| Objective:We sought to identify possible miRNAs able to regulate RBP2 expression in human gastric cancer, and explore its effect on RBP2 expression and cancer development.Methods:1. To identify miRNAs that specifically target RBP2, we used bioinformatic analyses available on the web, including TargetScan, miRanda, and miRBase.2. We first analyzed RBP2 and hsa-miR-212 expression level in tumor specimens and matched normal tissues from 19 patients with gastric cancer.3. We constructed pS-miR-212, anti-miR-212 plasmid, pMIR-REPORT/ RBP2/3'UTR1S and three mutants pMIR-REPORT luciferase constructs using standard DNA techniques. We transfected AGS cell with pS-miR-212 and anti-miR-212 plasmids. Then the expression level of miR-212 was measured by quantitative real-time PCR.4. To determine whether miR-212 reduces RBP2 expression, we transiently transfected pS-miR-212, anti-miR-212 plasmid or empty plasmid into AGS,BGC-823 and GES-1 cells and measured the levels of RBP2 mRNA and protein by quantitative real-time PCR and Western blotting.5. To determine whether the putative miR-212 target sequence in RBP2 3'-UTR could mediate translational repression, we constructed pMIR-REPORT luciferase constructs containing the putative miR-212 binding site of RBP2 3'UTR. This construct and pMIR-REPORTβ-gal control plasmid were co-transfected into GES-1 cells in the presence of pS-miR-212, anti-miR-212 plasmids or empty vector (control).Then, the luciferase activity were measured. GES-1 cells were co-transfected with three pMIR-REPORT luciferase mutants, pMIR-REPORTβ-gal and pS-miR-212. Then, the luciferase activity was measured.6. To determine whether miR-212 influences p21CIP1 and p27kip1 expression, we transiently transfected pS-miR-212, anti-miR-212 plasmid or empty plasmid into AGS, BGC-823 and GES-1 cells and measured the levels of p21CIP1 and p27kip1 mRNA by quantitative real-time PCR.7. To determine the functional role of miR-212 in gastric cancer cell lines, the colony formation assay was performed in AGS and GES-1 cell lines. These cell lines were transfected with pS-miR-212, anti-miR-212 plasmid or empty plasmid and then the number of colonies were counted.Result:1. Comparing the results obtained from the different searches, we found that miR-212 consistently showed the highest score of probability for targeting RBP2 3'UTR. RBP2 3'UTR contains two potential binding sites for miR-212.2. In Gastric cancer, the expression of miR-212 was inhibited whereas RBP2 was overexpressed. Conversely, in their matched normal gastric tissues, the level of miR-212 was high and RBP2 was low.3. All constructs were confirmed by direct DNA sequencing analyses. The expressionof miR-212 could be upregulated or downregulated by pS-miR-212 and anti-miR-212 transfection, respectively.4. The mRNA and protein level of RBP2 was decreased in the miR-212-transfected sample, suggesting that repression of RBP2 may be the result of mRNA degradation. Transfection of the anti-miR-212 into AGS, BGC-823 and GES-1 cells resulted in an increase in RBP2 mRNA and protein levels. Taken together, our results suggest that in gastric cancer cells, miR-212 inhibits RBP2 expression by direct cleavage of RBP2 mRNA.5. Transfectants with pS-miR-212 plasmid showed significantly decreased luciferase activity compared with negative control. This indicates that miR-212 modulates RBP2 expression by direct binding to its 3'UTR. Mutation of either predicted binding sites attenuated miR-212-mediated repression of luciferase activity, whereas mutation of both binding site abolishes this repression by miR-212. Our results suggest that both of the two predicted binding sites contribute to the miRNA-mRNA interaction.6. Cells transfected with pS-miR-212 exhibited elevated mRNA expression levels of p21CIPI and p27kipl. Transfection of the anti-miR-212 resulted in a reduction of the endogenous mRNA levels of p21CIPI and p27kipl for all 3 cell lines.7. Either enforced expression or knockdown of miR-212 could affect clony formation. Transfection of pS-miR-212 resulted in reduction in the number of colonies, whereas anti-miR-212 transfection resulted in increase in colony formation. These results indicate that miR-212 inhibits in vitro the proliferation of human gastric cancer cells.Conclusion:In this study, miR-212 was identified to modulate RBP2 expression. We found that miR-212 levels negatively correlated with the expression of RBP2 expression in hhuman gastric tissues species. Hsa-miR-212 was found to negatively regulate RBP2 expression at the mRNA and protein level by binding to RBP2's 3'UTR regions. Forced expression of miR-212 leads to the inhibition of gastric cancer cell proliferation. |
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