A total of 20 patients with CRC were enrolled in the present study. Inclusion criteria were as follows: i) CRC was diagnosed by pathological examination; ii) patients had no other severe diseases, including other cancers and high blood pressure with complications and iii) patients were willing to participate and signed the informed consents. Exclusion criteria were as follows: i) Incomplete information and ii) patients received chemo- or radiotherapy before collecting tissue samples. The fresh CRC samples were collected after the patients had been completed the surgery but within 10 min and stored in liquid nitrogen immediately. All fresh CRC samples were collected from November 2020 to December 2021 in the Affiliated Hospital of Southwest Medical University. The present study was approved (approval no. KY2023025) by the Ethics Committee of the Affiliated Hospital of Southwest Medical University (Luzhou, China), and informed consent was signed by all patients. Clinicopathological characteristics of patients are provided in Table I.

A total of 24 4-week-old female BALB/c nude mice (weight, 16±3 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. and housed in laminar flow cabinets under a specific pathogen-free condition (37°C; 12/12-h light/dark cycle; free access to food and water). Mice experiments were approved (approval no. swmu20230055) by the Ethics Committees of Southwest Medical University. The nude mice were divided into four groups randomly, and each group included six nude mice. A total of ~1×10⁷ cells resuspended in 50 µl PBS were injected into the subcutaneous tissues of each nude mice. A total of seven, 10, 13, 16, 19 and 22 days after the cells were injected, the formed xenografts were measured, respectively. The nude mice were sacrificed before the longest diameter of xenografts exceeded 15 mm. They were euthanized by cervical dislocation after receiving satisfactory anesthesia by injection of 50 mg/kg pentobarbital sodium. Then, the xenografts were taken out and weighted. The volumes of xenografts were calculated based on the following formula: V=π/6 × L × W × H, which is used to calculate the volume of an ellipse.

Human colon cell lines (SW480, SW620, HCT116 and T84) and the normal epithelial cell line, CD 841 CoN were purchased from the National Collection of Authenticated Cell Cultures and cultured with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; cat. no. SV30087.03; Hyclone; Cytiva), in 5% CO₂ at 37°C. Cell transfection was performed by using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Routinely, the transfection was carried out in six-well plates at room temperature. For each well, 2 µg overexpression/shRNA vector or 50 nM siRNA were used to incubate with cells for 24 h. After that, cells were harvested for subsequent analysis.

Total RNAs of transfected SW480 or SW620 cells were extracted by using an RNA Extraction kit (Chongqing Van der Waals Biotechnology, Co., Ltd.) according to the manufacturer's protocol. Briefly, 10⁶ cells were lysed using 100 µl Lysis Buffer I, and then 300 µl Lysis Buffer II was added. The solution was added into RNA Binding Columns and centrifuged at 13,000 × g for 30 sec at 4°C. After washing, 50 µl Elution Buffer was added into the RNA Binding Columns to dissolve the RNAs. The stability of mRNA was performed by incubating 1 µg total RNA with 2 µg/ml Actinomycin D (Sigma Aldrich; Merck KGaA) at indicated time points, and analyzed by PCR.

Total RNAs were reversely transcribed into cDNAs by using a PrimeScript™ RT reagent kit (Takara Bio, Inc.) and the cDNAs were amplified by using a SYBR^® Premix Ex TaqTM II reagent (Takara Bio, Inc.). Initial denaturation was performed at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 10 sec), annealing (60°C for 30 sec), and extension (72°C for 30 sec). The expression of miR-29c-3p and BVES was calculated by using the 2^−ΔΔCq method (17). ACTB served as the internal reference. The primers of miR-29c-3p were purchased from (Shanghai, GenePharma Co., Ltd.), and the primers of MBNL1-AS1, BVES and ACTB were shown in Table SII.

