miR-210在冷应激大鼠肝脏中的靶基因筛选及增殖凋亡研究

发布时间：2018-08-05 19:04

【摘要】：本文旨在研究冷应激条件下miR-210的靶基因,并以大鼠肝脏组织和肝细胞系为研究对象。通过qRT-PCR技术对大鼠肝脏中的miR-210表达量进行了分析,后续又通过生物信息学的方法,应用TargetScan、miRanda、PicTar和mirDB四种预测软件对miR-210的靶基因进行了分析。首先在细胞中筛选上调和下调miR-210的最佳条件,之后对miR-210的8个靶基因(ISCU、PSMB6、RAD52、GPD1、GPD2、ATP1b1、E2F3和Rap1b)进行筛选,并利用Western Blot技术对后续蛋白表达进行验证。根据细胞试验筛选出的靶基因,再对大鼠肝脏中的这些靶基因进行验证。最后对大鼠肝细胞进行凋亡和增殖实验。结果显示:大鼠肝脏中miR-210的表达量出现极显著(P0.01)上调。在后续下调和上调试验筛选中发现,最佳上调条件为细胞培养24 h转染mimics 100pmol,最佳下调条件为细胞培养24 h,转染inhibitor 150pmol。细胞中qRT-PCR结果显示GPD2、ISCU、PSMB6、RA52和E2F3均在miR-210上调时出现显著水平的表达下调,并在miR-210下调时出现显著水平的上调,并且其靶蛋白ISCU和PSMB6也有相同趋势的表达。在大鼠肝细胞系增殖和凋亡的试验中发现miR-210的升高会明显抑制细胞增殖,引起细胞死亡,而miR-210降低并未影响细胞死亡,但是会促进细胞增殖。后续的大鼠肝脏试验中又对GPD2、ISCU、PSMB6、RA52和E2F3五个靶基因进行了验证,发现ISCU、PSMB6、RA52和E2F3 4个靶基因出现了显著(P0.05)下调,GPD2并无明显变化,冷应激大鼠肝脏中的靶蛋白ISCU和PSMB6均出现显著(P0.05)或极显著下调(P0.01)。结论:miR-210的升高可能对冷应激大鼠肝脏造成一定损伤,尤其是影响大鼠肝细胞的增殖和凋亡;降低大鼠的免疫力;并抑制大鼠肝脏中的呼吸链电子传递;同时也减少了大鼠肝脏在过度动员时产生的氧化损伤。
[Abstract]:The aim of this study was to study the target genes of miR-210 under cold stress and to study the liver tissues and cell lines of rats. The expression of miR-210 in rat liver was analyzed by qRT-PCR technique, and the target genes of miR-210 were analyzed by means of Target ScanmimiRanda PicTar and mirDB. The optimal conditions for up-regulation and down-regulation of miR-210 were first screened in the cells, then the eight target genes of miR-210 (miR-210 PSMB6RAD52GPD1GPD1GPD2ATP-1b1A1E2F3 and Rap1b) were screened, and the subsequent protein expression was verified by Western Blot technique. The target genes in rat liver were screened by cell test. Finally, apoptosis and proliferation of rat hepatocytes were tested. The results showed that the expression of miR-210 in rat liver was significantly up-regulated (P0.01). In the subsequent down-regulation and up-regulation test screening, we found that the best up-regulation condition was that the cells were transfected with mimics 100pmol for 24 h, and the best down-regulation condition was the cell culture for 24h, and the optimal down-regulation condition was that the cells were transfected with inhibitor 150pmol. The results of qRT-PCR showed that both GPD2 / ISCUU PSMB6 RA52 and E2F3 were down-regulated when miR-210 was up-regulated, and significantly up-regulated when miR-210 was down-regulated, and the target proteins ISCU and PSMB6 showed the same trend. In the experiment of proliferation and apoptosis of rat liver cell line, it was found that the increase of miR-210 significantly inhibited cell proliferation and caused cell death, while the decrease of miR-210 did not affect cell death, but promoted cell proliferation. In the subsequent rat liver test, the five target genes of GPD2 / ISCUPSMB6ARA52 and E2F3 were verified. It was found that the four target genes of ISCU PSMB6, RA52 and E2F3 were significantly down-regulated (P0.05) and no significant changes were found in GPD2. The target protein ISCU and PSMB6 in the liver of cold stress rats were significantly (P0.05) or extremely down-regulated (P0.01). Conclusion the increase of miR-210 may damage the liver of cold stress rats, especially affect the proliferation and apoptosis of rat hepatocytes, decrease the immunity of rats, and inhibit the electron transfer of respiratory chain in rat liver. It also reduces the oxidative damage caused by excessive mobilization of the liver in rats.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.3

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