靶向切割HPV16E6 mRNA的核酶诱导宫颈癌细胞CaSKi凋亡的研究(英)

Ribozyme Targeted on HPV16E6 mRNA Induced Apoptosis on Human Cervical Carcinoma Caski Cells

背景与目的:人乳头瘤病毒与宫颈癌的发生相关,核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染了靶向切割HPV16E6mRNA核酶的宫颈癌细胞株的表型,以及核酶对宫颈癌细胞增殖与调亡的作用。方法:计算机设计特异性切割HPV16E6mRNA的核酶,构建抗HPV16E6核酶(HRz)的真核表达质粒。以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞。Northern杂交检测3种细胞中E6基因的表达。流式细胞仪检测3种细胞的凋亡率,将3种细胞在相差显微镜和荧光显微镜下观察,采用荧光(Hoechst33342)染色和TUNEL(TDT-mediateddUTPnickendlabeling)两种方法测定细胞凋亡。流式细胞术测定HPV16E6、c-myc、bcl-2、p53、Fas等蛋白的表达。结果:RNA点杂交证实核酶mRNA能稳定表达于CaSKi-R细胞中。Northern杂交证实CaSKi-R中E6mRNA表达水平较CaSKi细胞明显降低,而CaSKi-P和CaSKi细胞的E6mRNA表达水平无差异。CaSKi-R细胞凋亡率明显高于CaSKi和CaSKi-P细胞,细胞周期阻滞于G2期,S期细胞百分率下降。抗HPV16E6核酶能明显减少CaSKi-R细胞中HPV16E6、c-myc、bcl-2蛋白的表达,增高p53蛋白的表达;这种现象并不发生于CaSKi-P细胞中。Fas蛋白的表达在3种细胞中都相近。

BACKGROUND &OBJECTIVE: Human papillomavirus is related to cervical cancer. Ribozyme is special kind of RNA that can cleave target RNA. The aim of this study was to investigate the characterization of the cultured cervical cancer cell line transfected with anti HPV16E6 ribozyme (HRz) and the effect of ribozyme on proliferation and apoptosis of cervical cancer cell. METHODS: Ribozyme targeted on HPV16E6 mRNA was designed using computer. With the method of lipofectin transfection, the anti HPV16E6 ribozyme and empty eucaryotic expressing plasmids were transfected into the CaSKi cells, which named as CaSKi R and CaSKi P, respectively. The expression of E6 mRNA in the three kinds of cells was examined by Northern blot analysis. Cell cycle was determined using flow cytometry. Cell apoptosis rate was examined using fluorescent (Hoechst) staining and TUNEL (TDT mediated dUTP nick end labeling).The expression of certain proteins, including HPV16E6, c myc, bcl 2, p53, and Fas, were also determined using flow cytometry. RESULTS: RNA dot blot analysis demonstrated that HRz mRNA expressed stably in the CaSKi R cells. Northern blot analysis showed that the expression of E6 mRNA was much lower in the CaSKi R cells than that in the CaSKi cells, while there was no difference of E6 mRNA levels between the CaSKi cells and the CaSKi P cells. The apoptosis rate in the CaSKi R cells was much higher than that in the CaSKi cells and the CaSKi P cells. The cell cycle was arrested in G2 phase, with decrease in percentage of S phase cells. Anti HPV16E6 ribozyme can significantly reduce the expression of E6, c myc, and bcl 2 genes in the CaSKi R cells, and increase the expression of p53 compared with that in Caski cells. This phenomenon was not found in the CaSKi P cells. The expression of Fas was similar in the three kinds of cells. CONCLUSION: Ribozyme targeted on HPVE6 mRNA can induce apoptosis of human cervical cancer CaSKi cells. The reason may be the decrease of expression of E6 gene, and the successive changes of expression of some genes, including c myc, bcl 2, and p53 genes.