The Effect And Mechanism Of PiR-823 In Liver Fibrogenesis

Hepatic fibrosis is caused by chronic diseases such as viral infection,alcohol,cholestasis,metabolic diseases and parasitic infections.The structure and function of liver is abnormal because of the imbalance of extracellular matrix?ECM?synthesis,degradation and excessive deposition of ECM.It is the common and inevitable step in the development of various chronic liver diseases to liver cirrhosis and even liver cancer,which seriously threatens human health.Liver fibrosis is a reversible pathological change.Various studies have shown that hepatic stellate cells,hepatocytes,bile duct epithelial cells,Kupffer cells and other intrahepatic cells are involved in the occurrence of hepatic fibrosis.However,among them,hepatic stellate cells?HSCs?play the key role in the development of liver fibrosis.In normal liver,HSCs are in a quiescent state.When hepatic tissues encounter insult,the HSCs are activated via various cytokines.The activation of HSCs is essential to the fibrogenesis of the liver.The activated HSCs demonstrated characters of cytomegaly,synapses extension and proliferation,and they are the main source of myofibroblasts that synthesize the components of ECM.In-depth study of the mechanism of the activation of HSCs is the key to find an effective target for anti-fibrosis treatment and to prevent or even reverse to liver fibrosis.TGF?-SMAD3 pathway,PDGFR?pathway,hedgehog?Hh?pathway and other pathways can lead to activation of HSCs.However,there are still no good therapeutic targets in clinical practice.We still need to find other mechanisms which could promote the activation of HSCs in order to find potential therapeutic targets.Piwi-interacting RNA?pi RNA?is a novel regulatory RNA found in mammalian germ cells.It is one of the"star"molecules in small non-coding RNA which is single-stranded with a length of 23-35 nucleotides.It is named because it only binds to the PIWI subprotein family in the Argonaute protein family.Initial studies have shown that piRNAs are mainly found in mammalian germ cells and stem cells.It combined with piwi subfamily proteins to form pi RNA complexes?piRC?to regulate gene silencing pathways,maintain germline and stem cell functions and regulate translational mRNA stability,etc.Recently,studies showed that piRNAs are also present in normal somatic cells?such as cells of the heart,brain,liver,etc.?other than germline cells,or in cancer cells,and participate in the occurrence of various cancers such as gastric cancer,liver cancer,lung cancer,and lymphoma.It is also closely related to diseases of the central nervous system and heart regeneration.Studies have shown that there were abundant piRNAs expressed in liver.Rizzo F use small RNA-Seq found that approximately 1400mammalian germline piRNAs expressed in rat liver which participate in the regulation of the pathophysiological process of liver cell regeneration.Meanwhile,human liver is rich in piRNAs too.Pi RNAs expression characteristics are different in the development of normal liver to liver cirrhosis or liver cancer.piR-823 is a member of the piRNA family.Studies have shown that piR-823 can promote the proliferation of cancer cells in multiple myeloma and colorectal cancer.Its expression is elevated in liver cirrhosis,precancerous lesions,and liver cancer.The biological behavior of HSCs is similar to that of cancer cells:the proliferation ability of activated HSCs is enhanced and TGF-?1 can be secreted,while TGF-?1 is significantly increased in patients with liver cancer.Therefore,the role of piR-823 in promoting cancer cell proliferation may also apply to HSCs.We speculate that piR-823 expression is also increased during hepatic fibrosis,but there is currently no study of the role of pi R-823 in HSC.The purpose of this study was to observe the expression of piR-823during the activation of HSC and to elucidate the role of piR-823 in the regulation of HSC activation and its possible molecular mechanism.The expriment consists of the following three parts:Part?The expression of piR-823 in activated hepatic stellate cellsObjective:To investigate the expression of piR-823 in primary hepatic stellate cells from hepatic fibrotic mice and the expression of piR-823 during the activation of hepatic stellate cells in vitro.Methods:Liver fibrosis mice models were established by common bile duct ligation and intraperitoneal injection of carbon tetrachloride?CCl₄?.Hepatic stellate cells from normal and hepatic fibrotic mice were isolated by in situ perfusion to establish activation models of HSCs in vitro.HE staining was used to detect the histopathological changes of the liver,masson staining and Sirius red staining were used to detect collagen deposition.The Lys method was used to detect the serum levels of ALT,AST and TBIL.Immunohistochemistry?IHC?,western blot and real-time PCR were used to detect the expression of COL1a1 and?