs can not only drive the progression of CRC as oncogenes but also drive the progression of CRC as tumor suppressor genes. LINC01559 expression was downregulated in CRC. LINC01559 overexpression inhibited CRC cell growth and invasive capacity by sponging the miR-106b-5p to regulate PTEN expression.8 In addition, lncRNAs can also act as effective diagnostic biomarkers and early screening indicators of CRC.9, 10, 11
SVEP1 is a multi-structural domain extracellular matrix protein composed of Sushi, Von Willebrand factor type A, epidermal growth factor (EGF) and pentraxin domain-containing protein 1.12 Studies have suggested that SVEP1 plays a vital function in cell adhesion, normal lymphatic vessel development and epidermal differentiation.13, 14, 15, 16 SVEP1 and its alternative splice forms may regulate the invasive ability of breast cancer cells within the bone wall niche.17 A recent study revealed that SVEP1 levels in hepatocellular carcinoma (HCC) tissues were significantly downregulated, and high SVEP1 expression was correlated with better progression. Mechanistically, SVEP1 regulated the invasion and metastasis of HCC cells through the PI3K/ATK pathway.18, 19 However, the function of SVEP1 in CRC remains unknown.
LncRNA BVES-AS1, located in the q21 region of chromosome 6, is the antisense strand of the protein-coding gene BVES. BVES-AS1 has been found to have prognostic value in triple-negative breast cancer, with higher expression correlating with improved overall survival.20 A previous study indicated that BVES-AS1 expression was downregulated in colon cancer and can serve as a prognostic marker for colon cancer patients. However, the exact mechanism by which BVES-AS1 affects colon cancer is still not fully understood.21 In our previous analysis using The Cancer Genome Atlas (TCGA) database, we observed that the expression of BVES-AS1 in CRC tissues was significantly lower than that in paracancerous tissues. Further bioinformatic analysis revealed a significant positive correlation between BVES-AS1 and SVEP1 in CRC tissues. SVEP1 has been shown to exhibit tumor suppression effects in various cancers. Therefore, we hypothesize that BVES-AS1 may regulate the biological functions of CRC through its interaction with SVEP1.
Objectives
The purpose of this study was to investigate the role of BVES-AS1 in CRC and the mechanism by which BVES-AS1 regulates SVEP1.
Materials and methods
Clinical samples
Colorectal cancer and paired paracancerous tissues were collected from patients who underwent radical surgery at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) between September 2020 and March 2021. A total of 96 CRC patients were included. The collected samples were immediately placed in liquid nitrogen for freezing and then transferred to a –80°C refrigerator for permanent storage. The clinical data of CRC patients were obtained by consulting the electronic medical record system. All included patients were confirmed to have adenocarcinoma or mucinous adenocarcinoma by pathological examination and received no neoadjuvant therapy (including chemoradiotherapy, targeted therapy or immunotherapy). In addition, patients had no other malignancies. This research protocol was reviewed and approved by the Medical Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval No. 2020-358) and informed consent was obtained from all the study participants.
Cell culture and transfection
Cell culture and transfection were conducted as previously reported. The human CRC cell lines SW620, HT-29, LoVo, and SW480 were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The normal colonic epithelial cell NCM460 line was obtained from the American Type Culture Collection (ATCC; Rockville, USA). SW620, HT-29, SW480, and NCM460 cells were cultured with DMEM (Gibco, Waltham, USA), and the F12 medium (Gibco) was used to culture LoVo cells. All media were replenished with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin (Gibco). Small interfering RNA (siRNAs), mimics and plasmids were designed and synthesized by (RiboBio, Guangzhou, China) and transfected into SW480 and HT-29 cells using Lipofectamine 3000 (Lipo3000; Invitrogen, Waltham, USA). Lentiviruses of control and overexpression human BVES-AS1 were packaged by HanBio (Shanghai, China). Stable overexpression of BVES-AS1 CRC cells after lentivirus transfection was screened with puromycin (MedChemExpress, Monmouth Junction, USA). The siRNA and mimic sequences are shown in Supplementary Table 1.
