RNA Interference Mediated By Lentiviral Vector Suppresses Expression Of Connective Tissue Growth Factor (CTGF) In Hepatic Stellate Cells And Its Role In Anti-fibrosis

Chronic liver damage which was caused by various factors, such as viruses, alcohol, drug liver toxicity, etc, will lead to the liver fibrosis and probably liver cirrhosis. Currently, there are no effective methods to cure them. In China, there are a large number of persons who suffering chronic liver damage, therefore, it is an emergent problem to find effective anti-fibrosis ways and to prevent the diseases developments.Connective tissue growth factor (CTGF) is the main cytokine promoting liver fibrosis, and plays an important role in the pathogenesis of liver fibrosis. CTGF mainly mediates transforming growth factor-β(TGF-β) to activate hepatic stellate cells (HSC), leading to the accumulation of extracellular matrix (ECM), which induces liver fibrosis. The activation of HSC is the key step in the course of development of liver fibrosis, and the high expression of CTGF is a necessary condition of the activation of HSC. Therefore, to inhibit the expression of CTGF in HSC may prevent the activation of HSC, which may further prevent or block the process of liver fibrosis.RNA interference (RNAi) is the sequence-specific post-transcriptional gene silencing in biology. Lentiviral vector is one of viral vectors which are commonly used to mediate RNAi. It can efficiently infect both the cells with split or non-split growth, and does not induce the immune response from the host. It is a powerful tool for gene silencing with a longer expression duration.In this study, we select HSC as the target cell to find the mechanism of HSC activation using RNA interference mediated by lentiviral vector to silence CTGF. The main object is to demonstrate the pathological mechanism of liver fibrosis and to provide benefit support for prevention and treatment of liver fibrosis.1. Construction and identification of the lentiviral vector transfer plasmid mediating RNA interference of target gene.According to the rat CTGF (NM₀22266) gene sequence in GenBank, we designed the four siRNA and one negative control sequence. The synthesized sequences were formed into double - stranded DNA by annealing, and then cloned into the lentiviral vector transferred plasmid pGCL-GFP after double-directional enzyme digestion. The plasmids were extracted after transformation. The sequences specificity were confirmed by PCR identification and DNA sequencing and it is indicated that five lentiviral vector transfer plasmids to CTGF gene (including one negative control) have been successfully constructed.2. Packaging and screening of RNAi lentivirusThe successfully constructed transfer plasmid, together with the two auxiliary packaging vector plasmids were transfected into 293T cells, packaging into the four lentiviral particle expressing CTGFsiRNA and one negative control. The five recombinants were infected HSC-T6 cells to screen the most effective suppression recombinant by reverse transcript-ploymerase chain reaction (RT-PCR) and Western Blot. The inhibition efficiency of the most effective recombinant was 84.6%. This recombinant was named as Len/siRNA-CTGF and used in the following study.3. CTGF gene silence mediated by lentiviral vector and its effect on downstream geneHSC-T6 is the activated hepatic stellate cells, which characterized as the high level expression of the type I collagen (COL I), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. The influence of CTGF on the activation of HSC can be analyzed by observating the change of COL I, TIMP-1 and TIMP-2 mRNA expression before and after CTGF gene silencing.Len/siRNA-CTGF and the negative control virus were used to infect HSC-T6 cells respectively. Normal HSC-T6 cells without the virus infection served as negative control. Ninety-six hours after infection, TIMP - 1, TIMP-2, and COL I mRNAs level were analyzed by RT-PCR to detect the inhibition effect of CTGF gene silence by Len/siRNA-CTGF. The expression of CTGF was significantly inhibited by Len/siRNA-CTGF in HSC-T6 cells. The inhibition efficiency is about 70%. At the same time, the expressions of TIMP-1, TIMP-2, and COL I were dropped to 23%, 18% and 50% respectively, compared with the negative control.4.The relationship between CTGF gene silence and the proliferation and activation of HSC-T6The change of HSC-T6 DNA was analyzed before and after CTGF gene silence using propidium iodium (PI) staining flow cytometry DNA content analysis (PI / FCM) , The growth curve of the cells were analyzed by MTT. The proliferation and activation of HSC-T6 were inhibited after CTGF gene silence. Conclusion:We constructed and screened successfully the lentiviral vector mediating CTGF silencing.It was demonstrated that after it silences CTGF gene it also inhibits the expression of TIMP-1, TIMP-2 and COL I in HSC-T6.The proliferation and growth of HSC-T6 is also inhibited,which may contribute to prevent the occurrence of liver fibrosis.