抑制端粒酶逆转录酶基因表达对C-myc蛋白表达的影响

Influence of RNA interference targeting against human telomerase reverse transcriptase on expression of C-myc protein

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。

Objective：To investigate the effect of inhibiting human telomerase reverse transcriptase （hTERT） on expression of C-myc protein by RNA interference （RNAi） in the larynx cancer cell line, Hep-2. Method：The primary structures of hTERT cDNA were found in GeneBank, Then the structure analyses were done according to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid, One type of plasmid, pshRNA1, involved in fluorescein gene was synthesized based on the specific base sequence, Control pshRNA2-a random sequence-were also constructed. METAFECTENE was used as the transfect ion reagent. Cells were treated daily with pshRNA1-2 or normal culture medium respectively. After administration of pshRNA1-2, hTERT mRNA was detected by RT-PCR,hTERT protein and C-myc protein were examined by Western Blot, Resuit： The expression of hTERT mRNA and protein were both significantly decreased after treated by pshRNA1 （ P d0, 05）, The expression of C-myc protein was significantly increased after treated by pshRNA1 （ P〈0.01 ）. Conclusion：The inhibition of hTERT expression could increase the expression of C-myc protein in Hep-2 cells.