RhoC在肝癌细胞生长中的作用及分子机制

Effect of RhoC on hepatoceilular cardnoma cell growth and related molecular mecbanisms

目的研究RhoC在肝癌细胞生长中的作用及分子机制，为抑制肝癌细胞生长寻找分子靶点。方法构建siRNA．RhoC载体，建立RhoC基因沉默的肝癌细胞株。采用四甲基偶氮唑蓝（MTT）法检测细胞生长，硝酸银染色检测细胞增殖，平板克隆实验检测细胞克隆形成能力，流式细胞术（FACS）检测细胞周期，逆转录聚合酶链反应（RT-PCR）检测细胞周期蛋白表达。PcDNA3-RhoC转染肝细胞，建立RhoC高表达细胞株，检测RhoC基因对正常肝细胞生长的影响。结果siRNA-RhoC载体转染Bel7402细胞后，RhoC基因表达的抑制效率为82．3％。细胞培养第4天，RNAi组吸光度（A）值为0．4 ±sO．10，显著低于Bel7402细胞组（0．73±0．11，P〈0．05）和阴性对照组（0．71±0．07，P〈0．05）。此后，RNAi组的细胞增殖速度持续低于Bel7402细胞组和阴性对照组，直到第7天实验结束。RNAi组的细胞核平均银染颗粒数明显少于Bel7402细胞组和阴性对照组（分别为1．23±0．35、3．47±0．93和3．17±0．78，均P〈0．01）；Go／G，期细胞显著升高[分别为（73．14±5．93）％、（57．05±5．97）％和（52．99±4．80）％，均P〈0．05]；cyclinD1表达下降（分别为0．45±0．21、1．25±0．24和1．12±0．15，均p〈0．05）；CDK4表达下降（分别为0．55±O．08、1．18±0．32和1．10±0．29，均P〈0．05）；p16表达升高（分别为1．07±0．23、0．36±0．12和0．35±0．13，均P〈0．01）；p21表达升高（分别为0．42±0．12、0．174±0．06和0．19±0．08，均P〈0．05）。HL7702细胞转染PcDNA3-RhoC质粒后，Rhoc高表达，至转染的第3天，RhoC转染组A值为0．83±0．10，显著高于HL7702细胞组（0．54±0．11，P〈0．05）和空载体对照组（0．58±0．55，P〈0．05）。其后，RhoC转染细胞生长速度持续高于HL7702细胞组和空载体对照组，直至第7天实验结束。结论RhoC是调控肝癌细胞生长的关键分子，是抑制肝癌细胞生长的良好分子靶点。

Objective To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth. Methods siRNA- RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection. Results The inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Be17402 and negative control groups （0.41 ±0.10 vs. 0.73 ±0.11 and 0.71±0.07 respectively, P 〈 0.05 ）. AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Be17402 and negative control（ 1.23 ±0.35 vs. 3.47± 0.93 and 3.17± 0.78,P 〈 0.01 ）. Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [ （20.33±5.42）% vs. （70.58 ± 10.10）% and （69.83± 14.77）%, respectively, P 〈 0.01 ]. Cell cycle analysis by FACS showed that Go/Gl cell percentage in the RNAi group was significantly higher than that in the control group [ （73.14 ±5.93 ）% vs.（57.05± 5.97 ） % and （ 52.99±4.80 ） %, P 〈 0.05]. Compared with Be17402 and negative control groups, the expression of following growth associated genes was significantly decreased： cyclin D1 （0.45±0.21 vs. 1.25± 0.24 and 1.12 ± 0.15, respectively, P 〈 0.05 ） and CDK4 （0.55 ± 0.08 vs. 1.18 ± 0.32 and 1. I0 ±0.29, respectively, P 〈0.05） ; the following genes were notably increased： p16（ 1.07 ±0.23 vs. 0.36±0.12 and 0.35 ±0. 13, respectively, P 〈0.01）and p21 （0.42 ±0. 12 vs. 0. 17±0.06 and 0.19± 0. 08, respectively, P 〈 0.05 ）. RhoC was highly expressed in PcDNA3-RhoC transfeeted hepatoceUular cell line, From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PeDNA3 groups（0.83 ±0.10 vs. 0.54 ±O. 11 and 0.58 ±0.55, respectively, P 〈0.05 ）. Conclusions RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.