The Molecular Regulating Mechanism Studies Of MiR-21 And MiR-143 Genes On Human Cervical Carcinoma

Purpose:Based on miR-21 and miR-143 expression profiles in the cervical carcinoma, we hypothesized that these miRNAs might be involved in the pathogenesis of cervical carcinoma. Through manipulating the expression of miR-21 (down-regulated) and miR-143(up-regulated), we will observe their effects on cell proliferation and apoptosis activity of cervical carcinoma cell lines CaSki and Hela229. We hope to elucidate the role of miR-21 and miR-143 in the carcinogenesis of cervical carcinoma, and provide the theoretical basis for cervical carcinoma diagnosis and molecular treatment.Materials and Methods:The cervical squamous carcinoma cell lines CaSki and adenocarcinoma cell lines Hela229 were adopted, then synthetic antisense oligonucleotide AS-miR-21 and miR-143 mimics were transfected into CaSki and Hela229 respectively by lipofectamine2000. The expression changes of miR-21 and miR-143 in the CaSki and Hela229 were measured by Real time RT-PCR. Cellular growth and proliferation capacity were assayed by the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM).The apoptosis rates were tested by TUNEL assay and flow cytometry.Results:Contrast with the normal cervical tissue, the expression level of miR-21 in the cervical squamous carcinoma tissue, squamous carcinoma cell lines CaSki and adenocarcinoma cell lines Hela229 were significantly higher; while the miR-143 gene expression were prominently lower. AS-miR-21 could effectively knockdown the expression of miR-21 in CaSki and Hela229, miR-143 mimics upregulated the expression of miR-143 gene in the cervical cancer cells successfully. Both of them were dose-effect relationship. Cell proliferation capacity was inhibited obviously after miR-21 knockdown in CaSki and Hela229 cell lines; Cell cycle analysis showed that the cells in G0/G1 phase increased, accompanying with the S phase cells reduction in both CaSki and Hela229 cells following the down-regulation of miR-21, and the inhibition effects were positively related to the transfection concentrations and time. By flow cytometry and TUNEL methods, apoptosis rates were significantly higher than the blank control group or negative control group. The accrescence extend of apoptosis rates were associated with the transfection concentration, and dose-effects relation was recorded between them too. The miR-143 up-expression in the two cells could inhibit cell proliferation, and induce more CaSki and Hela229 cells arrested in GO/G1 phase, thus weakening cancer cell proliferation activities, the inhibition effects also obeying the relationship of dose-responsing effects. We finally observed the up-expression of miR-143 can induce apoptosis of the cervical squamous carcinoma cells; however, the phenomenon was not found in the cervical adenocarcinoma cell line Hela229. The further study on the apoptosis effect of miR-143 gene expression in the cervical adenocarcinoma cancer was needed.Conclusions:In the cervical cancer tissue, cervical squamous carcinoma cells lines CaSki and adenocarcinoma cell lines Hela229, miR-21 expression was up-regulated, while miR-143 expression was significantly reduced compared with the normal cervical tissue; miR-21 can promote cell proliferation and play an important role in anti-apoptosis as an oncogene in the cervical squamous carcinoma cells and adenocarcinoma cells; on the other hand miR-143 plays a role in inhibiting cervical cancer cell proliferation, intervening cell cycle and especially can induce the cervical squamous carcinoma cells apoptosis as a tumor suppressor gene.