2.1 Cell lines and cell culture

Human CRC cell lines SW620, HCT116, HT-29, SW480, Caco-2, normal colon cell line CCD841, HUVECs, and HEK293a were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, P.R. China). HCT116 and HEK293a cells were cultured in regular roswell park memorial institute (RPMI) 1640 medium (#CF0001, SparkJade, Shandong, P.R. China). HUVECs were cultured in ECM (#1001, ScienCell, New York, USA) medium. Other cell lines were cultured in complete dulbecco's modified eagle medium (DMEM) (Gibco, Grand Island, NY, USA). All cell cultures were supplemented with a mixture of 10% fetal bovine serum (FBS) (#04-001-1A, Biological Industries, Hubei, China) and 1% penicillin/streptomycin (#BL505A, Biosharp, Shanghai, China). All cells were incubated at 37°C in a 5% CO₂ atmosphere.

2.2 CRC patient samples and immunohistochemistry

Human CRC microarray (training cohort) for Mex3a staining were purchased from Shanghai Outdo Biotech, China. A validation cohort for Mex3a staining consisted of CRC and para-tumor tissues that were surgically resected from CRC patients at Qilu Hospital (Jinan, Shandong, China) between September 2017 and October 2019. Human CRC for RAP1GAP staining were also surgically resected from Qilu Hospital between September 2017 and October 2019. All diagnoses were confirmed by pathology. All experiments were approved and supervised by the Medical Ethics Committee of Qilu Hospital of Shandong University. TNM stages were classified according to the eighth American Joint Committee on Cancer (AJCC) edition. Immunohistochemistry was performed according to standard protocols using Mex3a and RAP1GAP antibodies from Abcam (Cambridge, UK). The expression levels of Mex3a and RAP1GAP were scored using the IHC scoring method [19] by a pathologist (KH) and an investigator (HXL). For Table 1–3, we divided patients into high expression or low expression groups according to IHC score.

2.3 EDU assay

All transfected with siRNA, plasmid, lentivirus, miR-6887-3p mimics or miR-6887-3p inhibitor cells were seeded into 24-well plates at 1 × 10⁵ cells/well and incubated for 24 h. The proliferation ability was evaluated with an EDU assay kit (C10310-1, RIBO Biotechnology, Guangzhou, Guangdong, China) according to the manufacturer's instructions. Images were taken under a microscope (Olympus, Shanghai, China), and the percentage of EDU-positive cells was calculated using the formula EDU-positive cell count/total cell count × 100%.

2.4 Migration and Matrigel invasion assays

For migration and Matrigel invasion assays, 5000 cells were seeded in the upper chamber with 300 µL serum-free medium, and 600 µL medium containing 10% FBS were added to the lower chamber, which acted as a chemoattractant. After incubation for 48 h, the cells were wiped off the upper chamber with a cotton swab, and the invaded cells to the underside of the membrane were fixed and stained with 0.1% crystal violet (#C0121; Beyotime, Shanghai, China), and counted in five randomly selected microscopic fields (200×) per filter.

2.5 Cell viability assays

For cell counting kit 8 (CCK8) assay (#BB-4221; BestBio, Nanjing, Jiangsu, China), SW620 cells transfected with Mex3a siRNA or HCT116 cells transfected with Mex3a overexpression plasmid were resuspended and counted, then cells were plated into 96-well plates at a density of 1 × 10⁴ cells per well and were treated with 5-fluorouracil (5-FU) (50, 100, 250, 500, 1000, 2000, 3000 µmol/L) (#F6627, Sigma, Shanghai, China). After 24 h of 5-FU treatment, the cell viability was evaluated by assessing the optical density (OD) at 450 nm following the manufacturer's instructions.

