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目的:本课题利用RNA干扰(RNAi)技术,研究短发夹RNA(siRNA)抑制人端粒酶逆转录酶(hTERT)基因表达对鼻咽癌细胞生长的抑制作用。方法:根据hTERT cDNA序列构建表达hTERT mRNA的特异的、含荧光素基因的真核表达载体,包装成慢病毒。实时定量PCR及Western blot分析染细胞hTERT mR-NA及蛋白表达水平。MTT实验检测转染细胞的体外增殖速度。流式细胞仪检测细胞的生长周期。体外细胞侵袭实验检测比较各组的侵袭能力。结果:RT-PCR和Western blot检测显示,hTERT siRNA能显著下调hTERT mRNA和蛋白水平,尤其hTERT siRNA1,siRNA处理后的肿瘤细胞增殖受到抑制,细胞迁移、侵袭能力也显著降低。siRNA组和空病毒组(NC组)分别与空白细胞组(control组)之间比较,siRNA组与control组瘤细胞生长差异有统计学意义(P<0.01),NC组与control组之间瘤细胞生长差异无统计学意义(P>0.05)。结论:慢病毒载体沉默hTERT基因可以显著抑制鼻咽癌细胞的生长;hTERT siRNA可有效抑制鼻咽癌细胞的增殖、迁移和侵袭;提示hTERT可能成为抑制鼻咽癌细胞增殖和转移的新的分子靶点。
OBJECTIVE: To study the inhibitory effect of short hairpin RNA (siRNA) on the growth of nasopharyngeal carcinoma cells by inhibiting the expression of human telomerase reverse transcriptase (hTERT) gene by RNA interference (RNAi) technique. Methods: According to the hTERT cDNA sequence, a specific eukaryotic expression vector expressing hTERT mRNA was constructed and packaged into lentivirus. Real-time quantitative PCR and Western blot analysis of hTERT mR-NA staining and protein expression levels. MTT assay was used to detect the in vitro proliferation rate of transfected cells. Flow cytometry to detect cell growth cycle. In vitro cell invasion assay tests the invasion ability of each group. Results: The results of RT-PCR and Western blot showed that hTERT siRNA significantly downregulated hTERT mRNA and protein levels. In particular, hTERT siRNA1 inhibited the proliferation of tumor cells and decreased the ability of cell migration and invasion. The difference between siRNA group and control group was statistically significant (P <0.01). There were significant differences between siRNA group and control group (NC group) and control group The difference of cell growth was not statistically significant (P> 0.05). CONCLUSION: Lentiviral vector silencing hTERT gene can significantly inhibit the growth of nasopharyngeal carcinoma cells; hTERT siRNA can effectively inhibit the proliferation, migration and invasion of nasopharyngeal carcinoma cells; hTERT may be a new molecule that inhibits the proliferation and metastasis of nasopharyngeal carcinoma cells Targets.