腺病毒介导的Akt／mTOR基因沉默抑制肾癌786-O细胞增殖并促进细胞凋亡

目的应用腺病毒技术下调Akt／mTOR表达水平，检测Akt／mTOR基因沉默对人肾癌786-O细胞增殖和凋亡的影响。方法收集人肾癌及癌旁样品，RealtimePCR检测两组样品中Akt和mTOR的mRNA水平。利用病毒包装细胞293A获得靶向Akt／mTOR基因的重组腺病毒，感染人肾癌786-O细胞株。实验分2组：加人靶向Akt／mTOR基因的腺病毒感染的肾癌细胞组（rAd5-Am组），未处理的腺病毒感染的。肾癌细胞组（NC组）。应用Real timePCR及Western blot检测干扰前后Akt和mTOR mRNA及蛋白表达水平的变化。用细胞增殖及凋亡试剂盒检测Akt和mTOR沉默后，肾癌786-O细胞增殖、凋亡及PCNA表达水平的改变。结果与癌旁组织相比，Akt和mTOR的mRNA水平在肾癌组织中分别上调了2．613和3．254倍。成功获得knockdownAkt／mTOR的rAd5-Am腺病毒及对照腺病毒，感染肾癌786一O细胞。Real time PCR显示rAd5-Am组与NC组相比Akt／mTOR的mRNA水平分别下调65．3％和77．1％，Westernblot结果显示rAd5-Am组与NC组相比Akt／mTOR的蛋白表达水平分别下调78．4％和89．2％。rAd5．Am能显著降低786-O细胞中Akt／mTOR基因的表达。细胞增殖与凋亡实验表明，Akt／mTOR基因沉默后能显著抑制肾癌细胞786-O的增殖，并促进其凋亡（rAd5-Am组细胞凋亡率是NC组的2．57倍）。进一步的实验结果表明，Akt／mTOR下调后能降低PCNA的蛋白量，从而抑制。肾癌细胞的增殖。rAd5-Am感染的。肾癌786-O细胞的增殖和PCNA蛋白量受到抑制，而凋亡率增加。结论构建出能稳定干扰Akt／mTOR基因表达的。肾癌786-O细胞株。Akt／mTOR腺病毒干扰载体可有效沉默肾癌786-O细胞的内源性Akt／mTOR基因，抑制肾癌786-O细胞的增殖，下调PCNA的表达，并促进细胞凋亡。

Adenovirus-mediated Akt/mTOR silence inhibits proliferation and promotes apoptosis in human renal carcinoma cell line 786-0 in vitro

Objective To investigate the effect of adenovirus-mediated gene silence targeting Akt/mTOR on prolifer-ation and apoptosis of human renal carcinoma cell line 786-O. Methods Compare the mRNA levels of Akt/mTOR via real time PCR in collected human renal carcinoma and juxta-tumor tissues. Recombinant adenoviruses targeting Akt/mTOR har- vested from 293A cells were used to infect human renal carcinoma cell line 786-O. There were two groups： in rAd5-Am group, renal carcinoma cells were infected with adenovirus targeting Akt/mTOR genes, while NC group was with negative adenoviral infection. Akt/mTOR mRNA levels and related protein expression in the 786-O cells were assayed via real time PCR and Western blot, respectively. MTT, flow cytometry and Western blot were applied to examine the effect of Akt/ mTOR silence on the proliferation and apoptosis of 786-O cells and PCNA expression. Results The mRNA levels of Akt/ mTOR were up-regulated in renal carcinoma compared to juxta-tumor tissues. With comparison to the control vector, rAd5- Am treatment resulted in reduced expression of Akt/mTOR mRNA and protein in the human renal carcinoma cell line 786-O, Akt/mTOR mRNA levels after adenovirus infection were down-regulated （by 65.3% and 77.1% respectively） and Akt/mTOR protein expression significantly decreased （by 78.4% and 89.2%, respectively）. Furthermore, in rAd5-Am group, cell proliferation at 3d after infection was significantly decreased （P〈 0.05） and apoptosis rate was 2.57 times of NC group （P〈 0.05）. PCNA was down-regulated in rAd5-Am group in contrast to NC group. The proliferation and protein level of PCNA were suppressed, while apoptosis was promoted in rAd5-Am infected 786-O cells. Conclusions Renal carcinoma cell line 786-O cells with stable genes silence of Akt/mTOR were established. Adenovirus-mediated gene silence can specifically inhibit endogenous Akt/mTOR expression, effectively inhibit proliferation, down-regulate PCNA protein level and promote apoptosis in human renal carcinoma cell line 786-O.