2.6 Co-culture angiogenesis assay

First, precooled u-Dish ^{35mm, high} Grid-500 ibiTreat were coated with 200 µL of Matrigel (#354248, Corning Technology, Shanghai, China) at 37°C for 30 min. HUVECs were seeded at 2.5 × 10⁴ cells/well, SW620 and SW480 cells were transfected with Mex3a siRNA or HCT116 cells were transfected with Mex3a overexpression plasmid, after 24 h of transfection, CRC cell supernatants were added to the HUVECs dish at a volume of CRC cell supernatants: ECM equal to 1:1. Tube formation was captured under a microscope (Olympus), and the number of branches and tube lengths were calculated using ImageJ software (National Institutes of Health).

2.7 Western blotting analysis

Cells transfected with siRNA, plasmid, lentivirus, miR-6887-3p mimics or miR-6887-3p inhibitor were harvested in sample buffer supplemented with Protease/Phosphatase Inhibitor Cocktail (#5872, Cell Signaling Technology, Shanghai, P.R. China). Protein levels were detected and calculated using the bicinchoninic acid (BCA) assay (#AR0197A, BOSTER, Shanghai, P.R. China) according to the manufacturer's instructions. Equal amounts (20 µg) of protein were added into sodium dodecyl sulfate (SDS) polyacrylamide gel, and the gel was transferred to polyvinylidene difluoride membranes (#10600023, GE, Shanghai, P.R. China). The membrane was incubated with primary antibodies overnight after blocking by milk. Then, the membrane was incubated with second antibody to be photographed. All used antibodies were Mex3a (1:1000 dilution; #ab79046, Abcam), RAP1GAP (1:5000 dilution; #ab32373, Abcam), MEK (1:500 dilution; #8727, Cell Signaling Technology, Shanghai, P.R. China), p-MEK (1:500 dilution #9154, Cell Signaling Technology, Shanghai, P.R. China), ERK (1:1000 dilution; #4695, Cell Signaling Technology, Shanghai, P.R. China), p-ERK (1:1000 dilution; #4370, Cell Signaling Technology, Shanghai, P.R. China), HIF-1α (1:1000 dilution; #ab51608, Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000 dilution; #2118, Cell Signaling Technology, Shanghai, P.R. China).

2.8 Co-immunoprecipitation (Co-IP) assays

Co-IP assays were performed according to standard protocols. When SW620 cells' fullness reached more than 90%, the cells were scraped off directly with a cell scraper using immunoprecipitation lysis buffer NP-40 (#P0013F, Beyotime Institute of Biotechnology, Shanghai, P.R. China) with phenylmethanesulfonyl fluoride (PMSF)and protease inhibitor. Then, the cells were centrifuged at 12,000 rpm for 15 min, and the supernatant was collected, followed by incubation with primary antibodies (RAP1GAP) or isotype immunoglobulin G (IgG) (#3900, Cell Signaling Technology, Shanghai, P.R. China), gentle rocking 2 h at 4°C. Then, 35 µL of protein A/G beads (#sc-2003, Santa cruz, Shanghai, China) was added to each immunoprecipitation mixture with gentle rocking overnight at 4°C. The next day, the mixtures were washed five times with cold 1×Co-IP buffer, and bound protein was denatured with 2× SDS sample buffer. The supernatants were collected and proceeded to SDS-PAGE Western blot analysis.

2.9 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from CRC cell lines using TRIzol (#15596-026, Invitrogen, CA, USA) following the manufacturer's instructions. cDNA was synthesized using a reverse transcription kit (#QP056, Genecopoeia, Guangzhou, Guangdong, P.R. China). GAPDH and U6 were used as internal parameters, and relative mRNA expression levels were calculated by comparing Ct method. The primers used were as follows: Mex3a-forward-5′-TGGAGAACTAGGATGTTTCGGG-3′; Mex3a-reverse-5′-GAGGCAGAGTTGATCGAGAGC-3′; hGAPDH-forward-5′-GGAAATCGTGCGTGACATTAA-3′; hGAPDH-reverse-5′-AGGAAGGAAGGCTGGAAGAG-3′; miR-6887-3p:5′-GAUCCUCCUUUCACCUCCCCU-3'; U6: HmiRQP9001; Genecopoeia, Guangzhou, China

2.10 Establishment of transient transfected cell lines

The cells were transiently transfected with siRNAs, plasmids, hsa-miR-6887-3p mimics and hsa-miR-6887-3p inhibitor using jetPRIME transfection reagent (#CPT114, Polyplus, New York, USA) according to the manufacturer's instructions. The cells were harvested to evaluate the transfection efficiency after 48 h. The sequence of Mex3a siRNA, RAP1GAP siRNA, hsa-miR-6887-3p inhibitor, hsa-miR-6887-3p mimics and control are shown in Table S1. Plasmid sequence of Mex3a and RAP1GAP are shown in Table S2.

