MiR-31Regulates The Function Of Lung Cancer Cells By Targeting SATB2

Objective To screen abnormal expression of miRNAs in the serum of lung cancerpatients with metastasis, then testing its expression in lung cancer cell lines and to verifyits target. To investigate the effects of miR-31on the migration and invasion capability ofhuman lung cancer cells, and to further explore its mechanism of action, which alters thebiological function of lung cancer cells.Methods First part Establish the detection method of miRNA in the serum of lungcancer by Real-time fluorescence quantitative PCR. Detect the expression of miR-20a,miR-21, miR-25, miR-29a, miR-31, miR-126, miR-129, miR-145, miR-205in61serumcases of lung cancer (including26cases with distant metastasis).Second part Software and websites of bioinformatics including TargetScan, PicTarand MirBase were used for target gene prediction. Recombined vector PGL3-control wasconstructed to express SATB23’UTR and dual-luciferase reporter assay was analyzed.Transfection of miR-31mimics/inhibitors was performed in95D (highly metastatic humanlung adenocarcinoma cells), Western blot was used to detect the expression of SATB2protein in95D cell line.Third part The expression of miR-31in lung bronchial epithelial cell (HBE) andlung cancer cell lines (A549, H1299,95C and95D) were determined using real-timepolymerase chain reaction (PCR) and SATB2was detected by Western blot analysis. Toselect one lung cancer cell line of the SATB2protein highest expression (95D). The lungcancer cell line95D which overexpressed or inhibition of miR-31was established. And95D cell line of the suppression SATB2was also established. Cell proliferation was detctedby CCK-8kit. Wound healing and Boyden chamber assays were performed to analyze themigration and the invasion capabilities of human lung cancer95D cells.Results First part The expression of miR-31decreased in the serum of lung cancer when distant metastasis, detected by real-time quantitative PCR. It was statisticallysignificant (t=3.320，P=0.002).Second part Fragments of3’UTR of human SATB2containing miR-31complementary sites were successfully cloned into PGL3-Control vector. The recombinedvector was named PGL3-SATB2. Dual luciferase reporter assay showed that miR-31caninhibit SATB2gene. By western blot, we found SATB2protein level increased in whichmiR-31is over-expressed and reduction in which miR-31is down-expressed in95D cells,compared with the control group. Further confirmed SATB2is a target gene of miR-31.Third part Detect the expression of miR-31and SATB2protein in lung bronchialepithelial cells HBE cell line and lung cancer cell lines (A549, H1299,95C,95D). Theresults of real-time PCR indicated that the expression of miR-31was the lowest (F=6.976,P=0.001) in the lung cancer cell line95D with high metastatic potential, whereas SATB2was the highest in the95D cell line by Western blot. This suggests that miR-31wasnegatively correlated with the expression of SATB2. Migration and invasion of95D cellswere reduced following overexpression of miR-31and inhibition of SATB2. Conversely,transfection of95D cells with miR-31inhibitor could promote cell migration andeffectively penetrate the chamber and travel to the other side of the membrane. Thealteration of miR-31expression was ineffective in cell proliferation in lung cancer95Dcell.Conclusion miRNAs associated with metastatic lung cancer were screenedprimarily, of which miR-31targeting SATB2might be one of the markers in metastaticlung cancer. MiR-31can suppress SATB2gene expression at the post-transcriptional levelby targeting the specific sequence of SATB23’UTR. MiR-31is downregulated in thehuman lung cancer cell line95D with high metastatic potential. Acting as a tumorsuppressor gene, miR-31suppressed migration and invasion by targeting SATB2. SATB2is a potential therapeutic target for the biological treatment of tumors.