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目的观察针对人乳头瘤病毒(human papillomavirus,HPV)16E6基因的RNA干扰(RNA interfering,RNAi)对人鼻咽癌(nasopharyngeal carcinoma,NPC)HNE-1细胞侵袭转移能力及细胞周期的影响,探讨HPV16E6基因作为鼻咽癌基因治疗标靶的可行性。方法根据基因库上的HPV16E6mRNA序列,设计合成针对HPV16E6的siRNA,转染HNE-1细胞,采用RT-PCR及Westernblot分析E6基因的表达,利用侵袭实验检测细胞的侵袭能力,MTT法检测细胞的生长增殖,流式细胞术检测细胞周期。结果针对HPV16E6的siRNA下调鼻咽癌HNE-1细胞株E6mRNA和蛋白表达水平,分别下调66.3%、56.7%(P<0.05);侵袭实验结果显示,siRNA转染组的穿膜细胞数为(45.3±4.0),显著低于阴性对照组[(147.7±4.7),P<0.05];转染组细胞的增殖受到抑制,与阴性对照组相比差异有统计学意义(P<0.05);流式细胞术显示细胞阻滞于G0/G1期。结论siRNA可以有效干扰鼻咽癌HNE-1细胞内HPV16E6基因的表达,进而影响HNE-1细胞的侵袭转移能力,并抑制肿瘤细胞的生长增殖,使细胞周期重新分布。
Objective To investigate the effect of RNA interference (RNAi) against human papillomavirus (HPV) 16E6 gene on the invasion and metastasis of human nasopharyngeal carcinoma (HNE-1) cells and the cell cycle of HPV16E6 Gene as a Gene Therapy Target for Nasopharyngeal Carcinoma. Methods siRNA targeting HPV16E6 was designed and synthesized according to the sequence of HPV16E6 gene on the gene bank. The expression of E6 gene was analyzed by RT-PCR and Western blot. The invasion ability of cells was detected by invasion assay. The cell growth was detected by MTT assay Proliferation, flow cytometry cell cycle. Results siRNA targeting HPV16E6 down-regulated the expression of E6 mRNA and protein in nasopharyngeal carcinoma HNE-1 cells, down-regulated by 66.3% and 56.7%, respectively (P <0.05). The results of invasion assay showed that the number of transmembrane cells in siRNA- ± 4.0), which was significantly lower than that in the negative control group [(147.7 ± 4.7), P <0.05]. The proliferation of the transfected cells was inhibited compared with the negative control group (P <0.05) Cytometry showed cells arrested in the G0 / G1 phase. Conclusion siRNA can effectively interfere with the expression of HPV16E6 gene in HNE-1 cells, and further affect the invasion and metastasis of HNE-1 cells, and inhibit the growth and proliferation of HNE-1 cells and redistribute cell cycle.