MiR-19A And MiR-19B Target IGF2R In Cervical Carcinoma Cells

[Objective] MicroRNAs (miRNAs) are a class of small non-coding RNAs that include18-26nucleotides, which post-transcriptionally regulate gene expression. MiRNAs are involved in many physiological and pathological progress. Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors and play an important role in cancer initiation and progression by regulating their target genes negatively. Study demonstrates that microRNA-19a (miR-19a) and microRNA-19b (miR-19b) were significantly up-regulated in human cervical carcinoma tissue samples, and the result was confirmed by real-time quantitative PCR.In this study, we focused on the effects of miR-19a and miR-19b on the phenotypes of cervical carcinoma cells as well as the identification of their direct target genes, in order to illuminate the molecular mechanisms of miR-19a and miR-19b in the initiation and progression of cervical carcinoma.[Methods] We detected the differencial expression of miR-19a and miR-19b in human cervical carcinoma and adjacent normal tissues by Real-time PCR assay. MiR-19a and miR-19b were overexpressed and knockdowned in cervical carcinoma cell lines respectively, and the changes of cell phenotypes were detected both with WST-1assay,colony formation assay,Transwell,etc. Subsequently, we combined bioinformatics of three target-predicted webs and identified the candidate target genes for miR-19a and miR-19b. Then fluorescent reporter assay was performed to confirm the reliability of the direct target genes. Furthermore, the mRNA levels and protein levels of target genes in miR-19a and miR-19b overexpressed cervical carcinoma cancer cells or tissues were detected with Real-time PCR and Western blot, in order to confirm the regulative role of miR-19a and miR-19b on IGF2R expression. Finally, the target gene was knocked down using RNA interference and changes of cell phenotypes were also detected with WST-1assay,colony formation assay, Transwell,etc.[Results] Real-time PCR results showed that the expression of miR-19a and miR-19b in human cervical carcinoma tissues were higher than those in adjacent normal tissues. We report that after overexpression of miR-19a and miR-19b, the proliferation activity, the colony formation activity and the invation activity of gastric cancer cells were inhanced. When knockdowned miR-19a and miR-19b,we got the opposite results. Subsequently, we identified oncogene IGF2R as congenerous candidate target genes for both miR-19a and miR-19b. The IGF2R mRNA3’untranslated region (3’UTR) contains the potential binding site both of miR-19a and miR-19b. The fluorescent reporter assay also confirmed that miR-19a and miR-19b can directly bind to the specific site of IGF2R mRNA3’UTR and negatively regulate the gene expression. When miR-19a and miR-19b function was overexpressed and knockdowned in cervical carcinoma cancer cells, mRNA level and protein level of IGF2R were both depressed and raised. We also discoved that when the target gene was knocked down, the proliferation activity, the colony formation activity and the invation activity of cervical carcinoma cancer cells were inhanceed apparently.[Conclusions] Our results show that in cervical carcinoma cells, miR-19a and miR-19b functions as tumor promoters and accelerate cell proliferation. The high-expression of miR-19a and miR-19b in cervical carcinoma cells lead to an abnormally low expression level of anti-oncogene IGF2R and result in the activation of cell proliferation. The elucidation of the mechanisms of miR-19a and miR-19b in cervical carcinoma helps us to further understand the mechanism of cervical carcinoma initiation and progression, and pave a way for clinical diagnosis and therapy of cervical carcinoma cancer.