| Objective To investigate the the effect of recombinant lentiviral mediated RNA interference (RNAi) targeting the fatty acid binding protein-5(FABP-5)gene on cell proliferation, apoptosis and invasiveness in human hepatocellular carcinoma cell line HepG2, the recombinant lentiviral vector for RNA interference on the expression of FABP-5 gene in hepatocellular carcinoma HepG2 cells, and to explore the possible underlying mechanisms and tumor formation in nude mice.Methods 1. The mRNA level of FABP-5 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in HepG2, SK-hep-1and SMMC-7721 of these there kinds of human liver cancer cells to screen experiments using cells.2. Three vectors carrying short hairpin RNAs (shRNA) targeting the FABP-5 gene (FABP5-shRNA expression vector) were constructed by RNA interference technology, then determined the interference efficiency and selected for the most effective one.3. HepG2 cells were divided into three groups:an experimemtal group, a normal control group and a negative control group. For the experimental group, human hepatocellular carcinoma HepG2 cells were transfected with the recombinant lentivirirus vector (LV-shRNA-FABP5), the negative control group was transfected with a control lentiviral vector (LV-shRNA-NC), and the normal control group did not undergo any treatment. The mRNA level of FABP-5 was analyzed by transcription polymerase chain reaction (RT-PCR), The relative detection of protein was analyzed by Western blot.4. Cell proliferation was detected by MTT assay; cell colony formation was detected by Giemsa staining; cell invasion ability was assessed using the cell invasion chamber method and the cell cycle and apoptosis were observed by flow cytometry (FCM).5. RNA interference (RNAi) lentiviral vector was used in the experiment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, the tumor volume and weight were determined 4 weeks after the cell inoculation. In vivo imaging technology was used to take images of tumor proliferation.The expression of FABP-5 was detected by RT-PCR, Western blotting and immunehistochemical staining.Results 1. The quantitive real-time polymerase chain reaction result revealed that the gene abundance of HepG2 cells expressed were most suitable for knocking subtraction verification.2. FABP5-shRNA expression vectors were transfected into human hepatocellular carcinoma cell line HepG2, and fluorescence analysis indicated that> 90% of cells showed fluorescence signal.3. Compared with the normal control group and negative control group, FABP-5 mRNA and protein expression was significantly down-regulated in cells transfected with the three shRNA carrying vectors, and were all above 50% than the other two groups. Among them with LV-shRNA-FABP5(1) having the highest efficiency (P<0.05), reduction of 88.28%, as an experimental group.4. Detected by MTT assay, the result suggested that the LV-shRNA-FABP5 group of cells at 490nm absorption luminosity values in 1,2,3,4 and 5 day after transfected was lower than the the normal control group (P<0.05) Giemsa staining analysis showed that cell colony formation of the LV-shRNA-FABP5 group (12±2), significantly lower than the normal control group (149±10) and the negative control group (146±11) (P<0.05). cell invasion chamber method showed that the LV-shRNA-FABP5 group of cell invasion ability rate of LV-shRNA-FABP5 group (0.87%±0.11%) was significantly reduced than the normal control group (3.45%±0.12%) and the negative control group(3.37%±0.06%) (P<0.05). Flow cytometry (FCM) analysis showed that the percentage of the LV-shRNA-FABP5 group cells in G1 phase reduced than the normal control group and negative control group (P<0.05), and the th percentage of cells in S and G2/M phase significantly increased in cells transfected with FABP5-shRNA (P< 0.05). The apoptosis rate of the LV-shRNA-FABP5 group was (26.88%±0.11%), significantly higher than the negative control group (2.13%±0.36%) (P<0.05).5. Transfection of the retroviral vector FABP5-shRNA obviously reduced FABP-5 gene expression in the HepG2 cells.Tumor formation was all positive in the 3 groups of nude mice inoculated of the cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller in the mean volume and the mean weight (P<0.05). The mean volume of normal control group, negative control group and LV-shRNA-FABP5 group was(100.51±47.45)mm3, (104.02±52.89)mm3 and (29.41±8.98)mm3; the mean weight was(0.632±0.203)g, (0.629±0.226)g and (0.204±0.067)g. FABP-5 expression in transplanted tumor tissues was significantly down-regulated at mRNA and protein level in tumor tissues in experimental group as compared with normal control group and negative control group (P<0.05).Conclusion 1. RNAi-induced down-regulation of FABP-5 can significantly down-regulated at mRNA and protein level in HepG2 cells.2. The high expression of FABP-5 gene could be silenced by RNAi, and RNAi-induced FABP-5 knockdown could effectively inhibit the proliferation and invasion of of HepG2 cells, block the cell cycle in G2/M phase, and significantly increase cell apoptosis.3. RNAi-induced down-regulation of FABP-5 effectively inhibits the growth of transplanted hepatocellular carcinoma.4. It suggesting that FABP-5 gene may be an effective target for gene therapy in treating liver cancer. |
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