The full-length sequence of MBNL1-AS1 was synthesized and inserted into pcDNA3.1 vectors (Addgene, Inc.) or pLV-Puro vectors (Addgene, Inc.,) to construct the MBNL1-AS1-overexpressing vectors or the cell lines that stably overexpress MBNL1-AS1. The short hairpin RNA (shRNA) sequence for MBNL1-AS1 was synthesized and inserted into pLV-Puro vectors (Addgene, Inc.) to construct the cell lines that stably low express MBNL1-AS1 expression. The wild-type or mutant sequences of 3′UTR of BVES mRNA or MBNL1-AS1 containing the binding sites of miR-29c-3p were inserted into pGL3-Basic vectors (Addgene, Inc.) to construct the wild-type pGL3-BVES 3′UTR vector, the mutant pGL3-BVES 3′UTR vector, the wild-type pGL3-MBNL1-AS1 vector, or the mutant pGL3-MBNL1-AS1 vector. The recombinant vectors were selected by ampicillin after transforming in receptive bacteria. The shRNA sequences were shown in Table II. The siRNAs for BVES were purchased from Thermo Fisher Scientific, Inc. (cat. no. AM16708) and sequences were unavailable due to confidentiality issues. The transfection of these vectors or siRNAs were performed as aforementioned.

A total of ~1 µg of the wild-type pGL3-BVES 3′UTR vectors, the mutant pGL3-BVES 3′UTR vectors, the wild-type pGL3-MBNL1-AS1, or the mutant pGL3-MBNL1-AS1 vectors, and 0.05 µg of pRL-TK vectors (Promega Corporation) which served as control, were transfected into HEK293T cells, respectively, while the cells were also transfected with NC or miR-29c-3p mimics using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 24 h after transfection at 37°C, the cells were lysed, and the luciferase activity was measured using GloMax 20/20 Luminometer (Promega Corporation). The relative luciferase activity was calculated by normalizing Firefly luciferase to Renilla luciferase activity.

For Cell Counting Kit-8 (CCK-8) assay, ~2,000 transfected SW480 or SW620 cells were seeded into 96-well plates and cultured for 1, 2, 3, 4, and 5 days, respectively. For each well, 10 µl CCK-8 reagent (MedChemExpress) were diluted into 100 µl medium to incubate the cell and analyzed their proliferation based on the absorbance at 450 nm. For colony formation assay, ~1,600 transfected SW480 or SW620 cells were seeded into six-well plates and cultured for 2 weeks. Then the cell plates were fixed by 4% paraformaldehyde for 10 min at room temperature and stained by 0.1% crystal violet for 10 min at room temperature. For 5-ethynyl-2′-deoxyuridine (EdU) assay, the transfected cells were cultured in 15-mm slides and incubated with EdU solution for 2 h at 37°C. The staining was performed by EdU-567 kit (Guangzhou RiboBio Co., Ltd.) for 30 min at 37°C and the nucleus was stained by 5 mg/ml DAPI.

A total of ~20,000 transfected SW480 or SW620 cells were cultured in six-well plate and were harvested by trypsin without EDTA. Cells were resuspended by binding buffer, containing 2.5 µl AnnexinV-FITC and 2.5 µl PI for 30 min in the dark (cat. no. BD 556570; BD Biosciences). The percentage of apoptotic cells was recorded using a Beckman Flow Cytometer (Beckman Coulter, Inc.) using count model and analyzed using FlowJo software (Version 10.0; FlowJo LLC).

Total proteins of transfected SW480 or SW620 cells extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology) and concentration were measured by BCA kit (Beyotime Institute of Biotechnology). For electrophoresis, 30 µg protein samples were loaded each lane and were separated using 10% SDS-PAGE gels. Then, the proteins in the gels were transferred onto PVDF membranes, which were then incubated in a blocking buffer (3% bovine serum albumin; Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. Then, the PVDF membranes were incubated with anti-BVES antibody (1:500; cat. no. 12920-1-AP; Proteintech Group, Inc.) or anti-GAPDH antibody (1:10,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.) at 4°C overnight. After washing by 0.1% TBST solution, the PVDF membranes were incubated with HRP-conjugated goat-anti-rabbit secondary antibody (1:1,000; cat. no. 7074;) or goat-anti-mouse secondary antibody (1:1,000; cat. no. 7076; both from Cell Signaling Technology, Inc.) at room temperature for 1 h. After washing, the PVDF membranes were exposed using SuperSignal West Dura Extended Duration Substrate Kit (Thermo Fisher Scientific, Inc.).

The enrichment of signaling pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). The correlation of the expression between two genes were acquired from TCGA database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) and analyzed using Spearman's correlation (two-tailed). The binding sites of miR-29b-3p and miR-29c-3p were predicted by TargetScanHuman 7.1 (https://www.targetscan.org/vert_71/).

