Study On The Role Of RhoA ShRNA In Biological Behaviour Of Primary Hepatocellular Carcinoma

Background and PurposePrimary hepatocellular carcinoma (PHC) is a serious risk to human health and malignant diseases. The mortality rate ranked second among all types of cancer. The high mortality rate of PHC mainly cause by the proliferation of malignant characteristic and prone to early transfer. Proliferation and apoptosis imbalance is the core areas, invasion and metastasis is the essential characteristic of Hepatoma cell. The invasion and metastasis of hepatoma cell first come from the migration of individual cells including deformation, adhesion, extracellular matrix degradation, activation of multiple genes, transcription, regulation of complex biological processes. The current study found that the transitional process of cancer was closely related to changes in the cytoskeleton. In recent years, people have found a new class of cytokines Rho in the regulation of cytoskeleton plays an important role. RhoA is a symbolic factor with the most extensive study of the Rho family. Depth study found that it over expressed in lots of cancer in human, suggesting it is playing an important role in tumor formation. The subjects was to be starting from the biological characteristics of liver cancer, to explore the role of RhoA in proliferation, invasion and metastasis of PHC in tissue and cell levels, in order to provide an experimental basis for further study on the molecular mechanisms of the intrinsic biological behavior of PHC.MethodsFirst collected 30 cases of PHC surgical resection specimens of Tongji Hospital, then detected RhoA mRNA and protein expression levels with RT-PCR and Western- blot method cancer. Also we analyzed the expression levels of RhoA in cancer tissue with the clinical and pathological features (sex, tumor diameter, cell differentiation, the number of nodules. hepatic vein any foreign invasion and lymph node metastases) to explore the role of RhoA gene in proliferating, invasion and metastasis of PHC. In vitro, first the RhoA mRNA and protein expression levels detected with RT-PCR and Western-Blot in hepatocellular carcinoma cell lines (HepG2, SMMC7721, HepG2.2.15 and Hep3B) and normal liver cell line L02; Then the vector which specific inhibited the expression of RhoA and corresponding control vector were constructed with Pgenesil-2 plasmid; Screening the stable hepatoma cell line in which with the low expression of RhoA after the transfection of RhoA shRNA plasmid vector to HepG2 cell line through liposome. The invasion and athletic ability of these cell lines was observed using transwell; and the cell proliferation measured by flow cytometry after the cell lines marked with Carboxyl Fluorescein Acetoacetoxy Esters (CFSE).Results1.The expression of RhoA mRNA and protein were both had detected in PHC cancer and adjacent tissues.RhoA mRNA and protein expression levels were higher in PHC cancer tissue than adjacent tissue, and the absorbance (A) ratios were 1.45±0.66 and 1.18±0.53; 35.74±2.38 and 16.50±1.53. Significant differences of this were statistically significant (P<0.05). 2. In PHC cancer tissue, the mRNA and protein expression levels of RhoA were related to cell differentiation, the number of nodules, any vein and lymph node invasion and metastases beyond of hepatic (P>0.05), but not gender and the size of tumor (P<0.05).3. In hepatoma cell lines (HepG2, SMMC7721, HepG2.2.15 and Hep3B), the mRNA and protein expression levels of RhoA were significantly higher than the normal liver cell line L02 (P<0.05).4. RhoA shRNA plasmid vector (pshRNA-RhoA1 and pshRNA-RhoA2)and control plasmid vector (pshRNA-HK)constructed successfully. After they were transfected to HepG2, shRNA plasmid vector(pshRNA-RhoA2) which could specifically inhibit the expression of RhoA was selected out.5. After pshRNA-RhoA2 and pshRNA-HK was transfected to these cells whom were cultured selectively with G418, the HepG2 cell line (pshRNARhoA-HepG2)which can express stably RhoA gene with low level and control cell line (pshRNAHK- HepG2)were established successfully.6. In the invasion and athletic ability test, the number of cells trans-membrane in each group were: pshRNARhoA-HepG2 group (34.1±3.2/25.2±2.1), pshRNAHK- HepG2 group (62.0±2.8/39.5±1.6) and blank cells HepG2 group (65.3±3.4/41.7±2.9) .The results showed that when compared with HepG2, the invasion and athletic ability was significantly decreased in pshRNARhoA-HepG2 (P<0.05); but it was not change significantly in pshRNAHK- HepG2 (P>0.05). This suggested that the invasion and athletic ability of cells were significantly lower after the expression level of RhoA decreased in HepG2.7. The changes in proliferation ability of the cells: After pshRNARhoA-HepG2, pshRNAHK- HepG2 and HepG2 cells were labeled with CFSE, flow cytometry testing results showed that the proliferation coefficient of pshRNARhoA-HepG2, pshRNAHK- HepG2 and HepG2 was 40.33, 61.26 and 66.15. These indicated that when compared with HepG2, the proliferation rate was significantly reduced in pshRNARhoA-HepG2 (P<0.05), but it was not change significantly in pshRNAHK- HepG2 (P>0.05). It was noted that the proliferation ability of cells was significantly inhibited after the expression level of RhoA decreased in HepG2.ConclusionThe expression level of RhoA gene was excessive both in hepatocellular carcinoma tissue and in hepatoma cell lines, and related to clinicopathological features which reflected the invasion and metastatic degree of liver cancer. When the expression of RhoA gene inhibited in hepatoma cell line, the ability of cell proliferation, invasion and campaign all reduced. This partly reversed the malignant phenotype of hepatoma cell and provided experimental basis for the further study on mechanisms of invasion and metastasis and proliferation of liver cancer. In addition, RhoA gene can be used as a new indicator of invasion and metastasis in PHC and also expected to become the new target for the treatment of liver cancer.