MiR-212/132 Modulates The Growth, Migration And Invasion Of Human Cervical Cancer Cells

【Object】Micro RNAs(mi RNAs)are a class of small non-coding RNAs that are about 22 nucleotides, which transcriptionally or post-transcriptionally regulate gene expression. mi RNAs are involved in many pathological and physiological progress, such as cell differentiation, cell proliferation, apoptosis, cancer initiation and progression. Recent studies have shown that mi RNAs are involved in the regulation of cell program of human cervical cancer and function as oncogenes or tumor suppressors through regulating their target genes. The aim of this paper is to detect if mi R-212/132 cluster contribute to the regulation of cervical cancer cell proliferation, migration, invasion and cell cycle and to predict and determine the potential target gene for illuminating the molecular mechanism of mi R-212/132 in human cervical cancer.【Methods】First, we detected mi R-212 and mi R-132 expression in human cervical cancer and matched adjacent non-cancer normal tissues by real-time RT-PCR assay. Second, using colony formation assay we detected the effect of mi R-212 and mi R-132 on the proliferation ability of cervical cancer cell. We used FACS to detect the change of cell cycle. Additionally, we use transwell migration and invasion assay to detect the effect of mi R-212 and mi R-132 on cervical cance cell migration and invasion. We also use western blot and real-time RT-PCR to detect the expression level of EMT molecular marker. Third, we integrated the bioinformatics-based predictions and selected SMAD2 as the common target of mi R-212 and mi R-132. The effect of mi R-212 and mi R-132 on m RNA and protein levels of SMAD2 were tested by real-time PCR and western blot. The EGFP fluorescent reporter experiment was used to determine the direct binding role of mi R-212 and mi R-132 on SMAD2 3’UTR. Finally, we overexpressed or knockdown SMAD2 in cervical cancer cell to observe the effect of SMAD2 on cell proliferation, cell cycle, migration and invasion. Rescued experiment was used to validate if SMAD2 partly rescue the effect of mi R-212 and mi R-132 on cervicel cancer.【Results】 The real-time PCR results showed that compared with adjacent non-cancer narmal tissues, mi R-212 and mi R-132 were downregulated while SMAD2 was upregulated in cervical cancer tissues. The overexpression of mi R-212/132 not only led to a delay in the G1/S-phase transition and repressed cell proliferation but also resulted in an increase in E-cadherin expression and a decrease in vimentin, suppressing the epithelial to mesenchymal transition and migration and invasion in cervical cancer cells. Subsequently, SMAD2 was identified as a common target of mi R-212/132 and was found to be negatively regulated by mi R-212/132 at the m RNA and protein levels. Furthermore, silencing SMAD2 led to the same effect on cervical cancer cells as mi R-212/132 overexpression. Importantly, SMAD2 overexpression partially reversed the cellular phenotypes induced by mi R-212/132 overexpression.【Conclusions】Our study indicated that mi R-212/132 function as tumor suppressors by targeting SMAD2 and might serve as new biomarkers for cervical cancer.