WWOX基因对肺癌细胞侵袭性的抑制作用及机制研究

发布时间：2018-05-06 11:03

本文选题：肺癌 + 含WW域的氧化还原酶基因　；参考：《江南大学》2017年硕士论文

【摘要】：目的:探究含WW域的氧化还原酶(WWOX)基因与不同恶化程度非小细胞肺癌组织和肺癌细胞的相关性、WWOX过表达/干扰对肺癌细胞侵袭性的影响、RUNX2在WWOX过表达/干扰肺癌细胞中的变化及RUNX2介导WWOX抑制肺癌细胞侵袭性的机制。方法:本研究选用临床非小细胞肺癌组织标本和多种不同恶化程度的肺癌细胞作为研究对象。应用脂质体法瞬时转染外源性WWOX和RUNX2过表达质粒;脂质体法介导小干扰RNA沉默WWOX和RUNX2表达;荧光实时定量PCR检测不同处理条件下的WWOX、RUNX2和MMP-9 mRNA的表达含量;免疫印迹法(western blot)检测WWOX和RUNX2蛋白的表达含量;Transwell小室检测肺癌细胞侵袭性和迁移性;划痕试验检测肺癌细胞迁移性的改变;免疫荧光检测肺癌细胞相关黏附因子如E-cadherin的表达情况及形态学观察。结果:临床组织样本实验表明,相较于肺癌旁正常组织,肺癌组织中WWOX的表达出现减少或缺失,且在高恶化组织(II期-III期肺癌)比低恶化组织(I期肺癌)中表达要减少。细胞实验表明,WWOX的表达量与肺癌细胞恶化程度成反比,在肺正常上皮细胞中表达最多。针对高侵袭性但WWOX低表达的H1299细胞株,转染WWOX过表达质粒后,其侵袭性和迁移性出现减弱,黏附性出现一定的上调,而再转染RUNX2过表达质粒后,其侵袭性出现回升;针对低侵袭性但WWOX高表达的NL9980细胞株,应用小干扰RNA沉默WWOX的表达后,其侵袭性和迁移性增强,黏附性下降,而再应用小干扰RNA沉默RUNX2表达后,其侵袭性出现回降。Western blot证实WWOX对RUNX2存在抑制作用,荧光实时定量PCR证实RUNX2下游因子MMP-9在WWOX过表达细胞中出现下调,而在WWOX沉默表达细胞中出现上调。结论:以上结果表明,WWOX与肺癌组织和肺癌细胞的侵袭性密切相关。RUNX2介导了WWOX对肺癌细胞侵袭性的抑制作用。
[Abstract]:Objective: to investigate the correlation between WW domain redox gene and lung cancer tissues and lung cancer cells with different degrees of deterioration. The effect of overexpression / interference of WWOX on the invasiveness of lung cancer cells: RUNX2 overexpression / interference in WWOX The changes in cells and the mechanism of RUNX2 mediated WWOX inhibiting the invasion of lung cancer cells. Methods: clinical non-small cell lung cancer (NSCLC) tissues and lung cancer cells with different degrees of deterioration were selected. Exogenous WWOX and RUNX2 overexpression plasmids were transiently transfected with liposome method, small interfering RNA silenced WWOX and RUNX2 expression was mediated by liposome method, and the expression of WWOX and RUNX2 was detected by real-time fluorescence quantitative PCR. Western blots were used to detect the expression of WWOX and RUNX2 protein in lung cancer cells. The invasion and migration of lung cancer cells were detected by Transwell chamber, and the changes of migration of lung cancer cells were detected by scratch test. The expression and morphology of E-cadherin in lung cancer cells were detected by immunofluorescence. Results: the clinical tissue samples showed that the expression of WWOX in lung cancer was decreased or absent compared with the normal tissues adjacent to lung cancer, and the expression of WWOX was decreased in stage II-III lung cancer than in stage I lung cancer. Cell experiments showed that the expression of WWOX was inversely proportional to the deterioration of lung cancer cells, and was most expressed in normal lung epithelial cells. For H1299 cell line with high invasiveness but low expression of WWOX, the invasiveness and mobility of H1299 cells transfected with WWOX overexpression plasmid were weakened, adhesion was up-regulated, and the invasiveness of H1299 cell line increased after retransfection of RUNX2 overexpression plasmid. For the NL9980 cell line with low invasion but high expression of WWOX, the expression of WWOX was silenced by small interfering RNA, the invasion and migration of WWOX were enhanced, and the adhesion was decreased, while the expression of RUNX2 was silenced by small interfering RNA. The inhibitory effect of WWOX on RUNX2 was confirmed by Western blot. The down-regulation of RUNX2 downstream factor MMP-9 was found in WWOX overexpression cells and up-regulated in WWOX silent expression cells by real-time quantitative PCR. Conclusion: these results suggest that WWOX is closely related to the invasion of lung cancer tissues and lung cancer cells. RUNX2 mediates the inhibitory effect of WWOX on the invasion of lung cancer cells.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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