摘要
目的观察RNA干扰沉默RRM2基因表达对人肝癌细胞HepG2细胞生物学影响，并探讨其分子机制。方法利用小RNA干扰（siRNA）技术沉默HepG2细胞中RRM2基因的表达，用采用实时定量聚合酶链反应（Real．timePCR）及Western blot技术检测RRM2mRNA及蛋白的表达；用细胞计数试剂盒（CCK．8）方法检测siRNA—RRM2对HepG2细胞增殖的影响；用Transwell细胞迁移系统检测siRNA-RRM2对HepG2细胞迁移能力的影响；采用Western blot技术检测抗凋亡蛋白B淋巴细胞／白血病-2（bcl-2），促凋亡蛋白bax和细胞色素C（Cyt—C）蛋白水平变化；建立成瘤模型每组10只，观察RRM2基因沉默后对移植瘤生长的影响。结果siRNA—RRM2有效沉默HepG2细胞中RRM2表达；CCK-8法结果显示与对照组比较下调RRM2基因抑制HepG2增殖[（41．20±2．13）％比（91．35±10．22）％]；TransweU细胞迁移系统检测显示下调RRM2基因后，显著抑制了HepG2的迁移能力（21．20±1．28比132．50±7．88）；Westernblot结果显示抗凋亡蛋白bcl-2下调，促凋亡蛋白bax上调，Cyt—C上调；体内实验证实下调RRM2组肿瘤体积明显小于对照组，到第8周时，siRNA-RRM2组肿瘤体积为（171．13±28．12）mm^3，对照组为（1487．41±168．20）mm^3。结论siRNA—RRM2能显著抑制RRM2基因在人肝癌细胞HepG2中的表达，部分逆转HepG2的恶性生物学行为并明显抑制HepG2在裸鼠体内的成瘤和生长。
Objective To investigate the effect of small interference RNA （siRNA） silencing RRM2 on the biological behaviors of human hepatoma cancer cell line HepG2 and and the molecular mechanisms. Methods RRM2 expression was knocked down in human hepatoma cancer cell line HepG2 by RRM2 siRNA. The expression level of RRM2 was detected in human hepatoma cancer cell line HepG2 and human normal liver cell line HL-7702 at mRNA and protein levels by real-time polymerase chain reaction （PCR） or Western blotting. The cell proliferation was measured by cell counting Kit-8 （CCK-8） . The migration was observed by using Transwell cell migration system. The expression levels of B lymphocytes/leukemia-2 （bcl-2）, bax and cytochrome C （Cyt-C） proteins were detected by using Western blotting. Ten male BALB/C nude mice were subcutaneously injected with siRNA-RRM2/HepG2, siRNA-NC/HepG2 or NEG/HepG2 cells respectively to study the growth of tumor in each group. Results siRNA-RRM2 could down-regulate the expression of RRM2 in HepG2 remarkably, and siRNA-RRM2 could inhibit both the proliferation [ （41.20±2. 13）% vs （91.35 ± 10. 22）% ] and migration ability of HepG2 cells （21.20 ± 1.28 vs 132. 50 ± 7. 88） as compared with the negative control group, siRNA-RRM2 could down-regulate the expression of bcl-2 and up-regulate the expression of bax and Cyt-C. Additionally, the volume of xenograft tumors in siRNA-RRM2 group was decreased significantly as compared with control group. The volume of xenograft tumors in siRNA-RRM2 group was （171.13 ±28. 12） mm^3 and that in siRNA-NC group was （ 1487.41 ± 168.20） mm^3 at 8th week. Conclusion siRNA-RRM2 can down-regulate the expression of RRM2 in the HepG2 cells and may play an important role in the regulation of biological behaviors of HepG2 cells, and also inhibit the tumor growth of nude mice model.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第10期1917-1920,共4页
Chinese Journal of Experimental Surgery
