Experimental Research On The Effects Of RhoC Gene In Hepatocellular Carcinoma Cell Feature And Its Mechanism

Rho protein is an important member of Ras superfamily, which plays a diverse role in cell biology, including cell growth, differentiation and metastasis. RhoC is an important member of Rho GTPases. Recent researches showed the overexpression of RhoC in a great number of malignant tumors, and its activation could lead to enhanced tumor metastasis through specific signal transduction pathway. The high level expression of RhoC is closely correlated to tumor metastasis. Many studies suggested RhoC as a promising index in assessing tumor malignancy, with a possibility of acting as a therapeutic target for cancer gene therapy. However, research proofs on the role of RhoC in tumors are not strong enough to support RhoC as a therapeutic target, because there are still a lot of evidences should be provided and a lot of important mechanisms should be elucidated. This research systemically studied the effects of RhoC gene silencing in hepatocellular carcinoma (HCC) develpoment and cell differentiation. Also the role that RhoC plays in biological features and drug sensitivity in HCC cells were investigated. The importance of this research was to investigate the possibility of RhoC as a therapeutic target in HCC gene therapy.Immunohistochemistry was applied to detect RhoC expression in HCC cells. Eukaryotic expression plasmid pU6mRFP was employed as RNA interference vector; specific DNA sequence was synthesized to construct pU6mRFP siRNA-RhoC and pU6mRFP siRNA-scramble as negative control. Human HCC cell line Bel7402 was transfected to detect RhoC inhibiting efficiency. Lipotransfect was conducted to transfect Bel7402 cells, and its efficiency was assessed through laser confocal microscope. Semi-quantitative RT-PCR was used to detect mRNA level, Western blot was applied to detect RhoC protein expression. G418 was used to select stable transfection clone. MTT, cell nuclear AgNORs stain, plate clone formation and soft agar clone formation test were conducted to determine cell proliferation and growth feature. FACS was applied to determine cell cycle, cell apoptosis, cell membrane and mitochondrial membrane potential. As2O3 induction test, alkaline phosphate stain and biochemical analysis were employed to determine cell differenciation level. Cell migration capacity was determined through injury healing and cell spreading test. Zymography was used to detect MMPs activity. Semi-quantitative RT-PCR was performed to determine the expression level of cell cycle proteins, apoptosis associating proteins, MMPs and VEGF. IC50 values were measured to determine the sensitivities of anticancer drugs to HCC cells. Normal hepatocellular cell line HL7702 was transfected with RhoC to test RhoC transformation capacity. The results showed as followings:1. RNA interfering vector targetting RhoC was successfully constructed, with inhibiting effeciency in transfected Bel7402 cells as high as 85%.2. The effects of RhoC gene silencing in the proliferation of HCC and its mechanism. (1)The cultured cell proliferation capacity was compared between RNAi group (Bel7402 cell transfected with pU6mRFP siRNA-RhoC), negative control group (Bel7402 cells transfected with pU6mRFP siRNA-scramble) and parental Bel7402 cells. From the fourth day on, the proliferation speed in RNAi group was notably lower than in parental Bel7402 and negative control groups (0.41±0.10 vs. 0.73±0.11 and 0.71±0.07,p<0.05). These phenomena lasted to the end of the 7 day experiment. There was not remarkable difference between the two control groups. (2)Cell cycle analysis showed that G0-G1 cell percentage in RNAi group was significantly higher than in parental Bel7402 group (73.14±5.93% vs.57.05±5.97% and 52.99±4.80%,p<0.05). G2-S cell percentage was notably lower in RNAi group than in parental Bel7402 cells (24.86±3.66% vs.38.81±3.77% and 47.00±4.80%,P<0.01).There was not significant difference between two control groups.(3)Plate clone formation test showed that there was rare clones in RNAi group under naked eye observation, and more clones were formed in control groups. Statistical analysis showed that both clone number and clone formation efficiency in RNAi group were notably lower than in control groups (20.33±5.42% vs. 70.58±10.10% and 69.83±14.77%,p<0.01), suggesting RhoC gene silencing could notably inhibit the potential of cell growth. (4)AgNORs stain test showed that average cell stain particle in RNAi group was significantly lower than in parental Bel7402 and negative control(1.23±0.35 vs. 3.47±0.93 and 3.17±0.78,p<0.01), demonstrating the reduced cell proliferation capacity. (5)Soft agar clone formation test showed that clone formation number and percentage in RNAi group were significantly higher than in the control groups(0.133±0.115% vs. 2.867±0.987% and 2.533±1.007%,p<0.01), while the differences between two control groups were not remarkable (p>0.05), showing that RhoC silencing changed the non-anchor growth capacity of tumor cells. (6)Compared with Bel7402 and negative control groups, the following growth associated gene expressions were significantly decreased: CyclinD1(0.45±0.21 vs. 1.25±0.24 and 1.12±0.15,p<0.05)and CDK4(0.55±0.08 vs. 1.18±0.32 and 1.10±0.29,p<0.05). The following genes were notably increased: P16 ( 1.07±0.23 vs. 0.36±0.12 and 0.35±0.13,p<0.01)and P21(0.42±0.12 vs. 0.17±0.06 and 0.19±0.08,p<0.05). No remarkable changes were found in the following genes: CyclinA, CyclinG1, P19 and P27.3.The effects of RhoC gene silencing on Bel7402 cell apoptosis and its mechanism.