RNA extraction and quantitative real-time PCR
The RNA extraction procedure was performed as previously described. Total RNA was extracted from CRC tissues and cells with RNAiso Plus (Takara, Shiga, Japan). A Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA) was used to analyze the extracted RNA concentration and quality. The RT Reagent Kit and SYBR Green Kit (Takara) were used for reverse transcription (RT) and quantitative real-time polymerase chain reaction (qPCR) detection, respectively. Specific stem-loop primers were designed for the reverse transcription of microRNA (Tsingke, Beijing, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls for mRNA and miRNA, respectively. The sequences of all primers are described in Supplementary Table 2. The relative RNA expression was calculated using the 2^–ΔΔCt method.
RNA sequencing and bioinformatics analysis
RNA-sequencing (RNA-Seq) data for CRC patients were obtained from the TCGA database (portal.gdc.cancer.gov/) in March 2021. A total of 602 cases were downloaded, of which 554 were derived from CRC and 48 cases from normal colorectal tissue. The differentially expressed lncRNAs and mRNAs were filtered using the “edgeR” packages of R software (R Foundation for Statistical Computig, Vienna, Austria) with |log2foldchange|≥2 and p < 0.05. The correlation of lncRNA-mRNA or mRNA-mRNA was verified using Spearman’s correlation analysis. The miRNAs targeting lncRNAs were predicted using the Lncbase (www.microrna.gr/LncBase) and miRDB (www.mirdb.org/) databases. The TargetScan (www.targetscan.org) database was used to predict the binding between mRNA and miRNA. RNA sequencing of the HT-29 vector group and BVES-AS1 group was conducted using Illumina (Personalbio, Nanjing, China). A human genome reference was constructed based on GRCh38/hg38 of the UCSC. The R software package was used to perform the cluster and differential gene analyses. The differential mRNA was screened with |log2foldchange|≥1 and p < 0.05.
Cell Counting Kit-8 assay
The Cell Counting Kit-8 (CCK-8) assay was performed according to previously described methods.7 A total of 3,000 logarithmic growth stage CRC cells were inoculated in a 96-well plate. Ten microliters of CCK-8 solution (APExBIO, Houston, USA) were added at 0 h, 24 h, 48 h, and 72 h, and then incubated at 37°C for 2 h. The optical density (OD) values were measured at 450 nm using a spectrophotometer Multiskan MK3 (Thermo Fisher Scientific).
EdU assay
The 5-ethynyl-2’-deoxyuridine (EdU) assay was performed according to the manufacturer’s protocol of the EdU Cell Proliferation Kit (Beyotime Biotechnology, Shanghai, China). Colorectal cancer cells were seeded into 96-well plates and maintained for 24 h. After adding 50 mM EdU solution to the culture plates, the cells were cultured at 5% CO2 and 37°C for 2 h. After fixing the cells with 4% paraformaldehyde (PFA), the cells were permeabilized with 1% Triton X-100. Azide 594 and Hoechst 33342 were used for cell staining. Photographs were taken with a fluorescence microscope (model TE2000; Nikon Corp., Tokyo, Japan).
Wound healing assay
Colorectal cancer cells were seeded into 6-well plates and maintained until more than 90% of the cell density was reached. A sterile pipette tip formed a manual scratch on the bottom of the culture plate. Phosphate-buffered saline (PBS) was used to wash the culture plates, and the exfoliated cells were removed. Serum-free Dulbecco’s modified Eagle’s medium (DMEM) was used to culture cells. A microscope (model TS2; Nikon Corp.) was used to take pictures at 0 h and 24 h.
Transwell assay
The transwell assay was conducted as previously reported.7 The cells were resuspended in serum-free DMEM. The upper chamber of the transwell (Corning Company, Corning, USA) was inoculated with 200 μL of cell suspension, while the DMEM with 15% FBS was added to lower chamber. After 48 h of incubation at 5% CO2 and 37°C, 4% PFA was used to fix cells, and 1% crystal violet (Beyotime Biotechnology) was used to stain cells. In the transwell invasion assay, Matrigel (BD Biosciences, Franklin Lakes, USA) was used to cover the Transwell upper chamber before inoculating resuspended cells.
