The Mechanism Of LncARSR In The Progression Of Non-alcoholic Fatty Liver Disease And Hepatocellular Carcinoma

Objective:Nonalcoholic fatty liver disease(NAFLD)is a series of liver diseases ranging from non-alcoholic fatty liver(NAFL)to non-alcoholic steatohepatitis(NASH).It is a common chronic liver disease among overweight and obese people.With the increase in obese people,the incidence and number of patients with NAFLD continue to rise.It has become one of the most important causes of liver disease worldwide.NAFLD can evolve into liver cirrhosis and even hepatocellular carcinoma(HCC)with the progress of the disease,and become the main reason for the increase in the number of liver cirrhosis and HCC.NAFLD-related HCC is due to insidious onset and lack of effective monitoring.When discovered,tumors are often staged late,and because patients have more complications such as heart disease and diabetes,the possibility of radical treatment including tumor resection,especially liver transplantation,is reduced.According to reports,lncARSR is involved in the metabolic process in the body,and is elevated in a variety of malignant tumors.It is involved in the regulation of tumor cytological behavior,including proliferation,migration,invasion and stem cell resistance,and is related to the drug resistance of some tumors.This study explored the influence of lncARSR and its potential target gene Yes-associated protein(YAP)on the NAFLD disease process and on the proliferation and invasion of NAFLD-related HCC cells,and explored possible regulatory mechanisms during the above process.Methods:1.Feed C57BL/6 mice with a high-fat diet to construct a NAFLD animal model,use oleic acid to cultivate Hep G2 cells to simulate the high fatty acid environment of hepatocytes,take mouse livers and cells,perform lipid deposition,and determine triglyceride content,and The RT-q PCR method was used to detect the expression level of lncARSR.2.Analyze the binding of lncARSR and YAP1 through RNA pull-down experiment,and conduct RIP analysis to further verify the targeted binding relationship between the two,use RNA FISH experiment to detect the subcellular localization of lncARSR and YAP1,and use Wsetern Blot to detect YAP1 and phosphate The expression level of YAP1 in liver tissues of NAFLD mice.In order to detect the effect of lncARSR on YAP1 nuclear translocation,Western Blot was used to detect the expression levels of YAP1 and phosphorylated YAP1 in the nucleus and cytoplasm of Hep G2 cells cultured with oleic acid.3.In order to further study the effect of lncARSR on YAP1 phosphorylation and nuclear translocation,Hep G2 cells were transfected with lncARSR overexpression and silenced lentivirus,and the transfection efficiency of lncARSR was detected by RT-q PCR method.After transfection,each group of cells The expression levels of YAP1 and phosphorylated YAP1 in the cells were detected by Western Blot,and the subcellular localization of YAP1 in each group of cells was detected by FISH.4.In the liver of NAFLD mice and Hep G2 cells cultured with oleic acid,the m RNA and protein expression levels of IRS2 were detected by RT-q PCR and WB,and the expression of AKT protein was detected by WB to further explore the role of IRS2/AKT in NAFLD.And by transfecting Hep G2 cells with lncARSR and co-transfecting lncARSR with YAP1,the m RNA and protein expression levels of IRS2 were detected by RT-q PCR and WB,and the expression levels of phosphorylated YAP1 and AKT proteins were detected by WB to further verify lncARSR The IRS2/AKT pathway is regulated by YAP1.5.Inject sh-lncARSR lentivirus into the tail vein of high-fat diet mice,take mouse liver tissue,and detect the expression of lncARSR by RT-q PCR,HE staining and oil red-O staining to detect changes in lipid deposition,and to detect glycerol Changes in triester content.RT-q PCR and WB were used to detect the m RNA and protein expression levels of IRS2,and WB was used to detect the expression levels of phosphorylated YAP1 and AKT proteins and adipogenesis-related proteins(Fasn,Scd1 and GPA).6.After overexpression or silencing of lncARSR on Hep G2 cells cultured with oleic acid,the changes in lipid deposition were detected by Nile Red staining of each group of cells,and the changes in triglyceride content were detected;WB was used to detect IRS2 and phosphoric acid Analyze the expression levels of YAP1 and AKT proteins and adipogenesis-related proteins(Fasn,Scd1 and GPA),and further study the effect of lncARSR on lipid deposition in NAFLD-related HCC.7.Hep G2 cells were cultured with oleic acid in each group after transfection with lncARSR,cell proliferation was detected by MTT method and soft agar colony method,flow cytometry was used for cell cycle analysis,and cell invasion was detected by Transwell chamber.8.To further study the role of lncARSR in tumor growth in NAFLD-related HCC by subcutaneously injecting Hep G2 cells transfected with sh-lncARSR into NAFLD mice.Results:1.The fat deposition of NAFLD mouse liver and Hep G2 cells cultured with oleic acid was significantly higher than