2.11 Establishment of stable Mex3a-overexpressing or -knockdown cell lines

Mex3a overexpression and knockdown lentiviruses were purchased from the Genecopia Company. Stable Mex3a-overexpressing HCT116 cell lines were generated by being infected with lentivirus multiplicity of infection (MOI): 5 and selected with 0.3 mg/mL puromycin (#540411, Sigma, Shanghai, China) for about 1 week. Stable Mex3a-knockdown SW620 and SW480 cell lines were generated by being infected with lentivirus (MOI: 50 and 20, respectively) and selected with 1 mg/mL puromycin for about 1 week. Finally, the stable cell lines were verified using Western blot.

2.12 In vivo tumor xenograft assays

Six-week-old male BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co, Ltd (Beijing, P.R. China). Animal experimental procedures were approved by the Medical Ethics Committee of Shandong University (ECAESDUSM 2014056). The six-week-old male mice were randomized into different groups. SW480, HCT116, and SW620 cells (5 × 10⁶ cells/mice) were implanted subcutaneously into the flank of nude mice. After tumor formation, tumor volume (mm³) was measured every other day and calculated by the formula (length × width × width)/2. When the tumors had reached a volume of approximately 600 mm³, the mice were euthanized, and the tumors were excised and embedded in paraffin for IHC analysis.

2.13 Transcriptome sequencing and bioinformatics analysis

Genome-wide transcriptional sequencing was performed by Baimaike (Beijing, China). Transcriptome sequencing (NEB, USA) was used to identify mRNA transcripts with differential expression between Mex3a-knockdown SW480 cells and control SW480 cells. The RNA preparation and library preparation for transcriptome sequencing were performed according to the manufacturer's instructions.

The gene expression profiles and clinical data of CRC patients from the The Cancer Genome Atlas (TCGA) were downloaded from the University of California Santa Cruz (UCSC) Xena Browser (https://xenabrowser.net/). The expression and OS analysis of miR-6887-3p was downloaded from the Starbase software (starbase.sysu.edu.cn). The gene expression and clinical data of CRC patients from the gene expression omnibus (GEO) datasets GSE39582 were obtained (https://www.ncbi.nlm.nih.gov/gds). R software was used to do hierarchical clustering analysis and KEGG pathway analysis.

2.14 Luciferase activity assay

The putative miRNA of Mex3a was predicted using TargetScan (www.targetscan.org) and miRDB (mirdb.org). For luciferase activity assay, the 3′ UTR region of the Mex3a gene was generated by standard polymerase chain reaction (PCR) techniques and inserted into the vector. A mutant type (MT) construct that disrupted the miR-6887-3p-binding sites of the Mex3a 3′ UTR segment was also amplified using a Quick change Site-Directed Mutagenesis Kit (#200518, Agilent, Shanghai, P.R. China). Wild type (WT) 3′ UTR or MT 3′ UTR plasmid of Mex3a was co-transfected with miR-6887-3p mimic into HEK293a cells using Lipofectamine 2000 (#11668-019, Invitrogen, Shanghai, P.R. China). Luciferase activity was detected by a Luciferase Reporter Gene Assay Kit (#GM-040501; Genomeditech, Shanghai, P.R. China) after transfected for 48 h.

2.15 Statistical analyses

All statistical analyses were performed using GraphPad Prism Software (version 6.0) or R software (version 3.6.1). Each assay was performed at least three independent replicates, and all data are presented as mean ± standard error of the mean (SEM). For comparisons, Student's t-test (two-sided), One-Way analysis of variance (ANOVA), Pearson's chi-square test, log-rank test, Kaplan–Meier survival analysis, Fisher's exact test, and Pearson's correlation analysis were performed as indicated. A P value < 0.05 was considered statistically significant.