Western blot
The protein extraction and western blot analysis were based on a previous report.7 The primary antibodies including the following: rabbit E-cadherin antibody (#3195, Cell Signaling Technology (CST), Danvers, USA), rabbit PI3K antibody (#4257; CST), rabbit N-cadherin antibody (#13116; CST), rabbit P-PI3K antibody (#17366; CST), rabbit vimentin antibody (#5741; CST), rabbit AKT antibody (#2938; CST), rabbit p-AKT antibody (#13038; CST), and rabbit SVEP1 antibody (R&D Biosystems, Minneapolis, USA). Rabbit GAPDH antibody was purchased from Proteintech (Wuhan, China). The analysis was performed using an electrochemoluminescence (ECL) detection system (Bio-Rad, Hercules, USA).
Apoptosis assay
The Annexin-V-FITC apoptosis detection kit (Thermo Fisher Scientific) was used for the flow cytometry apoptosis assay. The centrifuged cells were resuspended in flow cytometry binding buffer (100 μL), and Annexin V/FITC (5 μL) and propidium iodide (PI; 5 μL) were used for staining. Apoptotic cells were detected using flow cytometry (CytoFLEX, Pasadena, USA).
Fluorescence in situ hybridization
The fluorescence in situ hybridization (FISH) kit (RiboBio) and BVES-AS1 probe with Cy3 labeling (GenePharma, Shanghai, China) were used for the FISH assay. According to the protocol, Cy3-labeled BVES-AS1 probes were incubated with CRC cells for 16 h at 37°C. Different concentrations of saline sodium citrate buffer solution was used to wash CRC cells. The nuclei of CRC cells were stained with DAPI (4’,6-diamidino-2-phenylindole). A laser confocal microscope (model LSM700; Carl Zeiss, Oberkochen, Germany) was performed to take photographs.
Dual-luciferase gene reporter assay
BVES-AS1 and SVEP1 3’-untranslated region (3’-UTR) sequences wild-type (WT) and mutant (Mut), which contain miR-1269a and miR-1269b binding sites, were constructed on the pSI-Check2 plasmid vector (Hanbio, Shanghai, China). The miR-1269a mimics, miR-1269b mimics and negative control (NC) mimics were cotransfected with WT and Mut dual-luciferase gene reporter plasmids into CRC cells using Lipo3000. Luciferase activity was detected after 48 h of transfection.
Biotin-labeled miRNA pulldown assay
Biotin-coupled miR-1269a mimics, miR-1269b mimics and NC mimics were designed and made by Sangon Biotech (Shanghai, China). Colorectal cancer cells were lysed after the biotin-labeled miR-1269a mimics, miR-1269b mimics and NC mimics were transfected for 48 h. Simultaneously, washed streptavidin magnetic beads (Invitrogen) were blocked at 4°C for 2 h The cell lysate was added to the washed magnetic beads and incubated in the mixed solution for 12–16 h at 4°C. After washing streptavidin magnetic beads 5 times, 500 μL of TRIzol was added to extract the RNA that specifically interacts with miRNA. Finally, the relative expression of enriched RNA was measured with qPCR.
RNA-binding protein immunoprecipitation assay
The RIP Kit (MilliporeSigma, St. Louis, USA) was applied for RIP assays. Briefly, RIP lysis solution was used to collect and lyse CRC cells. Anti-AGO2 antibody (Proteintech and normal rabbit IgG (MilliporeSigma) were added into the lysate, and the mixture was incubated at 4°C for 12–16 h with rotation. Subsequently, 30 µL of Protein A/G Magnetic Bead was added and incubated with rotation for 6 h at 4°C. After washing, the enriched RNA was extracted using the TRIzol reagent.
Xenografts in mice
The procedure of xenografts in mice was described in a previous study.7 Briefly, 4–6-week-old female BALB/c nude mice were provided by Ensiweier Biotechnology Ltd. (Chongqing, China) and housed in an environment free of specific pathogenic bacteria. Ten mice were randomly assigned into 2 groups; 200 µL (5×106) of HT-29 cells stably overexpressing BVES-AS1 or empty vector was injected subcutaneously into the right axilla of nude mice. The long axes and short axes of subcutaneous tumors were measured on days 5, 10, 15, 20, and 25 after injection, and the formula: V = (width2+long)/2 was used to calculate the subcutaneous tumor volume. The mice were sacrificed on the 25th day after tumor injection. The subcutaneous tumors were extracted from the nude mice and weighed, and the mass and volume of the tumors were measured. Tumors were fixed in a 4% formalin solution for hematoxylin & eosin (H&E) staining and immunohistochemistry (IHC). All animal experiments were conducted according to the protocol approved by the Animal Experimental Research Ethics Committee of Chongqing Medicine University (approval No. IACUC-CQMU-2022-0019).
