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目的:探讨应用RNA干扰技术抑制Hep2细胞人端粒酶逆转录酶(hTERT)基因对Cmyc蛋白表达的影响。方法:根据hTERTcDNA序列构建表达hTERTmRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1,随机选取一段与人类基因无同源性的碱基序列构建表达shRNA的含荧光素基因的对照质粒pshRNA2,采用METAFECTENE作为转染试剂。分别以质粒pshRNA1、pshRNA2及空白培养液处理喉癌Hep2细胞。应用RTPCR法检测hTERT基因的表达,WesternBlot法检测hTERT和Cmyc蛋白表达水平。结果:质粒pshRNA1转染组hTERTmRNA及蛋白表达水平显著低于其他处理组及空白对照组(P<0.05);质粒pshRNA1转染组Cmyc蛋白表达水平显著高于其他处理组及空白对照组(P<0.01)。结论:RNA干扰抑制人端粒酶逆转录酶活性使喉癌Hep2细胞Cmyc蛋白表达升高,其确切的机制有待进一步研究。
AIM: To investigate the effect of RNA interference on the expression of Cmyc in human Hep2 cells transfected with human telomerase reverse transcriptase (hTERT) gene. Methods: According to the hTERT cDNA sequence, a shRNA expression plasmid pshRNA1 expressing hTERTmRNA specific shRNA containing fluorescein gene was constructed and a fragment of pshRNA2 containing fluorescein gene was constructed by randomly selecting a base sequence with no homology to human gene. METAFECTENE was used as a transfection reagent. The laryngeal carcinoma Hep2 cells were treated with plasmids pshRNA1, pshRNA2 and blank medium respectively. The expression of hTERT gene was detected by RTPCR method. The expression of hTERT and Cmyc protein was detected by Western blot. Results: The expression of hTERT mRNA and protein in plasmid pshRNA1 transfected group was significantly lower than that in other groups (P <0.05). The expression of Cmyc protein in plasmid pshRNA1 transfected group was significantly higher than that in other groups (P < 0.01). CONCLUSION: RNA interference inhibits the activity of human telomerase reverse transcriptase and increases the expression of Cmyc protein in Hep2 laryngeal carcinoma cells. The exact mechanism remains to be further studied.