title: [Effect of RhoC on hepatocellular carcinoma cell growth and related molecular mechanisms].
abstract: 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 Bel7402 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 Bel7402 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 G(0)/G(1) 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 Bel7402 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 1.10 ± 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 transfected hepatocellular 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 PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 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.
authors.affiliation: Department of General Surgery, the First Hospital of Jilin University, Changchun 130021, China. xieshuli6310@hotmail.com
authors.forename: Shu-li
authors.initials: SL
authors.lastname: Xie
authors.forename: Ming-guang
authors.initials: MG
authors.lastname: Zhu
authors.forename: Guo-yue
authors.initials: GY
authors.lastname: Lü
authors.forename: Guang-yi
authors.initials: GY
authors.lastname: Wang
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
chemical_list: C530104:CCND1 protein, human; C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D019938:Cyclin D1; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D000075024:rhoC GTP-Binding Protein