Immunohistochemistry
The standard methods of IHC have been described in a previous study by Lu et al.22 Briefly, the collected samples were fixed with a 4% formaldehyde solution. After embedding in paraffin, they were cut into 4-µm sections. After dewaxing and hydration, the tissue sections were placed into 3% hydrogen peroxide to block the endogenous peroxidase activity. Anti-SVEP1 antibody (R&D Biosystems) and anti-Ki 67 antibody (Proteintech) solutions were added to the sections and incubated for 12–16 h at 4°C with anti-SVEP1 antibody and anti-Ki 67 antibody. The brown precipitates produced by 3,3’-diaminobenzidine (DAB) visualized the results. Finally, the sections were restained using hematoxylin. Immunohistochemistry images were obtained using a microscope (Olympus BX51; Olympus Corp., Tokyo, Japan).
Statistical analyses
Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, San Diego, USA) and IBM SPSS statistical software v. 25.0 (IBM Corp., Armonk, USA). All experiments were performed independently in triplicate, with each independent assay containing at least 3 samples. Mean ± standard deviation (M ±SD) was used to describe normally distributed data. Normality of the data was assessed using the Shapiro–Wilk test (Supplementary Table 5), and the homogeneity of variance was tested using the Levene’s test (Supplementary Table 7). For non-normally distributed paired samples, Wilcoxon signed rank analysis was conducted. Student’s t-test was used to compare 2 samples with normal distribution, while one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was applied to compare 3 or more samples with normal distribution and homogeneity of variance. If the data did not follow a normal distribution or heterogeneous variance, or the sample size was less than 9, the Mann–Whitney U test was performed to examine the data of 2 groups, and the Kruskal–Wallis test followed by Dunnett’s post hoc test was used for the difference analysis of 3 or more groups (Supplementary Table 6). Spearman’s correlation coefficient analysis was performed to evaluate the association between genes. The relationship between BVES-AS1 and the baseline clinical characteristics of CRC patients was analyzed using the χ2 or Fisher’s exact test. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.
Results
BVES-AS1 is downregulated in CRC and related to pathological stage
The bioinformatics analysis of TCGA data revealed 720 lncRNAs that were upregulated, and 293 lncRNAs that were downregulated in CRC tissue based on |log2fold change|≥2 and p < 0.05. The clustering heatmap showed the dysregulated genes in 47 paired CRC and normal colorectal tissues (Figure 1A). The differential expression gene analysis showed that BVES-AS1 expression in paracancerous tissue was significantly higher than that in CRC tissue (Figure 1B,C). The 96 CRC tissues and paracancerous tissues were gleaned and analyzed. The results showed that BVES-AS1 expression in CRC tissues was significantly downregulated compared with that in paracancerous tissues (Figure 1D). The clinical baseline analysis of CRC patients indicated that BVES-AS1 expression was closely associated with the tumor infiltration (p = 0.024), lymph node positivity (p = 0.029) and tumor-nodule-metastasis (TNM) stage (p = 0.009) (Figure 1E–G, Supplementary Table 3). Furthermore, BVES-AS1 was significantly downregulated in LoVo, HT29, SW480, and SW620 cells compared with NCM460 cells (Figure 1H).