The data are presented as the mean ± standard deviation (SD). The differences between two groups were analyzed using unpaired Student's t-test or Mann-Whitney U test and among more than two groups were analyzed using one-way ANOVA followed by Tukey's HSD test. P<0.05 was considered to indicate statistically significant difference. Graphs were generated by GraphPad Prism 8.0.1 (Dotmatics).

To investigate the potential lncRNAs that regulate the development of CRC, TCGA database was used to explore the altered signaling pathways. Enrichment analysis of KEGG revealed that the PI3K-AKT signaling pathway had the highest number of dysregulated genes among other pathways, with 46 downregulated genes and 26 upregulated genes. Additionally, the Calcium signaling pathway showed 46 downregulated genes in CRC tissues compared with their corresponding expression levels in normal tissues (Table SI, Fig. 1A). Subsequently, the dysregulated lncRNAs implicated in these pathways were examined. Notably, the present analysis revealed that the lncRNA MBNL1-AS1 exhibited significantly high enrichScores in the Calcium, PI3K-AKT and cAMP signaling pathways (Fig. 1B, Table SII), indicating its potential involvement in CRC progression.

Subsequently, the expression levels of MBNL1-AS1 were analyzed in CRC using the TCGA database; the analysis included 620 human CRC tissue samples and 830 normal colorectal tissue samples. The results indicated that MBNL1-AS1 expression was significantly decreased in CRC tissues compared with that noted in normal tissues (Fig. 1C). However, MBNL1-AS1 expression did not correlate with the TNM stage, lymph node metastasis, or distal metastasis of CRC (Fig. 1D-F). Moreover, MBNL1-AS1 expression was also significantly decreased in four CRC cell lines, including SW480, SW620, HCT116, and T84 compared with that noted in the normal epithelial cell line, CD 841 CoN (Fig. S1). Taken together, these results suggested that MBNL1-AS1 expression is decreased in CRC and that it is associated with the PI3K-AKT signaling pathway.

Given that MBNL1-AS1 expression was not altered in the metastatic CRC tissues compared with that noted in the non-metastatic CRC tissues, the effect of MBNL1-AS1 was investigated on the proliferation of CRC cells. Given that SW620 and SW480 cells exhibited the lowest and highest, respectively, expression of MBNL1-AS1 than the other CRC cell lines (Fig. S1), forced expression of MBNL1-AS1 was established in SW620 cells (Fig. 2A); the stable knockdown variant of MBNL1-AS1 was also established in SW480 cells (Fig. 2B). By using CCK-8, colony formation and EdU assays, it was found that the ectopic expression of MBNL1-AS1 significantly inhibited the proliferation of SW620 cells, while knockdown of its expression significantly promoted the ability of SW480 cells to proliferate (Fig. 2C-L). Moreover, both overexpression or knockdown of MBNL1-AS1 expression displayed no apparent impact on cell apoptosis (Fig. 2M-P).

To further evaluate the roles of MBNL1-AS1 on the proliferation of CRC, CRC cells were subcutaneously injected into nude mice. One week after the injection, the xenografts were formed, and their sizes were measured and recorded. A total of four weeks after the injection, the nude mice were sacrificed, and the xenografts were removed (Fig. 3A and D). Ectopic expression of MBNL1-AS1 significantly decreased the sizes and weights of the xenografts (Fig. 3B and C), while knockdown of MBNL1-AS1 expression revealed the opposite effects (Fig. 3E and F). Collectively, the results indicated that MBNL1-AS1 acted as a tumor suppressor that inhibited CRC cell proliferation.

To further explore the underlying mechanisms by which MBNL1-AS1 suppresses CRC cell proliferation, co-expression analysis of lncRNAs was performed with mRNAs in CRC tissues based on the TCGA database (Table SIII). The data indicated that BVES and MBNL1-AS1 exhibited the highest correlation coefficient followed by cholinergic receptor muscarinic 2 (CHRM2), a crucial member in the PI3K-AKT signaling pathway (18) (Table SIV). However, the present study focused on BVES instead of CHRM2, since the former has been reported to be involved in the development of CRC. Correlation analysis revealed that BVES mRNA expression positively correlated with MBNL1-AS1 expression in CRC tissues based on the TCGA database (Fig. 4A).

