抗HPV16E6核酶对宫颈癌细胞端粒酶活性的影响及其机制

Effects of anti-HPV16E6-ribozyme on telomerase activity in cervical carcinoma cells and the related mechanisms

目的:研究抗HPV16E6核酶(Ribozyme)对宫颈癌CaSKi细胞端粒酶活性的抑制及其机制。方法:以脂质体法将抗HPV16E6-Ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R、CaSKi-P细胞。点杂交检测核酶在细胞中的表达,Western blotting法检测3种细胞HPV16E6蛋白的表达,用TRAP-ELISA法检测端粒酶活性,用RT-PCR法检测P53、c-myc、hTERT和hRT的表达。结果:点杂交证实核酶能在CaSKi-R细胞中稳定表达,Western blotting证实CaSKi-R中表达E6蛋白较CaSKi-P、CaSKi明显降低。CaSKi、CaSKi-P、CaSKi-R3种细胞的端粒酶活性值分别为(0.89±0.14)、(0.90±0.11)、(0.36±0.06),转染了抗HPV16E6核酶的CaSKi-R细胞端粒酶活性的抑制率为59.55%,与CaSKi、CaSKi-P细胞比较明显下降(P<0.01)。与CaSKi和CaSKi-P细胞相比,CaSKi-R细胞表达c-myc、hTERT mRNA明显减少,P53、hRT mRNA表达无明显变化。结论:抗HPV16E6核酶明显抑制CaSKi-R细胞端粒酶活性,其机制可能与HPV16的E6蛋白及c-myc、hTERT mRNA减少有关。

Objective： To investigate the effects of HPV16E6-ribozyme on telomerase activity in cervical carcinoma cell line CaSKi and the related mechanisms. Methods： Anti-HPV16E6-ribozyme and blank eucaryotic plasmids were transfected into CaSKi cells via lipofectin, and the resultant cells were named as CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blotting. The expression of E6 mRNA and protein in the 3 kinds of cells were detected by Northern blotting and Western blotting, respectively. Telomerase activity was determined by TRAP-Elisa method; the expression of P53, c-myc, hTERT and hRT mRNA were examined by RT-PCR. Results： RNA dot blotting showed that anti-HPV16E6-ribozyme was stably expressed in transfected CaSKi-R cells. Western blotting showed that the expression of E6 mRNA and protein in CaSKi-R cells was obviously lower than that in CaSKi and CaSKi- P cells. The telomerase activities in CaSKi,CaSKi-P and CaSKi-R cells were （0.89± 0.14）, （0.90 ±0.11 ） and（0.36 ±0.06） ,respectively. The inhibitory rate of telomerase activity in CaSKi-R cells was 59.55%, which was significantly lower than those in CaSKi and CaSKi-P cells （P 〈0. 01 ）, The expression of c-myc and hTERT mRNA in CaSKi-R cells was lower than that in CaSKi and CaSKi-P cells ; the expression of P53 and hRT mRNA remained unchanged. Conclusion ： Anti-HPVE6-rivozyme can obviously inhibit the telomerase activity in CaSKi-R cells, which might be associated with de- cline of HPV16 E6 protein and c-myc, hTERT mRNA expression in CaSKi-R cells.