3.1 Mex3a is highly expressed in CRC and correlates with poor prognosis

To clarify the role of Mex3a in CRC, we first analyzed Mex3a protein levels in a training cohort containing 101 CRC tissues and 79 normal tissues by IHC staining. Mex3a expression levels were found higher in tumor tissues than in adjacent normal tissues (Fig. 1A). IHC score indicated that Mex3a expression was significantly increased in CRC tissues compared to adjacent normal tissues (P < 0.001) (Fig. 1B). Kaplan-Meier analysis also revealed that patients with higher expression of Mex3a was associated with poorer OS (P = 0.003 (Fig. 1C). To confirm this finding, we detected Mex3a protein levels using Western blotting in eight randomly selected and paired CRC specimens. Six out of the eight specimens showed higher levels of Mex3a protein than the adjacent normal tissues (P = 0.049) (Fig. 1D).

Next, we analyzed the mRNA level of Mex3a in human CRC samples from the TCGA and GEO dataset (GSE39582). Our results showed that the Mex3a expression level was significantly higher in CRC tissues than in normal tissues (P < 0.001) (Fig. 1E, F). We then investigated the relationship between Mex3a expression and survival characteristics in 566 CRC patients from the GSE39582 dataset. The results revealed that patients with higher Mex3a expression levels had poorer OS and recurrence-free survival (RFS) (Fig. 1G, 1H). These results suggest that Mex3a was upregulated in CRC and was closely related to its poor prognosis.

We further investigated the relationship between Mex3a and the clinicopathological features of CRC. We divided 101 CRC patients into Mex3a low-expression (52.5%, 53/101) and high-expression (47.5%, 48/101) groups according to their IHC score. We found that high expression of Mex3a was positively correlated with histopathological grade and tumor pathological T stage (pT stage) (P = 0.025, Table 1), while there was no correlation between Mex3a expression and sex, age, pN stage, pM stage, and pTNM stage. These results further confirmed that Mex3a high expression in CRC was correlated with poor prognosis.

3.2 High expression of Mex3a promotes tumorigenic properties of CRC cells

To investigate the potential roles of Mex3a in the development of CRC, we first measured Mex3a expression levels in five CRC cell lines compared to one normal colon cell line (CCD841). Among the five CRC cell lines, SW620, SW480, and Caco-2 cells showed the highest Mex3a expression at mRNA and protein levels, while HCT116 and HT-29 cells showed similar Mex3a levels as normal control CCD841 cells (Fig. S1A, S1B). Therefore, SW620 and SW480 were chosen for Mex3a silencing, and HCT116 was chosen for Mex3a overexpression. The efficiencies of Mex3a silencing and Mex3a overexpression were validated by Western blotting (Fig. 2A and 2B).

Next, SW640 and SW480 were used as the cell models for RNA knockdown. EDU assay revealed that Mex3a knockdown significantly impaired CRC cell proliferation (Fig. 2C). Transwell assays were performed to detect the effects of Mex3a on cell invasion and migration. Our results revealed that Mex3a knockdown substantially attenuated the metastatic ability of SW620 and SW480 cells (Fig. 2D, E).

Angiogenesis experiments were also performed to detect the effect of Mex3a on angiogenesis regulation. SW620 and SW480 cells were transfected with Mex3a siRNA or Control siRNA. Their supernatants were then collected and used to treat HUVECs. Mex3a silencing was found to reduce the number of branches and tubule length of HUVECs compared to those of the control groups (Fig. S1C). Finally, over a range of drug concentrations, Mex3a knockdown significantly enhanced sensitivity to 5-FU in SW620 cells (Fig. S1D). As expected, overexpression of Mex3a in HCT116 cells resulted in the opposite results (Fig. 2F-H and Fig. S1E, S1F). These results indicate that Mex3a could promote proliferation, invasion, and angiogenesis in CRC cells in vitro.

