原发性肝癌survivin基因表达及靶向survivin治疗

Expression of survivin gene in hepatoceliular carcinoma and survivin gene targeting therapy

目的:研究原发性肝癌survivin基因表达及治疗靶点作用.方法:收集肝癌和癌旁组织40例.Westernblot法检测survivin蛋白表达,RT-PCR法检测survivinmRNA.原位末端标记法检测组织凋亡指数.设计合成survivin反义寡核苷酸(antisenseoligonucleotide,ASODN)转染hepG2细胞,设置空白对照组、脂质体组、正义链组和各浓度ASODN组.流式细胞术检测凋亡指数,MTT法检测化疗药物对细胞生长抑制率.建立原位荷肝癌鼠模型并分为3组:反义survivin链组、空白对照组及脂质体组.V=ab2/2计算肿瘤体积,以V治疗后/V治疗前×100%计算生长指数.结果:肝癌细胞株和34/40例肝癌组织表达survivin蛋白及mRNA,癌旁组织无survivin表达.肝内转移组(93.5%)和门静脉癌栓组(92.8%)survivin蛋白表达阳性率显著高于阴性组(55.6%)和无癌栓组(66.7%)(p<0.05)肝内转移组(1.105±0.396)和门静脉癌栓组(1.137±0.404)survivinmRNA水平显著高于阴性组(0.572±0.082)和无癌栓组(0.627±0.122)(P<0.05).Survivin阳性组织凋亡指数为1.15±0.33%,显著低于阴性组(4.50±0.83%)(P=0.00002).ASODN组survivin表达减弱,凋亡指数增高,200nmol/L组为10.50±0.76%,400nmol/L组为12.99±0.42%,600nmol/L组为22.21±1.26%,明显高于对照组(P<0.05).化疗药物对ASODN组生长抑制率增高,200nmol/L组为47.7±4.6%(5-FU)、59.7±3.5%(DDP),400nmol/L组为56.8±4.0%(5-FU)、68.2±2.1%(DDP),600nmol/L组为85.2±2.3%(5-FU)、79.9±3.7%(DDP),明显高于对照组(P<0.05).23/25裸鼠成功建立原位荷肝癌动物模型.ASODN组肿瘤生长指数为51.0±24.2%,显著低于空白对照组(70.2±23.4%)和脂质体组(168.0±28.6%)(P<0.05).ASODN组凋亡指数为21.97±2.11%,显著高于空白对照组(3.21±0.57%)和脂质体组(3.07±0.81%)(P<0.05).结论:survivin在HCC发生中有重要作用.survivinASODN转染能下调survivin表达,诱导细胞凋亡,增加HCC细胞对化疗药物敏感性.survivinASODN对荷HCC鼠有一定治疗作用.

AIM: To investigate the role of survivin in the pathogenesis of hepatocelullar carcinoma (HCC), and to evaluate the therapeutic effect of antisense oligonucleotide (AS ODN) targeting surviving gene. METHODS: Forty HCC specimens and their neighboring noncancerous tissues were obtained. Western blot were used to detect survivin protein. Semi-quantitative RT-PCR was used to detect survivin mRNA. TUNEL was employed to examine the apoptosis index (Al). Survivin AS ODN was designed and synthesized. HepG2 cells were divided into 6 groups: control group, lipofectin group, ODN group, and 200, 400 and 600 nmol/L AS ODN groups. Al was examined by flow cytometry. Rate of inhibition (IR) by chemo-therapeutic drugs (5-Fu and DDP) was determined by the colorimetric MTT cell viability/proliferation assay. Orthoto-pic implant model was developed in BALB/nu/nu nude mouse. Tumor-take nude mice were divided into 3 groups: control group, lipofectin group, and AS ODN group. The size of tumor in situ was examined and the growth index (Gl) was calculated. RESULTS: Survivin protein and mRNA were detected in 2 HCC cell lines and 34 out of 40 patients, but not in nontumor tissues. The positive rate in patients with intrahepatic metastasis was 93.5%, higher than those without intrahepatic metastasis (55.6%, P<0.05). The positive rate in the cases with portal vein invasion was 92.8%, higher than those without portal vein invasion (66.7%, P<0.05). The levels of survivin mRNA in patients with intrahepatic metastasis (1.105±0.396) or portal vein invasion (1.137±0.404) were significantly higher than those without intrahepatic metastasis (0.572±0.082) or portal vein invasion (0.627±0.122) (P<0.05). Survivin expression was significantly associated with reduced apoptosis index (1.15%±0.33% vs 4.50%±0.83%, P<0.05). Expression of survivin in AS ODN groups was significantly decreased than that in the control group. The Al of AS ODN group was significantly higher than that of other groups. The Al were 10.50±0.76%, 12.99±0.42%, and 22.21±1.26% (P<0.05)in the 200, 400, 600 nmol/L AS ODN group. The IR of chemotherapeutic drugs in AS ODN groups was higher than that of other groups. The IR of 200 nmol/L was 47.7±4.6% (5-FU), 59.7±3.5% (DDP), 400 nmol/L was 56.8±4.0% (5-FU), 68.2±2.1 % (DDP) and 600 nmol/L was 85.2±2.3% (5-FU), 79.9±3.7% (DDP) (P<0.05). Orthotopic implant model of hepatomacelluiar was developed in 23 of 25 nude mice. The Gl of AS ODN group was 50.96±24.20%, higher than that of other groups (P<0.05). The Al of AS ODN group was 21.97±2.11%, higher than those of control and lipofectin groups (P<0.05). CONCLUSION: Survivin may play an important role in HCC carcinogenesis. AS ODN targeting survivin induces apoptosis and sensitizes HCC cells to chemotherapy. In tumor-bearing nude mouse, survivin AS ODN may inhibit the growth of tumor and induce cell apoptosis.