The Function Of MiR-148a In NSCLC

Lung cancer is one of the most common malignant tumors, which ranks first in incidenceand mortality of all cancers. Non Small Cell Lung Cancer (NSCLC) accounts for80%~85%inthe total number of lung cancer. At present, the treatment of NSCLC mainly depends on surgeryand chemotherapy. However, the majority of patients was found in the advanced cancer and losesthe best chance of operation. Therefore, to seek new treatments becomes more and moreimportant. MicroRNAs (miRNAs) are endogenous non-coding single-stranded RNA, about thesize of22-24nucleotides, which are the important post-transcriptional adjustment factors.Numerous studies show that miRNAs play an important role in the occurrence and developmentof tumor. Therefore, this paper mainly researchs the impact of miRNA on NSCLC, including theimpact on cell proliferation, apoptosis and cell cycle, and looking for its target genes to study themode of action of miRNA in NSCLC, trying to provide the basis for the clinical treatment ofNSCLC.By Solexa sequencing we built a set of miRNA expression profiles and choose miR-148a asthe object of study because the significant differences in the expression. By qRT-PCR wedetected the expression levels of miR-148a were significantly different in human NSCLC celllines and NSCLC tissues relative to the normal cells line Beas-2B and normal lung tissue. Inorder to increase the expression of miR-148a in intracellular, we transfected miR-148a mimics inthe NSCLC cell line H1299. The results showed that miR-148a can inhibit cell proliferation,induce cell apoptosis and inhibit cell cycle at the G2/M phase. Indicating that miR-148a playsan important tumor suppress role in NSCLC.TargetScan and miRbase were used to predict the potential target genes of miR-148a. Weused the Dual-luciferase Report Analysis to validate the potential target genes DNMT1,ATP6AP2, MAP3K9, BCL2L11, CDKN1B, PTEN, CDK19, TMED7. The results showed thatthe3’-Untranslated Region (3’-UTR) of DNMT1, ATP6AP2, MAP3K9, BCL2L11, and CDK19were regulated to varying degrees by miR-148a, but the3’-UTR of CDKN1B、PTEN、TMED7 were not regulated by miR-148a. We mutated the binding site of DNMT1and miR-148a. Theresults showed that miR-148a have no effect on mutated site. qRT-PCR and Western Blot assaysverified that high expression of miR-148a in intracellular reduced DNMT1expression at bothmRNA and protein levels. These results indicate that DNMT1is the direct target genes ofmiR-148a, and miR-148a inhibits DNMT1occurred in mRNA levels.In order to inhibit the expression of DNMT1in intracellular, we transfected DNMT1siRNAin the NSCLC cell line H1299. The results showed that DNMT1siRNA can induce cellapoptosis and inhibit cell cycle at the G2/M phase, as the same result of over expression ofmiR-148a. These results suggest that inhibition of DNMT1is one of the mechanisms ofmiR-148a in NSCLC.