(1)The results of cell apoptosis detected through FACS showed that the apoptosis ratio in RNAi group was significantly higher than in parental Bel7402 and negative control groups (21.00±2.23% vs. 6.47±1.64% and 4.63±0.47%,p<0.01). (2) Typical DNA fragment"ladder"for cell apoptosis could be found in RNAi group in gel electrophoresis analysis, suggesting there was great amount of cell apoptosis in RNAi group. There was not such DNA fragment"ladder"be found in parental Bel7402 and negative control groups, suggesting that there was not great amount cell apoptosis in control groups. (3)Wright stain test showed that there was lot of apoptosis cells in RNAi group, showing RhoC silencing could induce tumor cell death. (4)The membrane permeability in RNAi group was remarkably higher than in parental 7402 and negative control ones(30.70±5.63% vs.13.73±1.30% and 14.97±1.74%,p<0.01). (5)In RNAi group, the mitochondrial membrane potential was notably lower than in control ones ( 35.30±2.26% vs.65.60±5.54% and 65.37±5.61% , p<0.01 ) . (6)Compared with Bel7402 and negative control groups, Bcl-2 gene expression in RNAi group was significantly lower(0.28±0.15 vs. 0.96±0.21 and 1.03±0.24,p<0.05),and Bax gene expression was notably higher(1.09±0.21 vs. 0.26±0.10 and 0.25±0.07,p<0.01).4.Effects of RhoC gene silencing on Bel7402 cell differenciation and induced-differenciation. (1) Alkaline phosphates (ALP) stain showed that the stain color in RNAi group was more than in control ones. ALP activity in RNAi group was significantly higher than in parental Bel7402 and negative control groups (65.33±11.93u/L vs. 35.67±6.03u/L and 37.33±6.11u/L,p<0.05) .γ-GT activity was notably higher than in control groups ( 12.67±3.06u/L vs. 23.33±5.03u/L and 24.33±5.13u/L,p<0.05).(2)Under As2O3 induction, ALP activity in RNAi group was significantly higher than in parental 7402 and negative controls(123.67±24.58u/L vs. 56.67±11.93u/L and 54.67±10.26u/L,p<0.05).γ-GT activity was notably lower than in control ones(6.33±1.53u/L vs. 11.67±2.52u/L and 10.67±2.08u/L,p<0.05).5.Influence of RhoC gene silencing on Bel7402 invasion and metastasis. (1)Twenty four hours after scratch, the relative cell migration in RNAi group was significantly lower than in parental Bel7402 and negative control ones(32%±6% vs. 64%±13% and 67%±11%,p<0.01). (2)After 2 hours of cell spreading, the spreading ratio in RNAi group was notably lower than in parental 7402 and negative control ones(32.67±12.01% vs. 65.67±11.93% and 63.33±13.87%,p<0.05). (3)Zymography analysis showed that gelatinase activities could be found in parental Bel7402 and negative control ones, but not in RNAi group, suggesting the less production of MMPs with the silencing of RhoC gene. (4)MMP2 expression in RNAi group was significantly lower than in parental 7402 and negative control ones(0.26±0.10 vs. 1.01±0.23 and 1.03±0.27,p<0.01). MMP9 expression could not be detected in RNAi group, but with considerable level expression in control ones.6.Influence of RhoC on Bel7402 cell drug sensitivity. The value of IC50 for cyclophosphamide in RNAi group was significantly lower than in parental 7402 and negative control ones ( 838.54±69.22μg/ml vs. 1178.72±116.73μg/ml and 1168.36±168.68μg/ml ,p<0.01).Significant decrement of IC50 for vincristine could be found in RNAi group than in control ones(9.57±2.42μg/ml vs. 47.51±12.62μg/ml and 56.96±7.56μg/ml,p<0.01).7.The transformation effects of RhoC on normal liver cell HL7702. (1) The RhoC expression level in PcDNA3-RhoCgroup(HL7702 cells transfected with PcDNA3-RhoC) was significantly higher than in HL7702 and PcDNA3(HL7702 cells transfected with PcDNA3) groups(1.11±0.23 vs. 0.35±0.07 and 0.34±0.09,p<0.01), and Western blot detection proofed this difference. (2)From the third day on of cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than in HL7702 and PcDNA3 groups(0.83±0.10 vs. 0.54±0.11 and 0.58±0.55,p<0.05). (3)The clone formation efficiency in PcDNA3-RhoC group was significantly higher than in HL7702 and PcDNA3 group(64.27±8.57% vs. 42.33±6.70% and 40.93±5.78%,p<0.01).(4) AgNORs stain test showed that the average stain particles in PcDNA3-RhoC group was notably higher than in HL7702 and PcDNA3 groups(2.77±0.40 vs. 1.07±0.23 and 1.13±0.25, P<0.01). (5)Soft agar clone formation in PcDNA3-RhoC group could be detected, but could not in the HL7702 and PcDNA3 groups. (6)The ALP stain was more intensive in PcDNA3-RhoC group than in HL7702 and PcDNA3 groups.In summary, the overexpression of RhoC in hepatocellular carcinoma cells can control tumor cell proliferation and metastasis through regulating the gene expressions involving cell growth and migration. RhoC gene silencing can inhibit proliferation and metastasis capacity of tumor cells, as well as enhancing the sensitivity of chemical therapy drugs such as cyclophosphamide and vincristine. This research lays a foundation to selecting RhoC as target molecule in tumor gene therapy both theoretically and experimentally.