The Function And The Regulation Of MiR-3653-3p And MiR-3200-5p In Human Non-small Cell Lung Cancer Cells

miRNAs are a class of endogenous non coding RNA with length of about 21-23 nucleotides,miRNAs involved in a variety of life process,the aberrant expression of several miRNAs were associated with the occurrence of disease,miRNAs have been recognized as a potential tumor diagnostic and prognostic markers.In this study,we compared the expression and the level of DNA methylation in the promoter region of miR-3653-3p and miR-3200-5p in non-small cell lung cancer cells.The regulation and the role of miR-3653-3p and miR-3200-5p were studied by gene overexpressing,gene knockouting method combined with the malignant phenotype observaiton.1、The function and the regulation of miR-3653-3p in human non-small cell lung cancer cell line A549 or H460qRT-PCR results showed that compared with the relative expression was 100%in normal lung cells,miR-3653-3p expression in three human non-small cell lung cancer cell H460,A549 and SK-MES-1 were 67.74%,28.99%and 11.72%,bisulfite sequencing results showed that miR-3653-3p promoter DNA methylation levels were 61.43%,80%and 100%.The overexpression vector miR-3653-pIRES2-EGFP and the knockout vector miR-3653-gRNA were constructed and transfected into cancer cells,respectively.After the overexpression vector transfected,the abilities of cell proliferation,colony formation,migration and invasion were significantly reduced,while the abilities were increased after the knockout vector transfected.BRWD1 was predicted to be a target gene of miR-3653-3p by the MirTarget2 software,and qRT-PCR results showed that the expression of BRWDl in A549 or H460 decreased 31.93%and 52.05%after miR-3653-3p overexpression,while increased 71.87%and 53.44%after miR-3653-3p knockdown.The above results indicated that there was a negative correlation between the expression of miR-3653-3p and the DNA methylation status in its promoter region,and miR-3653-3p may play a role in tumor suppression by targeting BRWD1.2.The function and the regulation of miR-3200-5p in human non-small cell lung cancer cell line A549 or H460Compared with the relative expression was 100%in normal lung cells,miR-3200-5p expression in three human non-small cell lung cancer cell H460,A549 and SK-MES-1 were 78.69%,47.98%and 15.6%,promoter DNA methylation levels were 37.5%,55.83%and 70%.The overexpression vector miR-3200-pIRES2-EGFP and the knockout vector miR-3200-gRNA were constructed and transfected into cancer cells,respectively.After the overexpression vector transfected,the abilities of cell proliferation,colony formation,migration and invasion were significantly reduced,while the abilities were increased after the knockout vector transfected.MGLL was predicted to be a target gene of miR-3200-5p by the MirTarBase software,and qRT-PCR results showed that the expression of MGLL in A549 or H460 decreased 57.29%and 74.89%after miR-3200-5p overexpression,while increased 34.73%and 56.7%after miR-3200-5p knockdown.The results indicated that miR-3200-5p may play a role in tumor suppression by targeting MGLL.Construction of over expression vector MGLL-pIRES2-EGFP and interference vector pRNAT-U6.1-shMGLL/Neo based on CDS sequence of MGLL.After the overexpression vector transfected,the expression of miR-3200-5p was significantly decreased and the expression of miR-3200-5p increased significantly after the knockout vector transfected.The proliferation,migration and invasion ability of the cells showed that MGLL were able to reverse the inhibitory effect of transfection of miR-3200-5p over expression vector alone on the malignant phenotype of A549 or H460 cells after co-transfected MGLL overexpression vector and miR-3200-5p overexpression vector.The above results show that DNA methylation could inhibit the expression of miR-3200-5p in non-small cell lung cancer cells,miR-3200-5p inhibit the malignant phenotype of non-small cell lung cancer by targeting MGLL,and there may be interaction between miR-3200-5p and MGLL.