Construction Of The RNA Interference Vector Of Tissue Inhibitor Of Matrix Metalloproteinase-1Gene And Its Inhibitory Effects

Liver fibrosis is a common pathological change of chronic liver diseases. Itsessence is the increased synthesis and decreased degradation of extracellular matrix(ECM), which would cause excessive deposition of ECM in the liver. Matrixmetalloproteinases (MMPs) plays a major role in the degradation of ECM, which can beinhibited by the tissue inhibitor, metalloproteinase (TIMPs).TIMP-1as one of the TIMPsfamily is closely related to liver fibrosis formation. Researches show that liver fibrosis isreversible, and the whole hepatic fibrosis process can be prevented by inhibitin of collagenover-expression through enhancing the activities of MMPs and inhibiting the effects ofTIMPs, especially TIMP-1. Based on this theory we hypothesized that liver fibrosis couldbe reversed by RNAi of TIMP-1gene and removing its inhibition effects on MMPs. The study presented have build a lentiviral vector for TIMP-1gene silencing firstly, and thenverify the effects of the RNAi in hepatic stellate cells-T6（HSC-T6）in vitro. It mayprovide a viable strategy target for furthen research and contribute to new therapauticmethod of liver fibrosis.Main contents and methods1. Construction of TIMP-1gene of RNA interference vectorFirstly, the TIMP-1gene sequence was obtained from GeneBank, and four19nt targetgene sequences were screened. After synthesizing a pair of complementaryoligonucleotide sequences, double-stranded DNA fragments were formed and linear izedusing Age Ⅰ and EcoR Ⅰ digestion of pGCSIL-GFP vector. Subsequently theconnected product was transferred to competent cells, and the grown clones wereidentified by PCR. When the positive clone sequence was recognised, the TIMP-1RNAinterference lentiviral vector(pGCSIL-GFP-shRNA) was successfuliy constructedSecondly, TIMP-1gene fragment was extracted from the plasmid containing TIMP-1target gene following amplification and digestion. Meanwhile, the pEGFP-N1-3FLAGvector was digested, purified and connected with TIMP-1sequence the ligation productwas transferred to competent cells. Finally, the TIMP-1target sequence recombinantplasmid (pEGFP-N1-3FLAG-TIMP-1) was obtained.Thirdly, the recombinant pEGFP-N1-3FLAG-TIMP-1plasmids were transfected to293T cells with four different pGCSIL-GFP lentiviral plasmid carrying anique shRNA.Forty-eight hours later, the cells were collected, and the protein was extracted. A effectivetarget was screened out by Western Blot, which can be applied for knocking out the targetgene TIMP-1.2. Inhibitory effect of TIMP-1gene RNAi vectors on liver fibrosis in rat HSC-T6cells.In HSC-T6cells the inhibitory effect of lentiviral vector pGCSIL-GFP-shRNA onthe transcriptional and proten level of TIMP-1gene was verified by real-time PCR andWestern Blot vespectively. Main results1. The TIMP-1gene RNAi lentiviral vector was successfully constructed.2. An effective TIMP-1interference target was found from the screening. Which waslocated at the position of the TIMP-1gene173～192nt. Its sequence was5’-GGAACGGAAATTTGCAC AT-3’.3. The TIMP-1gene RNAi vector was confirmed to has a significant inhibitory effect ofTIMP-1gene in HSC-T6cells.ConclusionWe successfully built the lentiviral vector which could silence TIMP-1gene byexogenous and endogenous validation, and further provided a viable strategy target forfurthen research and contribute to new therapautic method of liver fibrosis.