3.3 Mex3a enhances oncogenesis of CRC cells in vivo

To investigate the functional role of Mex3a in vivo, we constructed lentivirus-mediated Mex3a knockdown SW480 cells and SW620 cells, and Mex3a overexpression HCT116 cells (Fig. 3A, B). Thereafter, SW480 and HCT116 cells with stable knockdown or overexpression of Mex3a were transplanted into nude mice. Knockdown of Mex3a in SW480 slowed the in vivo tumor growth (Fig. 3C-E). IHC staining showed that the staining intensity and expression levels of Ki-67 were both decreased in the Mex3a knockdown group compared to normal tissues (Fig. S1G). Moreover, xenotransplant experiments showed that enforced Mex3a expression markedly increased the tumorigenicity of HCT116 cells (Fig. 3F-H). IHC staining showed that the staining intensity and expression levels of Ki-67 were both increased in the Mex3a overexpression groups (Fig. S1H). Collectively, these results indicate that Mex3a could promote oncogenesis in nude mice.

3.4 Mex3a acts as a co-activator of RAP1GAP/MEK/ERK/HIF-1α signaling in CRC cells

To explore the possible mechanisms accounting for the role of Mex3a in the development of CRC, we performed transcriptome sequencing with stably transduced cell lines (SW480 control-knockdown and SW480 Mex3a-knockdown) to identify the relevant signaling pathway. Hierarchical clustering analysis showed that Mex3a knockdown led to a clear and consistent difference in gene expression profile (Fig. 4A). We identified 752 upregulated genes and 352 downregulated genes (|fold change| > 1, data not shown). The KEGG pathway analysis showed that Mex3a knockdown led to the enrichment of the mitogen-activated protein kinase (MAPK) and Rap1 pathway (Fig. 4B). Considering the differentially expressed genes involved in this pathway, we speculated that Mex3a could affect colon cancer phenotype by regulating the RAP1GAP/MEK/ERK/HIF-1α pathway.

Rap1 is a small GTP-binding protein that cycles between the GTP-bound and GDP-bound states [20, 21]. RAP1GAP is lost in several tumor types, and thus, it may function as a tumor suppressor [22]. Overexpression of RAP1GAP has been reported to induce apoptosis [23], suppress cell proliferation [24, 25], inhibit cell cycle progression [26], and reduce tumor progression [27, 28]. Thus, RAP1GAP possesses most of the well-studied characteristics found in tumor-inhibiting factors, suggesting that RAP1GAP may play a role in the progression of CRC.

We, therefore, verified the expression of the proteins involved in the RAP1GAP signaling pathway in lentivirus-mediated Mex3a knockdown SW620 and SW480 cells by Western blotting. We found that in both cell lines, knockdown of Mex3a induced the upregulation of RAP1GAP and downregulation of phosphor-mitogen activated protein kinase kinase 1/2 (p-MEK1/2), phosphor-extracellular regulated protein kinase 1/2 (p-ERK1/2), and HIF-1α (Fig. 4C). We further performed a rescue experiment using U0126, a specific MEK/ERK inhibitor, and PX-478, an HIF-1α inhibitor. We discovered that using U0126 (20 µmol/L) partially attenuated the proliferation, invasion, and migration of HCT116 cells caused by Mex3a overexpression (Fig. 4D-F). In addition, U0126 reduced the expression of p-MEK1/2 and p-ERK1/2 and impaired the expression of HIF-1α induced by Mex3a overexpression (Fig. 4G). PX-478 (25 µmol/L) also inhibited HIF-1α expression induced by Mex3a overexpression (Fig. 4H). Interestingly, Mex3a expression was also reduced following U0126 and PX-478 treatment. Possibly, this is because the inhibition of these pathways altered the stability of the Mex3a protein through a feedback loop. These results indicated that Mex3a regulated tumor growth through the RAP1GAP/MEK/ERK/HIF-1α signaling in CRC cells.