BVES-AS1 inhibits CRC cell proliferation and invasion in vitro
The previous results indicated that BVES-AS1 expression was downregulated in HT-29 cells, but relatively high in SW480 cells (Figure 1H). Hence, SW480 and HT-29 cells were used in subsequent studies. To elucidate the effect of BVES-AS1 on CRC biological function, HT-29 and SW480 cells with stable overexpression of BVES-AS1 and vector were constructed by lentivirus infection (Figure 2A). In addition, BVES-AS1 expression in SW480 cells was knocked down by si-BVES-AS1#1, and si-BVES-AS1#2 (Figure 2B). Cell Counting Kit-8 and EdU assays showed that BVES-AS1 overexpression prominently suppressed HT-29 and SW480 cell proliferation while silencing BVES-AS1 promoted SW480 cell viability (Figure 2C–F, Supplementary Fig. 1A–C). Transwell and wound healing assay results indicated that the migration and invasion abilities of CRC cells were significantly suppressed by upregulated BVES-AS1 (Figure 3A,C, Supplementary Fig. 1D–G). In contrast, the migration and invasion abilities of SW480 cells were enhanced by BVES-AS1 knockdown (Figure 3B,D). Meanwhile, western blot analysis revealed that BVES-AS1 upregulation significantly downregulated N-cadherin and vimentin levels while silencing BVES-AS1 produced the opposite effects (Figure 4E,F). Flow cytometry apoptosis assays indicated that the overexpression of BVES-AS1 contributed to SW480 and HT-29 cell apoptosis (Figure 4A,B, Supplementary Fig. 1H,I). BVES-AS1 downregulation inhibited SW480 cell apoptosis (Figure 4C,D). In general, BVES-AS1 acted as a tumor suppressor gene to inhibit CRC cell biological roles in vitro.
BVES-AS1 functions as a sponge for miR-1269a and miR-1269b
To clarify the mechanism of BVES-AS1, we employed nuclear-cytoplasmic fractionation and FISH assays and observed that BVES-AS1 was mainly distributed in the cytoplasm of HT-29 and SW480 cells (Figure 5A,B). Next, we used the miRDB and LncBase databases to predict miRNAs that may bind to BVES-AS1. The predicted results revealed that BVES-AS1 contained miR-1269a and miR-1269b binding sites (Figure 5C). Unexpectedly, the seed sequences of miR-1269a and miR-1269b were identical (Figure 5J). The study also found that compared with normal colorectal tissue, the miR-1269a and miR-1269b levels in CRC were upregulated (Figure 5D,F). Correlation analysis demonstrated that miR-1269a and miR-1269b expression had a negative correlation with BVES-AS1 expression in CRC (Figure 5E,G). In addition, overexpression of miR-1269a and miR-1269b in HT-29 and SW480 cells significantly downregulated BVES-AS1 mRNA levels (Figure 5H,I). To further explain the relationship between miR-1269a, miR-1269b and BVES-AS1, the WT and Mut dual-luciferase reporter gene plasmids of BVES-AS1 were constructed and synthesized. (Figure 5J). The miR-1269a mimics, miR-1269b mimics or NC mimics were cotransfected with WT or Mut plasmids into HT-29 and SW480 cells to detect luciferase activity. The results indicated that the miR-1269a or miR-1269b mimics could significantly inhibit luciferase activity in the WT group compared with the NC mimics. In contrast, the luciferase activity in the Mut group was not significantly different (Figure 6A–D). Biotinylated miR-1269a mimic and miR-1269b mimic WT probes, and Mut probes were designed and synthesized for a biotin-coupled microRNA capture assay. The results indicated that BVES-AS1 in the WT probe group was significantly enriched in HT-29 cells (Figure 6E,F). In addition, RIP experiments further found that compared with the immunoglobulin G (IgG) group, the enrichment of BVES-AS1, miR-1269a and miR-1269b was significantly higher (Figure 6G). Our data suggested that BVES-AS1 may function as a sponge for miR-1269a and miR-1269b in CRC cells.
BVES-AS1 reverses the oncogenic effect of miR-1269a and miR-1269b
To evaluate whether BVES-AS1 can inhibit the biological function of CRC cells by sponging miR-1269a or miR-1269b, rescue experiments were performed. The results indicated that the miR-1269a and miR-1269b mimics promoted CRC cell proliferation and vitality while transfecting the BVES-AS1 plasmid partially eliminated the proliferation ability induced by miR-1269a mimics or miR-1269b mimics (Supplementary Fig. 2A–D). Transwell and wound healing assays indicated that compared with NC mimics, the overexpression of miR-1269a and miR-1269b advanced CRC cell migration and invasion ability. The migration and invasion mediated by miR-1269a and miR-1269b mimics were also attenuated by upregulated BVES-AS1 (Supplementary Fig. 2E–I). Western blot analysis showed that E-cadherin levels were partially downregulated in CRC cells after cotransfecting with miR-1269a mimics, miR-1269b mimics and BVES-AS1. In contrast, N-cadherin and vimentin protein levels showed the opposite results (Supplementary Fig. 2L). Furthermore, inhibition of apoptosis induced by miR-1269a mimics or miR-1269b mimics was also reversed by upregulated BVES-AS1 (Supplementary Fig. 2J,K). In summary, these results demonstrate that BVES-AS1 can reverse the oncogenic effect of miR-1269a and miR-1269b in CRC cells.
