| Objective:To detect the expression levels of microRNA-210(miR-210) in human colorectal cancer (CRC) tissues, analyze its clinicopathological and prognostic value, and investigate its potential effect on CRC cell metastasis.Methods:1. The expression levels of miR-210in193pairs of human CRC tissues and adjacent non-cancerous tissues were tested by quantitative real-time PCR (qRT-PCR). Then, we compared the expression difference of miR-210between the two groups, analyzed the association of miR-210expression levels with various clinicopathological characteristics in CRC tissues, and evaluated the prognostic significance of miR-210. Also, we examined the expression levels of HIF1α mRNA in CRC tissues and corresponding non-cancerous tissues, determined the correlation between miR-210and HIF1α by Pearson’s correlation analysis.2. The expression levels of miR-210were examined in CRC cell lines (HT-29, HCT-116, SW480and SW620). We performed hypoxia experiment to evaluate whether the miR-210levels of CRC cells were induced by hypoxia.3. Transient transfection of miR-210mimics/miR-210inhibitor or miR-negative control was carried out in HT-29and SW480cells using LipofectamineTM2000 according to the manufacturer’s instructions. The expression of miR-210was tested by qRT-PCR24h later to evaluate transfection efficiency. Transwell experiments with or without Matrigel were performed to test the effect of miR-210on CRC cell migration and invasion. We calculated the migration and invasion cells in miR-210over-expression group/miR-210low-expression group and negative control group using transwell assay in vitro, thereby to detect the migration and invasion ability of miR-210on CRC cell lines.4. We examined the migration and invasion ability of CRC cells under hypoxic conditions using transwell assay in vitro.5. Bioinformatics analysis by TargetScan demonstrates that VMP1is a potential target of miR-210. Luciferase reporter assay was performed using the vectors containing wild type (WT)-VMP13’-UTR sequences or mutant (MUT)-VMP13’-UTR sequences. The HEK293T cells were transiently co-transfected with miR-210mimics/miR-negative control and WT-VMP13’-UTR vector/MUT-VMP13’-UTR vector. Luciferase activities were evaluated with the Dual-Luciferase assay kit following manufacturer’s instructions48h after transfection. We performed qRT-PCR and Western blot assays after transfection with miR-210mimics or miR-negative control to detect the mRNA and protein levels of VMP1. Also, the expression levels of VMP1mRNA in CRC tissues were tested by qRT-PCR, and the correlation between miR-210and VMP1was determined by Pearson’s correlation analysis.6. Transient co-transfection of miR-210mimics/miR-negative control and PCMV-HA-VMP1plasmid was carried out in HT-29and SW480cells. Migration and invasion abilities were detected by transwell assay in vitro in all groups. The possible molecular mechanisms by which miR-210promote metastasis of CRC cells were evaluated.Results:1. The expression level of miR-210in CRC tissues was significantly up-regulated in comparison to that in the adjacent non-cancerous tissues (P<0.0001).2. The expression level of miR-210in CRC tissues was significantly associated with tumor size (P=0.014), local invasion (P=0.047), lymph node metastasis (P=0.001) and clinical TNM stage (P=0.005). However, no significant correlations were found between miR-210expression and gender, age, tumor location and differentiation (all.P>0.05). The Kaplan-Meier survival analysis showed that CRC patients with high miR-210expression had a significantly poorer overall survival rates than those with low miR-210expression (P<0.001). Moreover, Cox regression univariate and multivariate analysis showed that miR-210expression was significantly associated with poor prognosis, independent of other clinicopathological parameters (RR=2.131;95%CI:1.130-4.019; P=0.019).3. The expression of miR-210was detected in all CRC cells (HT-29, HCT-116, SW480and SW620). The miR-210levels increased in response to hypoxia in these cells. Moreover, the expression of miR-210greatly increased with prolonged exposure to hypoxia, with the highest expression level in48h after hypoxia.4. Compared to the control group transfected with miR-negative control, miR-210expression level was significant increased in group transfected with miR-210mimics and significant down-regulated in group transfected with miR-210inhibitor (P<0.0001), which indicated the transfection efficiency was very high in the HT-29and SW480cell lines.5. Transwell experiments in vitro indicated that the upregulation of miR-210level by the miR-210mimics enhanced the migration and invasion potential of CRC cells and transfection with the miR-210inhibitor led to a significant decrease in the migration and invasion potential of CRC cells compared to the cells transfected with the miR negative control.6. The migration and invasion potential of CRC cells under hypoxic conditions were significantly increased compared to cells under normoxic conditions. Moreover, transfection with the miR-210inhibitor dramatically decreased the migration and invasion potential of the hypoxic CRC cells, whereas transfection with the miR-210mimics further enhanced the migration and invasion ability of the hypoxic CRC cells7. Bioinformatics analysis by TargetScan revealed that3’-UTR of VMPlcontains predicted binding site for miR-210. Luciferase reporter assay showed that co-transfection of miR-210mimics in HEK293T cells significantly suppressed the luciferase activity of the reporter containing WT-VMP13’-UTR but did not suppress the MUT-VMP13’-UTR reporter (P<0.05). These findings showed that VMP1is a direct functional target of miR-210. We also found that transfection of miR-210mimics could significantly decrease the expression of VMP1in both mRNA and protein levels of HT-29and SW480cells. Moreover, the expression of VMP1was found to be inversely correlated with miR-210expression in CRC tissues (r=-0.318, P<0.01).8. To further confirm that miR-210mediates its metastatic effects by VMP1, we co-transfected CRC cells (HT-29and SW480) with miR-210mimics/miR-negative control and PCMV-HA-VMP1plasmid. Transwell experiments in vitro indicated that miR-210mimics could promote the metastatic potential of CRC cells and decreased metastatic ability was observed in VMP1-overexpressing cells compared with control cells (P<0.05). Moreover, co-transfection of miR-210and VMP1could partially abrogate miR-210-induced metastatic ability in CRC cells (P<0.05).Conclusion:1. The expression of miR-210in CRC tissues was significantly higher than that in corresponding adjacent non-cancerous tissues, and high miR-210expression was associated with large tumor size, local invasion, lymph node metastasis and TNM clinical stage. In addition, the expression of miR-210was an independent prognostic factor of overall survival rate in CRC patients. All the results implied that miR-210may play an important function in CRC development, and could be used in the prognostic screening of patients in CRC.2. Hypoxia induced miR-210could promote the metastatic potential of CRC cells by directly downregulating VMP1expression, thereby contributed to the progression of CRC. Taken together, these results provide important clues for the complicated molecular pathogenesis of CRC and may assist in the development of new therapeutic regimens against hypoxic tumors. |
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