The Effect Of SiRNA Interfering SPINK1 Gene On The Biological Behavior Of Rat Hepatic Stellate Cells HSC-T6 And Its Molecular Regulation Mechanism

Liver fibrosis is caused by the excessive accumulation of scar tissue during the long-term wound healing process.The process of liver fibrosis is also accompanied by severe distortion of the liver vascular structure,especially on the connective tissue diaphragm that connects the afferent and efferent blood vessels.These can lead to deterioration of liver function,such as impaired ability to remove toxins and synthesize key proteins,lipids and carbohydrates needed to maintain body function,and eventually develop into liver cirrhosis.In advanced fibrosis,the risk of developing primary hepatocellular carcinoma（HCC）without obvious clinical or laboratory chemical signs of liver failure is 1-7%.Many recent studies have found that the activation of hepatic stellate cells plays an important role in the occurrence of liver fibrosis.Therefore,removal or inactivation of activated HSCs from the liver may be a factor in the recovery of the liver from liver fibrosis important means.Kazal type I serine protease inhibitor SPINK1 is isolated from the pancreas by Kazal et al.The spatial structure of SPINK1 and epidermal growth factor（EGF）is very similar.The study found that SPINK1 can combine with EGFR to regulate cell proliferation,apoptosis,inflammatory response,tumor occurrence and development.In the gene expression profile of the rat liver fibrosis model induced by CCl4 in our laboratory,we found that the expression of SPINK1 was significantly up-regulated at 3 weeks,6 weeks,and 9 weeks after modeling,and then through gene addition and recombinant protein treatment to improve endogenous and after exogenous SPINK1 levels,it was found that the cell viability,proliferation,cycle,and migration capabilities of HSC-T6 cells were significantly improved.It has not been reported whether SPINK1 knockout has an effect on the activation of HSC-T6 cells.Therefore,this project intends to explore the effect of siRNA interference Spink1 gene on the biological effects of rat hepatic stellate cells HSC-T6 and molecular regulation mechanism.This project first designs three different siRNA interference sequences based on the m RNA of SPINK1,uses Western Blot and q RT-PCR to screen the three siRNAs to find the fragments with the best interference effect,and then uses immunofluorescence to detect the siRNA interference efficiency;CCK8,Ed U,Transwell and scratch test to detect the viability,proliferation,migration and invasion of HSC-T6 after interference with SPINK1;PI staining method to detect HSC-T6 cycle changes;Annexin Ⅴ-FITC/PI fluorescence double staining detection apoptosis,and then detect cell proliferation,signal pathways and the expression of HSC-T6 cell activation-related proteins by Western blot.The results show that the knockout of SPINK1 gene in HSC-T6 cells can significantly inhibit the activity,proliferation,cycle,migration and invasion of HSC-T6,and significantly increase the apoptotic rate of HSC-T6.Moreover,it is related to the proliferation of HSC-T6.The protein phosphorylation in the PI3K-AKT and JAK-STAT signaling pathways also shown a significant inhibitory effect.These results indicate that SPINK1 may regulate the proliferation of HSC-T6 through the PI3K-AKT and JAK-STAT signaling pathways.In addition,the low expression of α-SMA and COL1A1 related to the activation of HSC-T6 cells,and the decrease of protein phosphorylation in the TGF-β signaling pathway,shows that SPINK1 may regulate HSC-T6 cell activation through the TGF-βsignaling pathway.Therefore,we added LY294002,HY-40354 and SB-431542 signaling pathway inhibitors,and used CCK8,EDU,flow cytometry and Western Blot experiments to verify the above speculation.The results prove that SPINK1 controls cell growth through the PI3K-AKT signaling pathway in HSC-T6 cells,and regulates the cell activation of HSC-T6 through the TGF-β signaling pathway.