靶向hTERT的特异性siRNA抑制SMMC-7721肝癌细胞增殖机制研究

Mechanism on inhibiting SMMC-7721 hepatoma carcinoma cell proliferation by specific siRNA targeting human telomerase reverse transcriptase

目的筛选有效抑制肝癌细胞hTERT表达的小干扰RNA(siRNA)序列,并检测其对hTERT蛋白表达、细胞凋亡的影响。方法设计并合成针对hTERT的6条siRNA双链及一条阴性对照siRNA,以LipofectaminTM2000转染至肝癌SMMC-7721细胞。用磺基罗丹明B法(sulforhodamine B法,SRB法)测定干扰后细胞增殖的变化,RT-PCR测定hTERTmRNA的表达,筛选出有效序列。有效序列转染后,进一步采用Western blot法检测hTERT蛋白水平的变化,AnnexinV-FITC/PI双染、流式细胞术检测细胞凋亡情况。结果以SRB和RT-PCR法筛选出的两条有效siRNA:siRNA-2和siRNA-6,能明显抑制肝癌SMMC-7721细胞增殖,对细胞的抑制率分别为45.3%、38.2%,能高度沉默hTERT mRNA的基因,沉默率分别为80%、75%。二者还能明显下调hTERT蛋白表达(P<0.05),明显增加凋亡,凋亡率分别为20.23%、17.94%。结论 siRNA-2和siRNA-6能特异性沉默肝癌SMMC-7721细胞hTERT基因表达,抑制增殖,降低hTERT蛋白水平,诱导凋亡,为肝癌基因治疗提供了有力的实验依据。

Aim To design and identify potent siRNA sequences against hTERT in hepatoma carcinoma cells,and to test influences of the most potent siRNA on hTERT protein expression and cell apoptosis in vitro.Methods Six hTERT-specific siRNA duplexes were designed and synthesized,and transfected into Hepatoma SMMC-7721 cells with Lipofectamin^TM2 000.In order to identify the most potent sequences against hTERT,sulforhodamine B and RT-PCR methods were adopted to determine cell proliferation and hTERT mRNA level,respectively.Furthermore,cell apoptosis induced by the potent sequences was detected with Annexin V-FITC/PI assay by flow cytometry,and the protein expression level of hTERT was determined by Western blot.Results Two most potent siRNA sequences,siRNA-2 and siRNA-6,were identified based on the SRB and RT-PCR results.In hepatoma cells,both siRNA-2 and siRNA-6 significantly inhibited cell proliferation by 45.3% and 38.2%,respectively,and downregulated hepatoma hTERT mRNA expression by 80% and 75%,respectively.Moreover,either siRNA-2 or siRNA-6 significantly induced cell apoptosis with an apoptotic rate of 20.23% or 17.94%,respectively,and a reduced hTERT protein expression level（P〈0.05）.Conclusion Newly identified siRNA sequences can inhibit the proliferation of hepatoma SMMC-7721 cells,induce cell apoptosis and reduce the expression of hTERT gene,which provides a strong experimental evidence for the gene therapy of liver cancer by targeting hTERT.