RNA干扰技术阻抑survivin基因表达对子宫颈癌细胞放射和化疗敏感性的影响

目的探讨利用RNA干扰(RNAi)技术阻抑survivin基因的表达,对宫颈癌HeLa细胞放射敏感性和化疗敏感性的影响。方法通过脂质体介导,将含survivin基因小分子干扰RNA的重组真核表达质粒pSilencer2．1-s2、阴性对照质粒pSilencer2．1-NC和空载质粒pSilencer2．1-U6 neo转染宫颈癌细胞系HeLa,获得HeLa-s2、HeLa-NC和HeLa-U6 neo细胞,同时设未转染的HeLa细胞为阴性对照。RT-PCR技术、蛋白印迹法分别检测survivin mRNA和蛋白的表达水平,并计算survivin mRNA和蛋白表达抑制率;激酶活性检测法测定波长在405 nm处的吸光度(A405)值,表示半胱氨酸天冬氨酸蛋白酶3(caspase-3)活性;流式细胞仪检测细胞凋亡率;平板克隆形成实验观察细胞的放射敏感性变化,以克隆形成率表示;四甲基偶氮唑蓝比色法检测细胞存活率并计算顺铂的50％抑制浓度(IC50)。结果与HeLa-NC、HeLa-U6 neo及未转染的HeLa细胞比较,HeLa-s2细胞survivin mRNA和蛋白表达水平明显下降,survivin mRNA和蛋白表达抑制率分别为(62．8±0．3)％和(60．1±0．5)％。HeLa-s2细胞的A405值为1．261±0．043,未转染的HeLa细胞的A405值为0．314±0．012,两者比较,差异有统计学意义(P<0．05)。HeLa-s2与未转染的HeLa细胞相比,细胞凋亡率明显升高(P<0．05),分别为(29．23±1．41)％和(2．74±0．32)％。不同照射剂量下,HeLa-s2与未转染的HeLa细胞分别比较,克隆形成率均明显降低(P<0．05)。在同一顺铂浓度下,HeLa-s2与未转染的HeLa细胞比较,细胞存活率显著降低(P<0．05),HeLa-s2细胞对顺铂的IC50值较未转染的HeLa细胞下降显著(P< 0．05),分别为(0．873±0．021)和(9．212±0．033)μg／ml。结论利用RNAi技术可阻抑HeLa细胞中survivin基因的表达,增强caspase-3活性,诱导细胞凋亡,显著提高细胞的放射敏感性和对顺铂的化疗敏感性。

Influence of survivin gene repression by RNA interference on the radiosensitivity and chemosensitivity to cisplatin of cervical cancer cell HeLa

OBJECTIVE: To observe the effect of survivin gene repression by RNA interference (RNAi) on the radiosensitivity and the chemosensitivity to cisplatin of cervical cancer cell HeLa. METHODS: The recombined eukaryotic expression plasmid pSilencer2.1-s2 containing human survivin gene small interference RNA (siRNA) was transfected into human cervical cancer cell line HeLa by using lipofectamine 2000. The expression of survivin gene mRNA and its protein was detected by semi-quantitative RT-PCR and western blot respectively. The changes of caspase-3 activity was assessed by kinase activity test. Cell apoptosis was examined by flow cytometry. The changes of cell radiosensitivity was observed by plate clone formation assay. Cell viability was examined by methyl thiazolyl tetrazolium (MTT) assay and 50% inhibitive concentration (IC(50)) of cisplatin was examined also. RESULTS: Compared with the cells which were transfected with negative control plasmid (HeLa-NC), pure plasmid (HeLa-U6 neo) and un-transfected cells (HeLa), the expression level of survivin gene mRNA and protein declined evidently in the cells transfected with pSilencer2.1-s2 plasmid (HeLa-s2), the expression inhibitory rates were (62.8 +/- 0.3)% and (60.1 +/- 0.5)%; the caspase-3 activity was enhanced and A(405) was 1.261 +/- 0.043 (P < 0.05); the apoptotic rate was increased obviously (29.23 +/- 1.41)% (P < 0.05). At the same dose of radiation, clone formation rate declined significantly (P < 0.05); at the same concentration of cisplatin, cell viability declined sharply and the IC(50) of cisplatin was (0.873 +/- 0.021) microg/ml (P < 0.05). CONCLUSION: Survivin gene repression by RNAi can enhance caspase-3 activity, induce cell apoptosis, significantly increase the radiosensitivity and chemosensitivity to cisplatin in human cervical cancer cell line HeLa.