Stathmin对肝癌细胞HCCLM3增殖及其肿瘤相关基因表达的影响

The effects of stathmin on cell proliferation and tumor-related genes expressions in HCCLM3 cells

目的探讨人stathmin基因在肝癌发生、发展过程中的作用及其机制。方法化学合成stathmin序列特异性小干扰RNA，用脂质体Lipofectamine^TM2000转染人肝癌细胞株HCCLM3细胞。采用RT-PCR和Western blot技术检测stathmin的RNA干扰效率；用细胞计数试剂检测干扰细胞的生长增殖情况；用膜联蛋白V／碘化丙啶双标记流式细胞术检测细胞凋亡；并用荧光定量PCR检测肿瘤增殖凋亡的相关基因。均数两两比较用配对t检验，多样本均数比较采用单因素方差分析。结果RNA干扰HCCLM3细胞后，stathmin的表达被显著抑制（P〈0．05），抑制率达90％；在RNA干扰24、48h和72h时，HCCLM3细胞的增殖抑制率分别为13．04％±0．10％、28．10％±0．41％和37．36％±2．15％（F=4．21，P〈0．05）；RNA干扰后，细胞凋亡比例从9．20％±0．64％上升至25．11％±1．62％（F=44．67，P〈0．01）；且RNA干扰组细胞癌基因c-myc、c-fos和增殖相关基因ki-67的mRNA表达水平明显下降，促凋亡基因caspase-3、bax和p53的mRNA表达水平升高（P值均〈0．05）。结论stathmin可能通过调节肿瘤增殖相关基因的表达水平来促进细胞增殖、抑制细胞凋亡，参与肝癌的发生和发展进程。

Objective To explore the biological function and possible underlying mechanism of stathmin gene during hepatocarcinogenesis. Method Three pairs of chemically synthesized small interfering RNA （siRNA） targeting on stathmin were tmnsfected into HCCLM3 by Lipofectamine^TM 2000. After cortfirming the interfering effects of stathmin siRNAs through reverse transcription PCR and Western blotting, the HCCLM3 cells proliferation and apoptosis were detected by cell count kit-8 （CCK-8） and flow cytometry analysis, and the expressions of rumor-related genes （c-myc, c-fos, p53, etc） were observed by real-time PCR. Results Stathmin expression was effectively inhibited up to 90% by stathmin silencing in HCCLM3 cells （P 〈 0.05）. By using CCK8 assay, it was shown that HCCLM3 cells proliferation were obviously depressed by 13.04% ± 0.10%, 28.10% ±0.41% and 37.36% ±2.15% at the time point of 24 h, 48 h and 72 h with the comparison to Mock group （F = 4.21, P 〈 0.05）. The results of flow cytometry demonstrated that the percentage of apoptotic cells was increased to 25.11% ±1.62% in RNAi group, compared with 9.20 % ± 0.64 % in Mock group （F= 44.67, P 〈 0.01）. The results of real-time PCR showed that oncogenes c-myc and c-fos expressions were repressed, proliferation-associated gene ki-67 was down-regulated, and apoptosis-promoting gene caspase-3, bax and p53 were induced （P 〈 0.05）. Conclusions Stathmin may promote cell proliferation, inhibit cell apoptosis and induce malignant transformation of hepatocytes by regulating some tumor-related genes expressions.