SVEP1 is the common downstream target of miR-1269a and miR-1269b
To further elucidate how BVES-AS1 regulated CRC cell function through miR-1269a and miR-1269b, we performed RNA-seq on HT-29 cells transfected with BVES-AS1 overexpression plasmid and vector plasmid. The results showed that compared with the vector group, 35 upregulated genes and 59 downregulated genes were detected in the BVES-AS1 overexpression group (|log2-fold change|≥1, p < 0.05) (Figure 7A, Supplementary Table 4). The TargetScan database and RNA-seq data were applied to prognosticate the target genes of miR-1269a and miR-1269b. The predicted results indicated that SVEP1 had binding sites for miR-1269a and miR-1269b (Figure 7B). In the TCGA database, SVEP1 was significantly downregulated in CRC tissue (Figure 7C). The qPCR detection of 96 CRC samples and matched adjacent samples suggested that the mRNA level of SVEP1 in CRC was also decreased (Figure 7D). Correlation analysis indicated that SVEP1 in CRC tissues was significantly positively correlated with BVES-SA1 (R = 0.615) (Figure 7E). In contrast, SVEP1 was negatively correlated with miR-1269a (R = 0.545) and miR-1269b (R = 0.545) in CRC (Figure 7F,G). The qPCR and western blotting indicated that SVEP1 was upregulated at the mRNA and protein levels by BVES-AS1 overexpression in CRC cells (Figure 7I,K). As expected, miR-1269a and miR-1269b mimics in CRC cells significantly downregulated the expression of SVEP1 (Figure 7H,J). To verify the relationship among SVEP1, miR-1269 and miR-1269b, we constructed SVEP1 3’-UTR WT and Mut dual-luciferase reporter gene plasmids (Figure 8A,B). The luciferase assay revealed that the luciferase activity of CRC cells transfected with the SVEP1 WT plasmid was inhibited by miR-1269a and miR-1269b mimics. In contrast, no significant difference was detected in the SVEP1-Mut plasmid (Figure 8C–F). These results indicated that SVEP1 is the common target gene of miR-1269a and miR-1269b.
SVEP1 suppresses the function of CRC cells through the PI3K/AKT pathway
The biological role of SVEP1 in CRC is still unknown. We performed functional experiments in CRC cells by knocking down SVEP1 with siRNA to elucidate the role of SVEP1 (Figure 9A, Supplementary Fig. 3A). Subsequently, EdU and CCK-8 assays indicated that compared with si-Ctrl transfection, SVEP1 knockdown promoted HT-29 and SW480 cell proliferation (Figure 9B–E). The si-SVEP1#1 and si-SVEP1#2 also enhanced CRC cell migration and invasion (Figure 9F–H, Figure 10A, Supplementary Fig. 3B). Western blot analysis indicated that SVEP1 knockdown in CRC cells significantly promoted EMT (Figure 10C). Moreover, downregulating SVEP1 also inhibited the apoptosis of CRC cells (Figure 10B, Supplementary Fig. 3C). The results demonstrated that SVEP1 has a tumor suppressor function in CRC cells.
A previous study mentioned that SVEP1 knockdown promoted proliferation, bone infiltration and lung metastasis of HCC cells by activating the PI3K/AKT pathway.18 Therefore, we discussed whether BVES-AS1 could regulate the PI3K/AKT pathway through the miR-1269a/miR-1269b-SVEP1 axis in CRC cells. Western blotting revealed that BVES-AS1 upregulation decreased the levels of phosphorylated AKT and PI3K in CRC cells (Figure 10D). In CRC cells, transfection of miR-1269a and miR-1269b mimics also upregulated p-AKT and p-PI3K levels (Figure 10E). Furthermore, SVEP1 silencing significantly enhanced the levels of phosphorylated AKT and PI3K in CRC cells (Figure 10F). Rescue experiments showed that miR-1269a or miR-1269b could partially restore the downregulation of phosphorylated AKT and PI3K caused by the upregulation of BVES-AS1 (Figure 10G). Silencing SVEP1 also partially restored the decrease in p-AKT and p-PI3K in CRC caused by overexpression of BVES-AS1 (Figure 10H). The data indicated that BVES-AS1 regulated the PI3K/AKT pathway through the miR-1269a/miR-1269b-SVEP1 axis (Figure 11G).