-SMA on both protein and mRNA levels.The real-time PCR method was used to detect the difference of pi R-823expression.Results:1.Mouse liver fibrosis model was established successfully.Liver fibrosis was induced by intraperitoneal injection of CCL₄.Mice were divided into four groups:control group?injected with oil?,CCL₄ 2 w group,CCL₄ 4 w group and CCL₄ 6 w group.The mice in the control group had liver of a soft texture with smooth surface,and a bright color.Mice in the 2w group had a hepatic toughness with a fine-grained surface.As the administration time prolonged,the particles on the surface of the liver were apparently brownish and the volume was slightly reduced.HE,masson,and Sirius red staining showed that the hepatic plates of the mice in the control group were well-arranged,and the structure of the hepatic lobule was complete with few or no collagen fibers.The mice in group 2 w had hemorrhage,necrosis,vacuoles and fatty degeneration of hepatocytes,infiltration of inflammatory cells,a small amount of collagen deposition but the arrangement of hepatic plate and lobular structures were normal.At 4 w and 6 w,the arrangement of normal liver plate of mouse was disappeared,the lobular structure was disordered,and the collagen fibers were significantly hyperplastic.Immunohistochemistry showed that the number of positive cells of?-SMA and COL1a1 in liver tissue of mice in the 4 w and 6w groups was significantly increased and the staining was deepened,which was significantly higher than that of the control group.Western blot results showed that the protein expression of COL1a1 and?-SMA in mice of the 2w,4 w and 6 w groups increased sequentially compared with the control group.The levels of ALT and AST in mice treated with CCL₄ were significantly higher than those in the control group.The above results indicate that the CCL₄-induced hepatic fibrosis model was successfully established.In experiment liver fibrosis induced by common bile duct ligation,mice were divided into two groups:sham operation?SH?and bile duct ligation?BDL?.In the SH group,the mouse liver had a soft texture with a smooth surface,and normal gallbladder size.In the BDL group,the gallbladder of mice was significantly enlarged,the liver was tough,and the surface was fine-grained.HE,masson and Sirius red staining results showed that the liver plates of the SH group were well-arranged,the hepatic lobule was intact,the collagen fibers were few and the bile ducts were normal.In the BDL group,there were hemorrhage,necrosis,inflammatory cell infiltration,obvious expansion of the bile ducts,disappearance of the normal arrangement of the liver plate,disordered lobular structure and marked proliferation of collagen fibers.Immunohistochemistry results showed that the number of?-SMA and COL1a1 positive cells in the liver of BDL mice was significantly increased and the staining was deeper,which was significantly higher than that of the SH group.The results of western blot and real-time PCR showed that the mRNA and protein expression of COL1a1 and?-SMA were increased in the BDL group compared with the SH group.The levels of ALT,AST and TBIL in BDL mice were significantly higher than those in SH group.The above results indicate that the cholestatic liver fibrosis model was successfully established.2.Successfully extracted mouse primary HSCs.The hepatic portal vein was punctured and the liver was infused in situ with streptokinase and type IV collagenase.The primary mouse HSCs were isolated by density gradient centrifugation.Observed under the microscope,the newly isolated HSCs were not activated,round or elliptical,and had a large volume and high refractive index.When observed under an inverted fluorescence microscope with a wavelength of 328 nm,the cells can spontaneously turn blue-green fluorescence.After light microscope and fluorescence microscopy,the purity of the cells were about 80%and the cell yield was about 1×10⁵-1.5×10⁵/mouse.Cells were seeded in plastic flasks and all cells adhered 24 hours later.In this experiment,the primary HSCs cultured on the first,fourth,and seventh days after isolation,respectively,were tested.The cells in this period were quiescent,partially activated and activated,which was the in vitro activation model of HSCs.3.The expression of piR-823 increased during the activation of hepatic stellate cells.The expression of piR-823 in HSCs extracted from the liver of hepatic fibrotic mice was also significantly higher than that in the control group.In spontaneously activated HSCs during in vitro culture,the expression of piR-823 increased with the activation of HSCs.These data indicate that the expression of pi R-823 is increased in activated HSCs.Conclusions:The expression of piR-823 is increased during hepatic stellate cell activation?in vivo and in vitro?.Part?piR-823 activates hepatic stellate cells and promotes liver fibrosisin miceObjective:To investigate the effect of piR-823 on activation of hepatic stellate cells.Methods:The synthetic piR-823 sense sequences?piR-823 mimics?and antisense sequences?piR-823 antagomir?were transfected into LX-2 and mouse primary HSCs to establish high expression and low levels of piR-823.Real-time PCR method was used to detect the expression of piR-823.Real-time PCR and Western Blotting method were used to detect the mRNA and protein levels of?