that of the control group,and the content of triglycerides was also significantly increased(P<0.05).The expression of lncARSR in the liver of NAFLD mice and Hep G2 cells cultured with oleic acid was significantly increased(P<0.05).2.The RNA pull-down experiment showed that the full-length lncARSR probe pulled down YAP1.RIP analysis showed that lncARSR and YAP1 bound to each other,and YAP1 was the target protein of lncARSR;the FISH experiment showed that lncARSR and YAP1 co-localized in the cytoplasm;in the NAFLD group In mouse liver tissue,the level of phosphorylated YAP1 was significantly lower than that in the control group(P<0.05);in the cytoplasm of Hep G2 cells cultured with oleic acid,the level of phosphorylated YAP1 was significantly reduced(P<0.05),suggesting that lncARSR and YAP1 are specific Interaction to promote nuclear translocation of YAP1.3.RT-q PCR showed that the level of lncARSR in the oe-lncARSR group increased,and the level of lncARSR in the sh-lncARSR group decreased(P<0.05);Western blot results showed that the expression of phosphorylated YAP1 decreased in the oe-lncARSR group,while the expression in the sh-lncARSR group increased(P<0.05);FISH results showed that YAP1 expression decreased in the cytoplasm of the oe-lncARSR group and increased in the nucleus,while the performance of the sh-lncARSR group was reversed.It shows that lncARSR regulates inhibition of YAP1 phosphorylation and promotes nuclear translocation of YAP1.4.In the liver tissues of mice in the NAFLD group and Hep G2 cells cultured with oleic acid,the m RNA and protein expression levels of IRS2 and the phosphorylation level of AKT were significantly higher than those of the control group(P<0.05).The results of RT-q PCR and WB after transfection of Hep G2 cells showed that in the oe-lncARSR group,the level of IRS expression and phosphorylation of AKT increased,while the phosphorylation level of YAP1 decreased;in the oe-lncARSR+sh-YAP1 group,IRS expression,Phosphorylated AKT and YAP1 levels decreased;sh-lncARSR group,IRS and phosphorylated AKT levels decreased,phosphorylated YAP1 levels increased,and the effect was enhanced after co-transfection with YAP1S127D;sh-lncARSR+oe-YAP1 group,IRS and phosphorylated AKT Both YAP1 and YAP1 increased(P<0.05).It is concluded that lncARSR promotes nuclear translocation of YAP1 and activates the IRS2/AKT pathway.5.RT-q PCR showed that the expression of lncARSR in the liver of NAFLD mice after injection of sh-lncARSR lentivirus was significantly lower than that of the control group(P<0.05),and the lipid deposition and triglyceride content in the liver of mice were significantly decreased(P<0.05);in NAFLD mice,silencing lncARSR significantly reduced the expression level of IRS2,increased the level of phosphorylated YAP1,and significantly reduced the level of phosphorylated AKT(P<0.05);adipogenesis-related proteins(Fasn,Scd1,and GPA)The expression level was significantly reduced(P<0.05).It shows that lncARSR silencing can inhibit fat deposition in the liver of NAFLD mice through the IRS2/AKT pathway.6.In Hep G2 cells cultured with oleic acid,the lipid deposition and triglyceride content of the oe-lncARSR group were significantly increased,while the sh-lncARSR group was significantly decreased(P<0.05);WB results showed that the oe-lncARSR group was phosphorylated YAP1 was significantly reduced,IRS2 expression was significantly increased,and phosphorylated AKT levels were significantly increased,without affecting the expression level of total AKT,but the changes were opposite in the sh-lncARSR group(P<0.05);adipogenesis-related proteins(Fasn,Scd1,and GPA)The expression of PPAR? and peroxisome proliferator-activated receptor(PPAR?)was significantly increased in the oe-lncARSR group(P<0.05),which was consistent with the changes in the levels of triglycerides in the cells.It can be concluded that lncARSR promotes lipid deposition in NAFLD-related HCC cells.7.The oe-lncARSR group significantly increased the proliferation of Hep G2 cells cultured with high fatty acid,while the sh-lncARSR group significantly decreased(P<0.05);flow cytometry analysis showed that Hep G2 cells cultured with oleic acid in the oe-lncARSR group stayed in the S phase The number was significantly increased(P<0.05);the Transwell invasion experiment showed that the cell invasiveness of the oe-lncARSR group was significantly enhanced,while that of the sh-lncARSR group was decreased(P<0.05).Therefore,it is concluded that lncARSR promotes cell proliferation and invasion of NAFLD-related HCC.8.The volume and weight of tumors in NAFLD mice in the sh-lncARSR transfected Hep G2 cell group were significantly lower than those in the control group(P<0.05),suggesting that lncARSR can inhibit the growth of NAFLD-related HCC tumors.Conclusion:1.lncARSR enhances the activity of IRS2/AKT through targeted regulation of YAP1 to promote NAFLD disease progression.2.lncARSR can promote the proliferation,invasion and tumor growth of NAFLD-related HCC cells by inhibiting YAP1 phosphorylation and enhancing the activity of IRS2/AKT.