小干扰RNA沉默survivin基因表达对肿瘤细胞A549、Hela S3和K562的影响及小干扰RNA序列的筛选

Study on silence of survivin gene in cancer cells A549, Hela S3 and K562 by small interfering RNA and its sequence screening

目的 探讨针对survivin基因的小干扰RNA（small interfering RNA,siRNA）对人肺癌A549、宫颈癌Hela S3及急性髓系白血病K562细胞株的效应,筛选有效的siRNA序列.方法 设计并合成3条针对survivin基因的siRNA,通过Hiperfect脂质体转染试剂分别转染A549、Hela S3及K562细胞,培养48 ～ 72 h后,用SYBR Green Ⅰ的实时定量PCR （RT-PCR）方法检测肿瘤细胞survivin mRNA的表达,用WST-8细胞计数试剂盒检测肿瘤细胞的增殖.结果 转染了特异性siRNA的肿瘤细胞survivin基因的表达量明显下降,转染后48 h基因表达抑制率为57.47％～88.53％,72 h为69.94％～95.03％;转染后各组肿瘤细胞增殖受到明显抑制,转染后48 h的细胞增殖抑制率为27.88％～47.36％,72 h为42.59％～57.29％;其中序列1的效果最为显著,对3种肿瘤细胞survivin基因表达的抑制率48 h均在75％以上、72 h均在90％以上,对细胞增殖的抑制率48 h均在40％以上、72 h均达50％以上.结论 siRNA能有效沉默survivin基因在多种肿瘤细胞中的表达,抑制肿瘤细胞的增殖,所设计的3条survivin siRNA序列均有活性,以序列1作用最强.

Objective To investigate the biological response of survivin siRNA in A549 human lung cancer cells and Hela S3 human cervical cancer cells as well as K562 human erythroleukemia cells,in order to screen the working sequences of survivin siRNA.Methods Three sequences of survivin-targeted siRNA were designed and synthetized,and human cancer lines of A549,Hela S3,K562 were transfected with Hiperfect liposome entrapped survivin siRNA,respectively.The expression of survivin mRNA was detected by means of real-time polymerase chain reaction （RT-PCR） with SYBR Green Ⅰ.Cell proliferation was detected by way of WST-8 cell count kit at 48 hours and 72 hours after transfection.Results The expression of survivin mRNA in all 3 cancer cells studied was significantly inhibited by all siRNA at 48 hours and 72 hours after transfection,gene expression inhibition ratio were 57.47%-88.53% after transfection 48 hours and were 69.94%-95.03% after transfection 72 hours.Cell proliferation was also significantly inhibited 48 hours and 72 hours after transfection,cell multiplication inhibition ratio were 27.88%-47.36% after transfeotion 48 hours and were 42.59%-57.29% after transfeciton 72 hours.Sequence 1 had the most inhibition efficacy on survivin gene expression and proli-feration in tumor cells.Inhibition rate of the three tumor cell gene expression of survivin at 48 hours are above 75%,72 h all over 90%,48 hours of cell proliferation inhibition rate are above 40%,72 hours of more than 50%.Conclusions The chemo-synthesized siRNAs can significantly down-regulate survivin mRNA expression in cancer cell studies.Survivin siRNA is capable of inhibiting the proliferation of tumor cell in vitro and it might be a new strategy for tumor-targeted therapy.Sequence 1 is the most efficacious working survivin siR-NA in the study.