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目的:研究靶向人端粒酶逆转录酶(hTERT)的短发夹RNA(shRNA)对鼻咽癌细胞系(CNE)端粒酶活性及PCNA、Caspase-3蛋白表达的影响。方法:构建表达靶向hTERT mRNA特异的shRNA的质粒pEG-FP-shTERT。将pEGFP-shTERT转染CNE细胞,TRAPPCR-ELISA法检测端粒酶活性,MTT法测定细胞增殖活性,TUNEL法检测细胞凋亡,Western blotting检测蛋白表达。结果:pEGFP-shTERT成功转染入CNE细胞后,端粒酶活性显著下降;细胞增殖活性明显受抑制;大量细胞凋亡;处理后24及48h,PCNA蛋白下调至64.41%、58.67%(P<0.05),Caspase-3上调到1.55、1.60倍(P<0.05)。结论:shRNA靶向抑制hTERT基因在鼻咽癌CNE细胞系中的表达,能显著地抑制细胞的端粒酶活性,调节PCNA和Caspase-3蛋白表达,从而抑制细胞增殖,并诱导细胞凋亡。端粒酶、PCNA和Caspase-3共同参与了鼻咽癌的发生、发展过程。
Objective: To investigate the effect of short hairpin RNA (shRNA) targeting human telomerase reverse transcriptase (hTERT) on the telomerase activity and the expression of PCNA and Caspase-3 in human nasopharyngeal carcinoma cell line (CNE). METHODS: Plasmid pEG-FP-shTERT was constructed to express shRNA targeting hTERT mRNA. The pEGFP-shTERT was transfected into CNE cells. The telomerase activity was detected by TRAPPCR-ELISA. The cell proliferation was measured by MTT assay. The apoptosis was detected by TUNEL assay. The protein expression was detected by Western blotting. Results: After transfection of pEGFP-shTERT into CNE cells, the telomerase activity was significantly decreased; the cell proliferation activity was significantly inhibited; a large number of apoptotic cells were detected; PCNA protein was down-regulated to 64.41% and 58.67% at 24 and 48 h after treatment (P < 0.05), Caspase-3 up to 1.55,1.60 times (P <0.05). CONCLUSIONS: shRNA targeting inhibits the expression of hTERT gene in CNE cell lines, which can significantly inhibit the telomerase activity, regulate the expression of PCNA and Caspase-3 protein, thereby inhibiting cell proliferation and inducing apoptosis. Telomerase, PCNA and Caspase-3 are involved in the occurrence and development of nasopharyngeal carcinoma.