Subsequent studies determined whether MBNL1-AS1 could regulate BVES expression and the data indicated that the ectopic expression of MBNL1-AS1 significantly increased the expression levels of BVES mRNA and protein (Fig. 4B and C), while knockdown of MBNL1-AS1 expression caused a significant decrease in these levels (Fig. 4D and E). Then, the forced expression of MBNL1-AS1 was established in SW620 cells (Fig. 4F); the stable knockdown variant of MBNL1-AS1 was also established in SW480 cells (Fig. 4G). However, BVES did not alter MBNL1-AS1 expression in CRC cells (Fig. 4H and I). These data suggested that MBNL1-AS1 was an upstream regulatory factor of BVES. Subsequent studies were performed to assess the mechanism by which MBNL1-AS1 increased BVES mRNA expression. Actinomycin D was used to inhibit RNA transcription in CRC cells and the data indicated that knockdown of MBNL1-AS1 significantly decreased BVES mRNA expression while the ectopic expression of MBNL1-AS1 increased BVES mRNA expression (Fig. 4J and K, respectively), suggesting that it regulated BVES mRNA expression at the post-transcriptional instead of the transcriptional level. Collectively, these results suggested that MBNL1-AS1 increased the stability of BVES mRNA. Moreover, their expression levels indicated a positive correlation in CRC tissues.

A large number of studies have shown that lncRNAs serve as ceRNAs to sponge miRs, which target and regulate the mRNA molecules (19). To explore whether MBNL1-AS1 serves as a ceRNA to sponge miRs, co-expression analysis was performed for MBNL1-AS1, miRs and BVES in CRC tissues based on the TCGA database. The results indicated that miR-29b-3p and miR-29c-3p were candidate miRs (Table SV), and their expression levels were both upregulated in CRC tissues compared with those observed in normal tissues (Fig. 5A and B). TargetScanHuman 7.1 was used to predict the binding sites of miR-29b-3p and miR-29c-3p with BVES mRNA and MBNL1-AS1. The data indicated that the seed sequences of the two miRs were both complementary to the 3,217-3,223 nt of BVES mRNA and to the 1,214-1,222 nt of MBNL1-AS1 (Fig. 5C). Then, the forced expression of miR-29b-3p and miR-29c-3p were established in SW620 cells (Fig. 5D and E). Subsequent experiments demonstrated that miR-29c-3p mimics significantly decreased the mRNA and protein expression levels of BVES, whereas miR-29b-3p mimics exhibited a weaker effect compared with that of miR-29c-3p mimics (Fig. 5F-I). Therefore, the present study focused on miR-29c-3p. Furthermore, luciferase reporter vectors were constructed that contained the binding sites of miR-29c-3p to BVES mRNA and MBNL1-AS1, as well as the corresponding mutant vectors. The results revealed that miR-29c-3p mimics significantly decreased luciferase activity levels in 293 cells transfected with the wild-type vectors compared with those of the mutant vectors (Fig. 5J). Furthermore, a significantly negative correlation was observed between the expression levels of MBNL1-AS1 and miR-29c-3p, and between those of BVES and miR-29c-3p (Fig. 5K and L). Collectively, these results suggested that MBNL1-AS1 increases BVES expression by serving as a ceRNA to sponge miR-29c-3p in CRC cells.

The present study further determined whether MBNL1-AS1 inhibited CRC cell proliferation by regulating miR-29c-3p/BVES signaling. BVES siRNAs were used to suppress the MBNL1-AS1-mediated increase in BVES expression (Fig. 6A and B). Furthermore, miR-29c-3p mimics also suppressed the MBNL1-AS1-mediated increase in BVES expression (Fig. 6C and D). CCK-8, colony formation and EdU assays indicated that significantly reversed the MBNL1-AS1-mediated decrease in the proliferation of SW620 cells, while miR-29c-3p mimics significantly suppressed the MBNL1-AS1-mediated decrease identified in the proliferation of SW620 cells (Fig. 6E-J) without affecting cell apoptosis (Fig. 6K and L). Collectively, these results suggested that MBNL1-AS1 inhibited CRC cell proliferation by regulating miR-29c-3p/BVES signaling.