本文選題：人非小細(xì)胞肺癌細(xì)胞系 + miR-3653-3p　； 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：miRNAs是一類內(nèi)源性約21-23個(gè)核苷酸長度的非編碼RNA,研究表明miRNAs參與多種生命過程的調(diào)節(jié),其異常表達(dá)常與疾病發(fā)生相關(guān),是潛在的腫瘤診斷及預(yù)后標(biāo)志物。本研究比較了正常細(xì)胞和非小細(xì)胞肺癌細(xì)胞中miR-3653-3p和miR-3200-5p的表達(dá)和啟動(dòng)子區(qū)DNA甲基化水平,并通過基因過表達(dá)和敲除技術(shù)結(jié)合腫瘤惡性表型檢測,對miR-3653-3p和miR-3200-5p在肺癌中的作用及調(diào)節(jié)機(jī)制進(jìn)行了研究。1、miR-3653-3p在人非小細(xì)胞肺癌細(xì)胞系A(chǔ)549或H460的中作用及調(diào)節(jié)qRT-PCR結(jié)果顯示,與肺正常細(xì)胞的相對表達(dá)量為100%相比,miR-3653-3p在三種非小細(xì)胞肺癌細(xì)胞H460、A549和SK-MES-1中的表達(dá)量分別為67.74%、28.99%和11.72%,亞硫酸氫鹽測序結(jié)果表明,miR-3653-3p啟動(dòng)子區(qū)DNA甲基化水平分別為61.43%、80%和100%。通過構(gòu)建和分別轉(zhuǎn)染miR-3653-3p過表達(dá)載體miR-3653-pIRES2-EGFP或敲除載體miR-3653-gRNA,結(jié)果表明,過表達(dá)miR-3653-3p后細(xì)胞增殖能力、克隆形成能力、遷移和侵襲能力顯著降低,而敲除miR-3653-3p后結(jié)果相反。經(jīng)MirTarget2軟件預(yù)測BRWD1為miR-3653-3p的靶基因,qRT-PCR結(jié)果顯示,過表達(dá)miR-3653-3p后A549或H460中BRWD1表達(dá)量分別降低了 31.93%和52.05%,敲除miR-3653-3p后BRWD1表達(dá)量分別升高了 71.87%和53.44%。綜合以上結(jié)果表明,miR-3653-3p在非小細(xì)胞肺癌細(xì)胞中表達(dá)量與其啟動(dòng)子區(qū)DNA甲基化水平負(fù)相關(guān),miR-3653-3p可能通過靶向作用BRWD1發(fā)揮抑癌作用。2、miR-3200-5p在人非小細(xì)胞肺癌細(xì)胞系A(chǔ)549或H460中的作用及調(diào)節(jié)與正常細(xì)胞的相對表達(dá)量為100%相比,miR-3200-5p在H460、A549和SK-MES-1中的表達(dá)量分別為78.69%、47.98%和15.6%,啟動(dòng)子區(qū)DNA甲基化水平分別為37.5%、55.830%和70%。構(gòu)建miR-3200-5p過表達(dá)載體miR-3200-pIES2-EGFP及敲除載體miR-3200-gRNA。過表達(dá)miR-3200-5p后細(xì)胞增殖能力、克隆形成能力、遷移和侵襲能力顯著降低,而敲除miR-3200-5p后結(jié)果相反。利用MirTarBase軟件預(yù)測MGLL為miR-3200-5p靶基因,qRT-PCR驗(yàn)證顯示過表達(dá)miR-3200-5p 后 A549 或 H460 中 MGLL 表達(dá)量分別降低了 57.29%和 74.89%,敲除miR-3200-5p后MGLL表達(dá)量分別升高了 34.73%和56.7%的結(jié)果表明MGLL可能是miR-3200-5p的靶基因。根據(jù)MGLL的CDS序列構(gòu)建過表達(dá)載體MGLL-pIRES2-EGFP和干擾載體pRNAT-U6.1-shMGLL/Neo。過表達(dá)載體 MGLL-pIRES2-EGFP 轉(zhuǎn)染后 miR-3200-5p的表達(dá)量顯著降低,干擾載體pRNAT-U6.1-shMMGLL/Neo轉(zhuǎn)染后miR-3200-5p的表達(dá)量顯著升高。細(xì)胞增殖、遷移和侵襲能力等檢測表明,MGLL過表達(dá)載體與miR-3200-5p過表達(dá)載體共轉(zhuǎn)A549或H460細(xì)胞后,MGLL可逆轉(zhuǎn)miR-3200-5p過表達(dá)載體單獨(dú)轉(zhuǎn)染對細(xì)胞惡性表型的抑制作用。綜合以上結(jié)果表明,miR-3200-5p在非小細(xì)胞肺癌細(xì)胞中低表達(dá)與其啟動(dòng)子區(qū)DNA甲基化水平負(fù)相關(guān),miR-3200-5p可能通過靶向作用MGLL抑制肺癌惡性表型,且miR-3200-5p和MGLL之間可能存在相互調(diào)節(jié)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉換新;張國祥;郭琳瑯;唐松山;;miR-139-5p在小細(xì)胞肺癌組織中的表達(dá)及其臨床意義[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2016年05期

，

本文編號(hào)：1800898