BVES-AS1 inhibits the proliferation of CRC cells in vivo
To verify the impact of BVES-AS1 on CRC cell proliferation in vivo, HT-29 cells stably overexpressing BVES-AS1 or the vector were xenografted into nude mice by right axilla subcutaneous injection to form an animal model. The size of the xenograft tumor was measured 5, 10, 15, 20, and 25 days after tumor cell injection. The xenograft tumors were harvested on the 25th day (Figure 11A). Compared with the vector group, the BVES-AS1 group had smaller tumor volumes and slower tumor growth in xenografts (Figure 11B–D). The xenograft tumor weight of the BVES-AS1 group was lower than that of the vector group (Figure 11E). To evaluate the ability of BVES-AS1 to induce tumor growth inhibition, H&E staining and IHC (Ki-67, SVEP1) were performed on subcutaneous xenograft tumors. The results showed that in xenograft tumors with BVES-AS1 upregulation, Ki-67 expression was decreased, and SVEP1 expression was increased (Figure 11F). Consistent with the in vitro experiments, the xenograft tumor assay indicated that BVES-AS1 could suppress CRC cell growth in vivo.
Discussion
To further understand the role of lncRNAs in the progression of CRC, we conducted bioinformatics analysis using TCGA data on paired tumor tissues and adjacent normal tissues from CRC patients. Our study revealed that BVES-AS1 expression was significantly downregulated in human CRC. The low expression of BVES-AS1 was associated with tumor infiltration depth and lymph node positivity in CRC patients. The overexpression of BVES-AS1 suppresses the proliferation, invasion and EMT capability of CRC cells. BVES-AS1 functioned as a sponge for miR-1269a and miR-1269b, regulating the expression of SVEP1. Additionally, SVEP1 inhibits CRC cell proliferation and metastasis through the PI3K/AKT pathway. Ultimately, our study uncovered a complex regulatory network involving BVES-AS1, miR-1269a/b, SVEP1, and the PI3K/AKT pathway, collectively governing CRC progression.
BVES-AS1 belongs to the antisense lncRNA family derived from the BVES antisense strand. LncRNAs can mediate biological functions through epigenetic, transcriptional and post-transcriptional regulation. The subcellular localization of lncRNAs determines their biological roles. LncRNAs localized in the cytoplasm regulate target genes mainly through competitive binding to miRNAs.6, 23, 24 The results indicated that most of BVES-AS1 was distributed in the SW480 and HT-29 cytoplasm, indicating that BVES-AS1 can regulate the function of CRC through the ceRNA (competing for endogenous RNA) mechanism. Bioinformatics analysis showed that miR-1269a and miR-1269b shared the same seed region sequence and had potential binding sites to BVES-AS1. The miR-1269a expression was reportedly elevated in various cancers, and miR-1269a overexpression promoted tumor cell proliferation, metastasis and EMT.25, 26, 27, 28 Bu et al. revealed that miR-1269a enhanced the TGF-β signaling pathway by competitively binding to HOXD10 and Smad7, and miR-1269a-HOXD10/Smad7-TGF-β built a positive feedback loop promoting CRC growth and metastasis.29 CircASS1 can sponge miR-1269a to regulate VASH1 expression, inhibiting CRC cells growth and invasion ability.30 Similarly, miR-1269b was upregulated in HCC, lung cancer and oropharyngeal squamous cell cancer.18, 31, 32 However, overexpression of miR-1269b induced downregulation of METTL3, suppressing stomach cancer cell proliferation and invasion.33 Notably, there was no report on the expression and function of miR-1269b in CRC. Our experimental data indicated that miR-1269a and miR-1269b were elevated in CRC and had a negative relationship with BVES-AS1 expression. Furthermore, as verified through molecular interaction experiments, BVES-AS1 could serve as a miRNA sponge to interact with miR-1269a and miR-1269b.
