The Study Of The Roles Of MiR-21/miR-140/miR-206in Lung Adenocarcinoma

In this study, we investigated the role of miRNA in lung adenocarcinoma. This research included two major parts. One is the study of the role of miR-21regulating MSH2expression in A549Lung adenocarcinoma. The other part is the study of the roles of miR-140and miR-206in Lung adenocarcinoma.1. The study of the role of miR-21regulateing MSH2expression in A549Lung adenocarcinoma(1) miR-21regualting MSH2expressionmicroRNA (miRNA) analysis softwares showed that MSH23’-untranslated region (3’-UTR) was targeted by miR-21. To further explore the effects of miR-21and MSH2on A549proliferation, we constructed pcDNA-GFP-msh-UTR vector (including MSH23’-UTR), which was tansfected into A549cells with miR-21. The GFP positive cells were estimated under a fluorescence microscopy and by flow cytometry. We found miR-21could obviously downregulate the expression of MSH2, which was further proved by western blotting.(2) The roles of miR-21and MSH2in A549cell growth and apoptosismiR-21acts as an oncogene in many kinds of tumors. Overexpression of MSH2can promote tumor cells apoptosis. Cisplatin was one of the effective anticancer drug for lung cancer therapy. We treated A549cells with cisplatin in vitro and in vivo. Real-time PCR analysis showed that miR-21expression was decreased. Western blotting revealed that the MSH2expression was increased both in A549cells and A549cancer xenografts. These results showed that Cisplatin might upregulate MSH2expression by reducing miR-21. We further detected the A549cell prolifertion after cisplatin increased MSH2expression. The MTT assay showed that overexpression of MSH2could effectivley inhibit A549cell growth on cellular level. Annexin V-FITC/PI dual-dye was used to detect cell apoptosis rate. We found that the apoptosis rate of overexpression of MSH2groups significantly higher than controls. In vivo study, after cisplatin treatment, the expression of MSH2was higher, and the volumes as well as weight of A549lung cancer xenografts were much smaller than those of control. These results demonstated that upregulation of MSH2through supressing miR-21expression could inhibit A549cell growth.2. The effects miR-140/miR-206om Lung adenocarcinoma cell growth(1) miR-140/miR-206take part in TGF-β1/Smad3signaling pathwaymicroRNA (miRNA) analysis softwares predicted that MSH23’-untranslated region (3’-UTR) was targeted by miR-21. pcDNA-GFP-smad3-3’UTR vector (including Smad33’-UTR) was constructed and transfected Lung adenocarcinoma cells with miR-140/miR-206.48h after transfection, the results of fluorescence microscopy and flow cytometry showed that the GFP expression was lower in miR-140/miR-206transfected cells compared to control oligo-treated cultures. Western blotting further confirmed that miR-140/miR-206could supppress expression of p-Smad3, respectively, indicating that miR-140/miR-206negatively regulating Smad3dependent TGF-β1signaling. After A549cells treated with TGF-β1, we detected the expresssion of miR-140/miR-206in cells by real-time PCR. The result showed that the expresssion of miR-140/miR-206were both reduced. The above results showed that miR-140/miR-206participate into Smad3dependent TGF-β1signaling.pathway(2)The role of miR-140/miR-206in A549cell proliferation and metastasisAfter reducing the expression of p-Smad3by miR-140/miR-206treatment, we further examined the expression of TRTB2using western blot. The results showed that TRIB2expression was inhibited significantly compared to control oligo-treatment. Considering TRIB2is an oncogene, we further detected A549cell proliferation after down-regualtion of TRTB2by miRNAS. The MTT assay showed that miR-140/miR-206could effectivley inhibit A549cell growth; Annexin V-FITC/PI dual-dye results showed miR-140/miR-206could effectivley promote cell apoptosis. The epithelial-to-mesenchymal transition (EMT) is important to the conversion of early stage tumors into invasive malignancies. Smad3is a center signaling molecule of TGF-β1pathway to induce EMT. Western blotting results showed epithelial markers E-cadherin expression was higher and mesenchymal marker a-SMA expression was lower. Cell migration assay showed that the number of metastasis cells treated with miR-140/miR-206was fewer. These results indicated that miR-140/miR-206could inhibit Lung adenocarcinoma cell metastasis by regulating E-cadherin and a-SMA expression.In summary, miR-21could regulate MSH2expression. Over-expresion of MSH2through inhibiting miR-21expression can inhibit lung adenocarcinoma cells proliferation. miR-140/miR-206, suppressing TREB2expression, can inhibit the lung adenocarcinoma cell proliferation by regulating Smad3dependent TGF-β1signaling.