microRNA抑制鼻咽癌hTERT基因表达的实验研究

Inhibition of expression of hTERT gene in nasopharyngeal carcinoma cells by microRNA

目的观察microRNA对鼻咽癌CNE-2细胞hTERT基因表达的调控效应,探讨microRNA在鼻咽癌基因治疗的应用前景。方法构建靶向hTERT基因的microRNA表达质粒,脂质体法转染鼻咽癌CNE-2细胞,RT-PCR检测转染细胞中hTERT基因的mRNA水平,Western Blotting观察microRNA对hTERT蛋白表达水平的调控效应,CCK-8比色法检测microRNA对细胞生长的抑制作用,流式细胞学检测miRNA诱导细胞凋亡的作用。结果在mcroiRNA表达质粒转染CNE-2细胞后,hTERT的mRNA表达水平下调,蛋白表达水平降低,细胞周期在G0～G1期受阻,并诱导细胞凋亡。结论microRNA可在鼻咽癌中有效下调hTERT基因的表达水平,抑制鼻咽癌细胞增殖并诱导其凋亡,为鼻咽癌基因治疗研究奠定了一定的基础。

Objective To explore the regulatory effect of microRNA on expression of hTERT gene in nasopharyngeal carcinoma, and to discuss the future application of microRNA to the gene therapy of nasopharyngeal carcinoma. Methods The recombination plasmid vectors which target hTERT gene were constructed and the plasmids were transfected into CNE-2 cells using Lipofectamine2000 reagent. The expression of hTERT mRNA and hTERT protein was detected with reverse transcriptase polymerase chain reaction （ RT-PCR ） and Western Blot respectively. CCK-8 assay was used for determining the inhibitory effect of microRNA on cell growth and flow cytometry was used for observing the apoptosis induced by microRNA. Results After the transfection of CNE-2 cells, the expression of hTERT mRNA and hTERT protein was down- regulated at a different degree. Meanwhile, the cell cycle was suffocated at G 0 - G 1 stage and the cell apoptosis was induced. Conclusion MicroRNA can effectively interfere nasopharyngeal carcinoma cells by down-regulating the expression of the hTERT mRNA and its protein, therefore can inhibit the growth of the cells and induce their apoptosis. Future application of microRNA to the gene therapy of nasopharyngeal carcinoma might be expected.