Zheng Yan-Fang, Zhang Ji-Ren
Oncology Center, Zhujiang Hospital, The First Military Medical University, Guangzhou, Guang dong, PR China.
Ai Zheng. 2003 May;22(5):458-62.
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.
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.
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.
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.
人乳头瘤病毒与宫颈癌相关。核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染抗HPV16E6核酶（HRz）的培养宫颈癌细胞系的特性，以及核酶对宫颈癌细胞增殖和凋亡的影响。
利用计算机设计针对HPV16E6 mRNA的核酶。采用脂质体转染法，将抗HPV16E6核酶和空真核表达质粒分别转染入CaSKi细胞，分别命名为CaSKi-R和CaSKi-P。通过Northern印迹分析检测三种细胞中E6 mRNA的表达。使用流式细胞术测定细胞周期。使用荧光（Hoechst）染色和TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）检测细胞凋亡率。还使用流式细胞术测定包括HPV16E6、c-myc、bcl-2、p53和Fas在内的某些蛋白质的表达。
RNA斑点印迹分析表明HRz mRNA在CaSKi-R细胞中稳定表达。Northern印迹分析显示，CaSKi-R细胞中E6 mRNA的表达远低于CaSKi细胞，而CaSKi细胞和CaSKi-P细胞之间E6 mRNA水平无差异。CaSKi-R细胞的凋亡率远高于CaSKi细胞和CaSKi-P细胞。细胞周期停滞在G2期，S期细胞百分比降低。与CaSKi细胞相比，抗HPV16E6核酶可显著降低CaSKi-R细胞中E6、c-myc和bcl-2基因的表达，并增加p53的表达。在CaSKi-P细胞中未发现此现象。三种细胞中Fas的表达相似。
靶向HPVE6 mRNA的核酶可诱导人宫颈癌CaSKi细胞凋亡。原因可能是E6基因表达降低，以及包括c-myc、bcl-2和p53基因在内的一些基因表达的相继变化。
