抑癌基因PTEN在肝癌组织中的突变及其对肝癌细胞增殖和凋亡的调控作用

目的 探讨抑癌基因PTEN在人原发性肝癌组织中的突变及其对肝癌细胞增殖和凋亡的调控作用。方法 （1）聚合酶链反应（PCR）-单链构象多态性（SSCP）法和序列分析法检测42例人原发性肝癌组织中抑癌基因PTEN第5、8外显子的突变。（2）脂质体介导的基因转染法将野生型PTEN基因、突变型PTEN基因的真核表达载体pEGFP-wt-PTEN、pEGFP-PTEN;G129R分别转染不表达内源性PTEN蛋白的人肝癌细胞系HHCC,G418筛选稳定表达PTEN蛋白的克隆,MTT比色实验分析测定细胞的增殖能力。以未转染基因的HHCC细胞和转染空载体pEGFP-C1的HHCC细胞为对照。（3）TNF-α诱导上述细胞凋亡,流式细胞仪测定凋亡细胞比例;Western印迹法检测细胞内磷酸化Akt（Ser473）的表达。结果 （1）在4例肝癌组织中检测出PTEN基因第5外显子的异常突变条带（9．5％,4／42）。（2）转染野生型PTEN基因的HHCC细胞生长明显抑制,而转染突变型PTEN基因的HHCC细胞的增殖能力与对照组比较差异无统计学意义。（3）TNF-α诱导分别转染野生型、突变型PTEN基因、空载体的HHCC细胞和未转染基因的HHCC细胞凋亡,细胞凋亡率分别为13．8％、8．1％、4．6％、3．3％,与转染空载体的HHCC细胞比较,转染野生型PTEN基因的HHCC细胞凋亡率增高（P〈0．05）;而转染突变型PTEN基因的HHCC细胞凋亡率差异无统计学意义（P〉0．05）。Western印迹检测显示未经基因转染的HHCC细胞内源性Akt水平较低;HHCC细胞经TNF-α作用,其内源性Akt水平增高;转染野生型PTEN基因,可降低TNF-α诱导的肝癌细胞内信号分子Akt（Ser473）的磷酸化水平。结论 （1）首次发现原发性人肝癌组织中抑癌基因P1EN发生突变;（2）野生型PTEN基因可抑制肝癌细胞增殖,而突变型PTEN基因丧失对肝癌细胞增殖的调控作用;（3）野生型PTEN基因可降低TNF-α诱导的肝癌细胞内重要的信号分子Akt（Ser473）的磷酸化水平,即野生型PTEN基因通过抑制TNF-α诱导的肝癌细胞Akt磷酸化（活化）而抑制细胞增殖,促进细胞凋亡。

Mutations of tumor suppressor gene PTEN mutations in hepatocellular carcinoma and its implications in tumor proliferation and apoptosis

OBJECTIVE: To study mutations of tumor suppressor gene PTEN in human hepatocellular carcinomas and its effects on the proliferation and apoptosis of hepatocellular carcinoma cell line HHCC. METHODS: (1) PCR-SSCP and sequence analysis were used to detect the mutations of the 5th and 8th exon of PTEN in 42 cases of human primary hepatocellular carcinoma. (2) Eukaryotic expression vectors of the wild-type (pEGFP-wt-PTEN) and the mutant type (pEGFP-PTEN, G129R) of PTEN were constructed. Lipofectamine 2000 mediated gene transfection was used to transfect hepatocellular carcinoma cell line HHCC, in which the PTEN protein is not expressed. Culture medium containing G418 was used to select stable transfectants. MTT colorimetry was used to analyze the proliferation ability of selected cell lines. Naive HHCC cells and HHCC cells transfected with empty vector (pEGFP-C1) served as controls. (3) TNF-alpha was used to induce apoptosis of selected cell clones. RESULTS: (1) Point mutation involving the 5th exon of PTEN was detected in 4 of 42 primary hepatocellular carcinomas. (2) Compared with the control groups, the proliferation of hepatocellular carcinoma cells was significantly inhibited by the transfection of wild-type PTEN gene, while the transfection with mutant PTEN construct did not significantly change the proliferation. (3) The apoptosis indices of cells transfected with the wild-type and the mutant PTEN genes were 13.8% and 8.1% respectively. Compared with the control, the apoptosis index of HHCC cell transfected by the wild type PTEN was significantly lower (P < 0.05). There were no significant differences between HHCC cells transfected with mutated PTEN gene and the control (P > 0.05). The expression of internal 473-phospharylated Akt of HHCC was weak, but was enhanced when the cells treated with TNF-alpha. However, it was down regulated by the wild type PTEN. CONCLUSIONS: (1) First time report that PTEN mutations can be found in 9.5% human primary hepatocellular carcinomas. (2) The expression of the wild-type PTEN can suppress the proliferation of HHCC cells, and such suppression was lost when PTEN gene was mutated. (3) PTEN inhibition of the proliferation and the enhancement of apoptosis of hepatocellular carcinoma cells is likely related to a down-regulation of the TNF-alpha induced activation of protein kinase Akt pathway.