-SMA and COL1a1 in HSCs.CCK-8 and Brdu methods were used to detect the activity and proliferation of LX-2 and primary mouse HSCs.The mice were transfected with piR-823 antisense using an AAV virus carrying a GFAP-specific initiation sequence to knock down the expression of piR-823 in the intrahepatic HSCs.HE staining was used to detect the histopathological changes of the liver,masson staining and Sirius red staining were used to detect collagen deposition.The Lys method was used to detect the serum ALT,AST and TBIL levels.Immunohistochemistry,western blot,and real-time PCR were used to detect the expression of COL1a1 and?-SMA protein and mRNA,and to confirm the liver fibrosis in mice.Results:1.Successful establishment of piR-823 overexpression and low expression of hepatic stellate cell line.We transfected piR-823 mimics and piR-823 antagomir into primary mouse HSCs and LX-2,which enhanced or inhibited piR-823 expression,and successfully established pi R-823 overexpressing and low expressing hepatic stellate cell lines.2.Upregulation of piR-823 expression promotes activation of HSCs.?1?The results of Western Blotting and real-time PCR showed that the expression of?-SMA and COL1a1 protein and mRNA were increased in primary HSCs and LX-2 cells transfected with piR-823 mimics compared with NC group.?2?The results of CCK-8 assay and Brdu assay showed that the OD values of primary HSCs and LX-2 cells transfected with piR-823 mimics were significantly higher than those of cells transfected with mimics-NC at the same detection time point.These data indicate that upregulation of piR-823expression promotes the synthesis,secretion and proliferation of HSCs,as well as increases the activity of HSCs.It means that upregulation of piR-823expression promotes the activation of HSCs.3.Knock down piR-823 expression inhibit HSCs activation.?1?The results of Western Blotting assay and real-time PCR showed that the expression of?-SMA and COL1a1 on both protein and mRNA levels were decreased in the primary HSCs and LX-2 cells transfected with piR-823antagomir compared with the control group.?2?The results of CCK-8 method and Brdu assay showed that the OD values of the primary HSCs and LX-2cells transfected with piR-823 antagomir were significantly lower than those of the control group at the same detection time point.These data suggest that knockdown the expression of piR-823 inhibits the synthesis,secretion and proliferation of HSCs,attenuates the activity of HSCs.It means that downregulation of piR-823 expression inhibits the activation of HSCs.4.Knockdown the expression of piR-823 in primary HSCs in mice attenuated liver fibrosis.After knocking down expression of piR-823 in primary HSCs in mice injected with AAV-pGFAP-piR-823 antisense,compared with the control group,the mice in the experimental group had reduced liver inflammation,decreased intrahepatic collagen deposition and intrahepatic alpha-SMA,decreased COL1a1 expression and the serum level of ALT was significantly reduced.The liver fibrosis of mice was weakened.Conclusions:The increase of pi R-823 expression promotes the activation of HSCs.Conversely,downregulation of piR-823 expression inhibits the activation of HSCs and attenuates hepatic fibrosis.Part?piR-823 activates hepatic stellate cells through promoting theexpression of TGF-?1Objective:To explore the molecular mechanism of piR-823 promoting hepatic stellate cell activation.Methods:Search for proteins,which the piR-823 specifically binds to,in the HSCs using the RNA pull down technique and LC-MS analysis.Results:1.piR-823 specifically combined with EIF3B.piR-823 was incubated with the LX-2 cell protein lysate and the proteins which piR-823 specifically bind were obtained by the RNA pull down technique combined with mass spectrometry.The EIF3B was further studied.The results of Western Blotting detection showed that there was more EIF3B protein in the pi R-823-bound protein solution.However,there was almost no EIF3B in the control group.RIP results showed that EIF3B protein bind a large number of piR-823 compared with IgG protein.These data indicate that piR-823 specifically bind to EIF3B.2.EIF3B regulates the translation of TGF-?1 protein.Real-time PCR and ELISA showed that knockdown of EIF3B reduced the expression of TGF-?1 protein,but its mRNA expression was not affected.It suggests that EIF3B influences the translation of TGF-?1 protein.3.piR-823 promotes the translation of TGF-?1 protein.Real-time PCR and ELISA showed that after up-regulation of piR-823expression,protein expression of TGF-?1 was increased but its mRNA expression was not affected.Knocking down the expression of piR-823,the protein expression of TGF-?1 was reduced compared with the control group,but there was no significant difference in mRNA expression.These data indicate that pi R-823 promotes TGF-?1 protein translation.4.piR-823 combines with EIF3B to promote TGF-?1 protein translation.Simultaneously transfected LX-2 cells with mimics-823 and siEIF3B to observe the effect of EIF3B on piR-823.Real-time PCR showed that both EIF3B and piR-823 had no effect on TGF-?1 mRNA expression.ELISA results showed that knockdown of EIF3B inhibited the stimulatory effect of piR-823 on TGF-?1 protein translation.The experimental results show that piR-823 promotes the translation of TGF-?1 protein by binding to EIF3B.Conclusions:In HSCs,piR-823 can combine with EIF3B to promote TGF-?1 protein translation,thereby affecting the activation of HSCs.