3.5 Mex3a physically interacted with RAP1GAP in CRC cells

To elucidate the molecular mechanism by which Mex3a promotes tumorigenic properties in CRC cells, we next identified proteins that interact with Mex3a in CRC cells. Several lines of evidence have revealed that Mex3b could bind to RAP1GAP [29, 30]. Mex3a and Mex3b are Mex3 family homologous proteins that have the same functional domain. Therefore, we determined whether Mex3a interacts with RAP1GAP. Co-IP was used to confirm the protein association between Mex3a and RAP1GAP in SW620 and SW480 cells (Fig. 5A). Furthermore, we analyzed Mex3a protein levels in validation cohorts containing 100 cases of CRC and 6 normal tissues by IHC staining. The results showed that high expression of Mex3a was accompanied by low expression of RAP1GAP in tumor tissues (Fig. 5B). Similar to the training cohort, the IHC score indicated that Mex3a expression was significantly increased in CRC tissues compared with normal tissues (P < 0.001) (Fig. 5C). RAP1GAP expression was also significantly decreased in CRC tissues compared to normal tissues (P = 0.026) (Fig. 5D). The available data suggested that the protein level of Mex3a was negatively correlated with RAP1GAP (r = -0.235; P = 0.020) (Fig. 5E).

Growing evidence suggests that the overexpression of RAP1GAP in human colon cancer cells impairs cell migration, invasion, and cell-matrix adhesion in vitro[31, 32]. We then performed a rescue experiment to explore whether Mex3a promoted the expression of MEK/ERK/HIF-1α through RAP1GAP. We found that RAP1GAP partially reduced the expression of p-MEK, p-ERK, and HIF-1α induced by Mex3a overexpression and that RAP1GAP knockdown reversed this decrease caused by Mex3a knockdown (Fig. 5F, G). Interestingly, Western blotting and qPCR showed that the RAP1GAP protein level was decreased but mRNA did not change after overexpression of Mex3a (Fig. 4G and Fig. S2A). These results indicated that RAP1GAP was lowly expressed in CRC and physically interacted with Mex3a in CRC cells.

We further investigated the role of RAP1GAP in CRC. First, we analyzed the mRNA level of RAP1GAP in human CRC samples from TCGA. The results showed that the RAP1GAP expression level was significantly lower in CRC tissues than in normal tissues (P < 0.001) (Fig. S2B). We then investigated the relationship between RAP1GAP expression and survival characteristics in 299 CRC patients from the TCGA. Our results revealed that lower RAP1GAP expression levels were associated with poorer OS (Fig. S2C). Moreover, we explored the relationship between the protein level of RAP1GAP and the clinicopathological features of CRC through tissue microarray. We divided 100 CRC patients into RAP1GAP low-expression (50%, 50/100) and high-expression (50%, 50/100) groups according to IHC score. We found that there was no correlation between high RAP1GAP expression and age, pT, pN, pM, pTNM stage, and histopathological grade except for gender (Table 2). In addition, the validation cohort confirmed that high Mex3a expression was positively correlated with pT stage (P = 0.043) and pN stage (P = 0.033) (Table 3). These results provided an insight that RAP1GAP may play an inhibitory role in CRC.

3.6 Mex3a is a target of hsa-miR-6887-3p in CRC cells

Now that we identified the oncogenesis role of Mex3a, we decided to find out the upstream regulatory mechanism of Mex3a. miRNAs are known to function by regulating target genes. The putative miRNA of Mex3a was predicted using TargetScan (www.targetscan.org) and miRDB (mirdb.org). The results showed that the 3′ UTR region of Mex3a possessed potential complementary sites for the miR-6887-3p seed region (Fig. 6A). A luciferase reporter assay was performed to further investigate the direct binding and inhibitory effects of miR-6887-3p and Mex3a. The Mex3a 3′ UTR-WT and corresponding MT were amplified and cloned downstream of a luciferase reporter gene in the pGL3 basic vector. In HEK293a cells, co-transfection of pcDNAmiR-6887-3p and Mex3a-3′ UTR-WT reduced luciferase activity compared with the Mex3a-3′ UTR-WT and mimics NC groups. However, no difference in luciferase activity was observed in cells co-transfected with Mex3a-3′ UTR-MT and pcDNAmiR-6887-3p (Fig. 6B). These results indicated that Mex3a was a target of hsa-miR-6887-3p in CRC cells. Analysis using the Starbase software (starbase.sysu.edu.cn) revealed that miR-6887-3p was under-expressed in colon cancer compared to the normal group (Fig. S2D). The high expression of miR-6887-3p correlated with high OS rate (Fig. S2E).