As ceRNAs, lncRNAs perform biological functions mainly by targeting downstream mRNAs. We conducted RNA-seq, and the RNA-seq data indicated that BVES-AS1 overexpression markedly upregulated SVEP1 expression in CRC cells. The online database and dual-luciferase experiments predicted and validated that SVEP1 was the common target of miR-1269a and miR-1269b. SVEP1 is an extracellular matrix protein associated with epidermal differentiation and lymphatic vessel development and can act as a cell adhesion molecule involved in intercellular adhesion.34 Its silencing enhanced cell chemotaxis and decreased epithelial marker expression and cell adhesion capacity.16, 35 Cell adhesion molecule degradation reduces the tumor adhesion ability, contributing to tumor cell separation from the primary region and metastasis.36 SVEP1 was expressed at relatively low levels in liver cancer and corresponded with invasion and metastasis. The proliferation, metastasis, bone invasion, and lung metastasis abilities were promoted by SVEP1 downregulation in liver cancer cells.18, 19 We first revealed the biological function of SVEP1 in CRC cells. The results suggested that SVEP1 downregulation promoted CRC cell proliferation, migration, invasion, and EMT. The PI3K/AKT pathway is crucial in the proliferation, invasion, and apoptosis of various cancers.37 SVEP1 knockdown induced the upregulation of phosphorylated AKT, thus promoting HCC cell proliferation and metastasis.18 Our research also demonstrated that SVEP1 downregulation could activate p-PI3K and p-AKT expression. In addition, overexpression of BVES-AS1 partially reversed the upregulation of phosphorylated PI3K and AKT induced by SVEP1 silencing. These data suggested that BVES-AS1 overexpression suppressed CRC cell proliferation and invasion by sponging miR-1269a/miR-1269b to upregulate SVEP1 expression and inhibit the activation of the PI3K/AKT pathway.
Limitations
Although the tumor suppressive effect and mechanism of BVES-AS1 were revealed in CRC, some limitations must be considered. First, the study showed that BVES-AS1 was expressed in the nucleus and cytoplasm of CRC cells. We only investigated the molecular mechanism of BVES-AS1 in the cytoplasm; the function of nuclear BVES-AS1 needs to be further verified. Second, BVES-AS1 was able to serve as a ceRNA to sponge miR-1269a and miR-1269b. However, in CRC cells, the relationship between miR-1269a and miR-1269b remains unclear. Finally, we suggest that SVEP1 knockdown contributes to the activation of the PI3K/AKT pathway. Nevertheless, the specific mechanism by which SVEP1 regulates the PI3K/AKT pathway needs to be further elucidated.
Conclusions
This study suggests that BVES-AS1 functions as a tumor suppressor gene inhibiting the growth and metastasis of CRC cells in vivo and in vitro. Mechanistically, BVES-AS1 acts as a miRNA sponge, attenuating the effects of miR-1269a and miR-1269b on SVEP1, which in turn inhibits the PI3K/AKT pathway. Therefore, our study reveals that the BVES-AS1-miR-1269a/b-SVEP1-PI3K/AKT axis is a key regulator in suppressing CRC progression. These findings provide new insights for potential treatment strategies in CRC.
Supplementary data
The supplementary materials are available at https://doi.org/10.5281/zenodo.10069707. The package contains the following files:
Supplementary Table 1. Sequences of oligonucleotides and probes used in this study.
Supplementary Table 2. Primer sequences.
Supplementary Table 3. Clinical characteristics of colorectal cancer patients.
Supplementary Table 4. Differentially expressed genes in RNA-seq.
Supplementary Table 5. Results of normality test.
Supplementary Table 6. Statistical analysis results of models.
Supplementary Table 7. Results of Levene’s test.
Supplementary Fig. 1. BVES-AS1 inhibits CRC cell proliferation, migration and invasion in vitro.
Supplementary Fig. 2. BVES-AS1 reverses the oncogenic effect of miR-1269a or miR-1269b.
Supplementary Fig. 3. SVEP1 inhibits CRC cell proliferation, migration, and invasion in vitro.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Not applicable.
