miR-3653-3p和miR-3200-5p在人非小细胞肺癌细胞中作用及调节研究

发布时间：2018-04-25 10:15

本文选题：人非小细胞肺癌细胞系 + miR-3653-3p　；参考：《内蒙古大学》2017年硕士论文

【摘要】：miRNAs是一类内源性约21-23个核苷酸长度的非编码RNA,研究表明miRNAs参与多种生命过程的调节,其异常表达常与疾病发生相关,是潜在的肿瘤诊断及预后标志物。本研究比较了正常细胞和非小细胞肺癌细胞中miR-3653-3p和miR-3200-5p的表达和启动子区DNA甲基化水平,并通过基因过表达和敲除技术结合肿瘤恶性表型检测,对miR-3653-3p和miR-3200-5p在肺癌中的作用及调节机制进行了研究。1、miR-3653-3p在人非小细胞肺癌细胞系A549或H460的中作用及调节qRT-PCR结果显示,与肺正常细胞的相对表达量为100%相比,miR-3653-3p在三种非小细胞肺癌细胞H460、A549和SK-MES-1中的表达量分别为67.74%、28.99%和11.72%,亚硫酸氢盐测序结果表明,miR-3653-3p启动子区DNA甲基化水平分别为61.43%、80%和100%。通过构建和分别转染miR-3653-3p过表达载体miR-3653-pIRES2-EGFP或敲除载体miR-3653-gRNA,结果表明,过表达miR-3653-3p后细胞增殖能力、克隆形成能力、迁移和侵袭能力显著降低,而敲除miR-3653-3p后结果相反。经MirTarget2软件预测BRWD1为miR-3653-3p的靶基因,qRT-PCR结果显示,过表达miR-3653-3p后A549或H460中BRWD1表达量分别降低了 31.93%和52.05%,敲除miR-3653-3p后BRWD1表达量分别升高了 71.87%和53.44%。综合以上结果表明,miR-3653-3p在非小细胞肺癌细胞中表达量与其启动子区DNA甲基化水平负相关,miR-3653-3p可能通过靶向作用BRWD1发挥抑癌作用。2、miR-3200-5p在人非小细胞肺癌细胞系A549或H460中的作用及调节与正常细胞的相对表达量为100%相比,miR-3200-5p在H460、A549和SK-MES-1中的表达量分别为78.69%、47.98%和15.6%,启动子区DNA甲基化水平分别为37.5%、55.830%和70%。构建miR-3200-5p过表达载体miR-3200-pIES2-EGFP及敲除载体miR-3200-gRNA。过表达miR-3200-5p后细胞增殖能力、克隆形成能力、迁移和侵袭能力显著降低,而敲除miR-3200-5p后结果相反。利用MirTarBase软件预测MGLL为miR-3200-5p靶基因,qRT-PCR验证显示过表达miR-3200-5p 后 A549 或 H460 中 MGLL 表达量分别降低了 57.29%和 74.89%,敲除miR-3200-5p后MGLL表达量分别升高了 34.73%和56.7%的结果表明MGLL可能是miR-3200-5p的靶基因。根据MGLL的CDS序列构建过表达载体MGLL-pIRES2-EGFP和干扰载体pRNAT-U6.1-shMGLL/Neo。过表达载体 MGLL-pIRES2-EGFP 转染后 miR-3200-5p的表达量显著降低,干扰载体pRNAT-U6.1-shMMGLL/Neo转染后miR-3200-5p的表达量显著升高。细胞增殖、迁移和侵袭能力等检测表明,MGLL过表达载体与miR-3200-5p过表达载体共转A549或H460细胞后,MGLL可逆转miR-3200-5p过表达载体单独转染对细胞恶性表型的抑制作用。综合以上结果表明,miR-3200-5p在非小细胞肺癌细胞中低表达与其启动子区DNA甲基化水平负相关,miR-3200-5p可能通过靶向作用MGLL抑制肺癌恶性表型,且miR-3200-5p和MGLL之间可能存在相互调节的作用。
[Abstract]:MiRNAs is a kind of endogenous non-coding RNAs with about 21-23 nucleotide length. It has been shown that miRNAs is involved in the regulation of many life processes and its abnormal expression is often associated with the occurrence of disease and is a potential tumor diagnosis and prognostic marker. In this study, we compared the expression of miR-3653-3p and miR-3200-5p and the level of DNA methylation in promoter region in normal and non-small cell lung cancer cells, and combined with the detection of malignant phenotype by gene overexpression and knockout techniques. The role of miR-3653-3p and miR-3200-5p in lung cancer and its regulatory mechanism were studied. The role of 1 miR-3653-3p in human non-small cell lung cancer cell line A549 or H460 and the regulatory mechanism of qRT-PCR were studied. Compared with the normal lung cells, the relative expression of miR-3653-3p was 67.7428. 99% and 11. 72% in H460A549 and SK-MES-1, respectively. The DNA methylation level in the promoter region was 61.4380% and 100%, respectively. MiR-3653-3p overexpression vector miR-3653-pIRES2-EGFP or knockout vector miR-3653-gRNAs were constructed and transfected respectively. The results showed that the ability of cell proliferation, clone formation, migration and invasion were significantly decreased after overexpression of miR-3653-3p, but the results were opposite after knockout of miR-3653-3p. The expression of BRWD1 in A549 or H460 was decreased by 31.93% and 52.05%, respectively, and the expression of BRWD1 increased by 71.87% and 53.44% after miR-3653-3p was knocked out by MirTarget2 software. The above results suggest that the expression of miR-3653-3p in non-small cell lung cancer cells is negatively correlated with the level of DNA methylation in its promoter region. The inhibitory effect of miR-3653-3p on human non-small cell lung cancer cell line A549 or H460 may be mediated by targeting BRWD1. The relative expression of miR-3200-5p in H460 A549 and SK-MES-1 was 78.69% and 15.6%, respectively. The DNA methylation level in promoter region was 55.830% and 70%, respectively. MiR-3200-5p overexpression vector miR-3200-pIES2-EGFP and knockout vector miR-3200-gRNA were constructed. After overexpression of miR-3200-5p, the ability of cell proliferation, clone formation, migration and invasion were significantly decreased, but the results were opposite after knockout of miR-3200-5p. MirTarBase software was used to predict that MGLL was the target gene of miR-3200-5p by qRT-PCR. The results showed that MGLL expression in A549 or H460 after overexpression of miR-3200-5p decreased by 57.29% and 74.89%, and MGLL expression increased by 34.73% and 56.7% after miR-3200-5p was knocked out. The results showed that MGLL might be the target gene of miR-3200-5p. The overexpression vector MGLL-pIRES2-EGFP and the interference vector pRNAT-U6.1-shMGLL / Neo. were constructed according to the CDS sequence of MGLL. The expression of miR-3200-5p decreased significantly after transfection of overexpression vector MGLL-pIRES2-EGFP, and the expression of miR-3200-5p increased after transfection of interference vector pRNAT-U6.1-shMMGLL/Neo. The results of cell proliferation, migration and invasion showed that miR-3200-5p overexpression vector and miR-3200-5p overexpression vector co-transfected A549 or H460 cells could reverse the inhibitory effect of miR-3200-5p overexpression vector alone on the malignant phenotype of A549 or H460 cells. These results suggest that the low expression of miR-3200-5p in NSCLC cells is negatively correlated with the level of DNA methylation in its promoter region. MiR-3200-5p may inhibit the malignant phenotype of lung cancer by targeting MGLL, and there may be an interregulatory effect between miR-3200-5p and MGLL.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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