To explore whether miR-6887-3p negatively regulated Mex3a expression in the CRC cellular environment, we first measured the expression level of miR-6887-3p in CRC cell lines and a normal colon cell line, CCD841. Among the three CRC cell lines, SW620 presented the lowest miR-6887-3p expression levels while HCT116 had relatively high miR-6887-3p expression levels (Fig. S2F). Thus, we used SW620 cells to overexpress miR-6887-3p by transfection with miR-6887-3p mimics. As HCT116 cells possessed relatively high miR-6887-3p levels, we knocked-down the endogenous miR-6887-3p by treating them with an miR-6887-3p inhibitor in HCT116 cells. Transfection efficiency was confirmed by qRT-PCR (Fig. 6C, D). mRNA and protein levels of Mex3a were measured. The results showed that ectopic expression of miR-6887-3p significantly decreased the Mex3a mRNA and protein levels in SW620 cells (Fig. 6E, F), whereas miR-6887-3p knockdown increased the Mex3a mRNA and protein expression in HCT116 cells (Fig. 6G, H). These results suggested that Mex3a could be a target of hsa-miR-6887-3p in CRC cells.

3.7 hsa-miR-6887-3p represses Mex3a to inhibit CRC growth

The above results prompted us to examine whether miR-6887-3p could inhibit CRC growth by inhibiting Mex3a. We first assessed whether miR-6887-3p could reverse the effect of Mex3a in CRC cells.

In SW620 cells transfected with miR-6887-3p mimics, Western blotting validated that overexpression of miR-6887-3p by transfection with miR-6887-3p mimics suppressed Mex3a expression (Fig. 7A). Compared with the control group, SW620 cells transfected with miR-6887-3p mimics displayed notably reduced cell proliferation and invasion (Fig. 7B, C), This was similar to the result of the low expression of Mex3a. HCT116 cells transfected with the miR-6887-3p inhibitor showed opposite results (Fig. 7D-F). To examine whether the function of miR-6887-3p depended on the expression of Mex3a in CRC cells, SW620 cells were transduced with miR-6887-3p mimics and Mex3a overexpression plasmid. Interestingly, ectopic Mex3a expression could at least partially rescue the proliferation and invasion ability inhibited by miR-6887-3p (Fig. 7C, D). HCT116 cells were transduced with miR-6887-3p inhibitor and Mex3a siRNA. Mex3a knockdown also partially rescued the proliferation and invasion ability promoted by inhibition of the expression of miR-6887-3p (Fig. 7E, F). Altogether, these results showed that hsa-miR-6887-3p could inhibit the proliferation and invasion of CRC cells by suppressing Mex3a.

3.8 Ectopic expression of miR-6887-3p suppresses CRC cell growth in vivo

To investigate the role of miR-6887-3p in tumor growth in vivo, SW620 cells were stably transfected with LV-miR-6887-3p to overexpress miR-6887-3p. Transfection efficiency was confirmed by qRT-PCR (Fig. 8A). The transfected cells were then injected into the flanks of 6-week-old male nude mice. The tumor was measured every other day. The mice were euthanized, and the tumors were collected after 18 days. Our results showed that the tumor size of the SW620-Lv-miR-6887-3p group was significantly smaller than that of the SW620-Lv-miR-NC group (Fig. 8B-D). Moreover, in the SW620-Lv-miR-6887-3p group, the staining intensity and expression levels of Ki-67 were decreased (Fig. S2G). Taken together, these results indicate that miR-6887-3p could suppress tumorigenesis in nude mice.