人工microRNA抑制PAR4表达载体的构建和应用

Construction and application of an artificial microRNA expression vector for inhibiting PAR4

目的:构建靶向PAR4的人工microRNA表达载体并用于抑制人肠癌细胞SW620中PAR4表达。方法:设计针对人PAR4的人工microRNA序列,利用2次PCR扩增获得目的片段,克隆于pMD-19T载体。经DNA测序证实后,将8个串联连接的PAR4人工microRNA序列亚克隆于真核表达载体pcDNA3.1(+),构建成pcDNA3.1(+)-8xPAR4-microR-NA真核表达载体。用脂质体将表达载体转染SW620细胞,经G418筛选出稳定转染细胞系,Western blot检测PAR4的表达水平。结果:DNA测序结果表明,克隆的针对人PAR4的人工microRNA序列正确。Western blot检测结果显示,稳定转染pcDNA3.1(+)-8xPAR4-microRNA载体的SW620中PAR4的表达较对照组明显受到抑制。结论:成功地构建了靶向PAR4的人工microRNA的表达载体,并能明显抑制靶基因的表达,为进一步研究PAR4的功能及探讨以PAR4为靶点的基因治疗打下了基础。

AIM： To construct artificial microRNA expression vector targeting PAR4 and suppress the expression of PAR4 in human colorectal cancer SW620 cells with the artificial microRNA.METHODS： Artificial microRNA was designed and amplified by two rounds of PCR and cloned into pMD-19T vector.The sequence of the cloned artificial microRNA was verified by DNA sequencing.Eight tandemly-repeated artificial microRNAs were subcloned into mammalian expression vector pcDNA3.1（＋） to make the artificial microRNA-expressing vector pcDNA3.1（＋）-8xPAR4-microRNA.The vector was transfected into human colorectal cancer SW620 cells,and stable transfectants were selected by G418.The expression of PAR4 was examined by Western blot.RESULTS： DNA sequencing showed that the sequence of the cloned artificial microRNA targeting PAR4 was correct.Western blot result showed that the expression of PAR4 in SW620 cells stably transfected with pcDNA3.1（＋）-8xPAR4-microRNA was markedly downregulated when compared to SW620 parental cells.CONCLUSION： Artificial microRNA expression vector targeting PAR4 is successfully constructed with significant suppression effect on PAR4 expression in SW620 cells.This provides the basis for future studies on the function of PAR4 and potential cancer gene therapy targeting PAR4.