The Mechanism Of IGFBPrP1-Induced Mouse Liver Fibrosis Based On The LncRNA NEAT1/MiR-29b/Atg9a Pathway

Background:Liver fibrosis is a wound-healing response caused by many factors and the final common pathway of chronic hepatic diseases developing into liver cirrhosis or even liver cancer.Hepatic stellate cell(HSC)activation is the central link in liver fibrosis.When HSCs are activated,a-smooth muscle actin(a-SMA)is produced and extracellular matrix(ECM)is depositedAutophagy is a metabolic process that degrades damaged and aged organelles,and misfolded macromolecular proteins in cells,providing energy for HSC activation Autophagy releases lipid that promotes fibrogenesis activated HSCs.Many autophagy related(Atg)proteins participate in autophagy process,among which microtubule-associated protein 1 light chain 3(LC3B)is the marker protein on autophagosome membrane,and p62 is the substrate of autophagy.Atg9a participates in the formation of autophagy membrane and plays an important role in the initiation of autophagyGiven their important roles in many biological processes,increased attention has been paid to noncoding RNAs(ncRNAs).One type of ncRNAs is the microRNA(miRNA),a short ncRNA,whose length is about 22 nt,induces mRNA degradation or represses mRNA translation by targeting the 3’-untranslated region(3’-UTR)of mRNAs.The miR-29 family includes three subtypes:miR-29a,miR-29b,and miR-29c.It has been found that miR-29 family is downregulated and miR-29b is down-regulated most significantly during CC14-induced liver fibrosis.Importantly,miR-29b is highly expressed in HSCs compared to other hepatic cell types.Therefore,we focus on miR-29b for our study.In addition,there is a binding site between miR-29b and 3’ UTR of Atg9a in mouse and human by using bioinformatics analysisLong noncoding RNAs(LncRNAs),whose length are more than 200 nt,regulate miRNAs,thus ultimately regulating target mRNA levels.LncRNA nuclear enriched abundant transcript 1(NEAT1)is highly expressed in liver fibrosis,and contains a target site for miR-29b in mouse and human by using bioinformatics analysisNEAT1 contains a target site for miR-29b,and Atg9a is a predicted target of miR-29b In addition,Atg9a is involved in autophagy.Thus we speculate that NEAT1,miR-29b,and Atg9a may form a NEAT1/miR-29b/Atg9a pathway to regulate autophagyInsulin-like growth factor binding protein-related protein 1(IGFBPrPl)is a new profibrotic factor reported by our group supported by several National Natural Science Foundation of China in the recent ten years.Then we found that IGFBPrPl accelerates liver fibrosis and interacts with transforming growth factor beta 1(TGβ1)which is the most profibrotic cytokine.In addition,IGFBPrPl can promote HSC autophagy and activation.Different researchers have found that NEAT1 was upregulated,miR-29b was downregulated,or Atg9a was upregulated after HSCs were treated with TGFβ1 in vitro The evidences are as follows:① miR-122 is downregulated thus NEAT1 increasing after primary mouse HSCs are treated with TGFβ1;② TGFβ1 decreases miR-29b level during activating LX-2 and increasing ECM in vitro.Overexpression of miR-29b inhibites LX-2 activation and ECM increase induced by TGFβ1.③ Atg9a is upregulated during primary mouse HSCs are activated by TGFβ1 in vitro.Hence,the partial mechanism of TGFβ1-induced liver fibrosis is that TGFβ1 regulates levels of NEAT1,miR-29b or Atg9a in HSCs.Therefore,whether IGFBPrβ1 affects NEAT1,miR-29b,and Atg9a just like TGFβ1 is not clear.In mice and humans,NEAT1 contains a target site for miR-29b,and Atg9a is a predicted target of miR-29b.In addition,Atg9a is necessary for autophagy.IGFBPrPl can promote HSC autophagy thus inducing liver fibrosis.However,what is the relationship among NEAT1,miR-29b,Atg9a,and HSC autophagy during IGFBPrPl-induced liver fibrosis?At present,there is no relevant literature at home and abroadIn the study,we focus on NEAT 1,miR-29b,Atg9a and HSC autophagy.The study is divided into four parts.In the first part,levels of NEAT1,miR-29b,Atg9a,and autophagy were detected in adenovirus-mediated IGFBPrPl(AdIGFBPrPl)-treated mouse liver tissue In the second part,levels of NEAT1,miR-29b,Atg9a,and autophagy were detected in immortalized mouse hepatic stellate cell line JS1 transfected with either AdIGFBPrP1 or siIGFBPrP1.In the third part,autophagy,activation,and ECM level were detected after altering NEAT1,miR-29b,or Atg9a levels in AdIGFBPrP1-treated JS1 cells.In the fourth part,relationships among NEAT1,miR-29b,and Atg9a were explored by using dual-luciferase renorter assays.Western blot.qRT-PCR.and immunofluorescence in AdIGFBPrP1-treated JS1 cells.The The study is to elucidate the mechanism of IGFBPrPl-induced liver fibrosis,and to provide new ideas and targets for the diagnosis and treatment of liver fibrosisPart 1 Level changes of IncRNA NEAT1,miR-29b,Atg9a,and autophagy during IGFBPrPl-induced liver fibrosis in miceExperiment 1 Dynamic changes of IncRNA NEAT1,miR-29b,Atg9a,and autophagy during IGFBPrPl-induced mouse liver fibrosisObjective:To determine whether there are NEAT1,miR-29b,Atg9a,and autophagy changes in IGFBPrPl-induced mouse liver fibrosisMethods:A total of 120 male C57BL/6 male mice were randomly divided into the AdIGFBPrP1 group(n=40),to which 0.1 mL AdIGFBPrP1(2 × 109 pfu/mouse)was injected via the tail vein,the CAd group(n=40),to which 0.1 mL control adenovirus(adenoviral vectors carrying no cDNA,2 × 109 pfu/mouse)was injected via the tail vein,or the control group(n=40),to which 0.1 mL saline was injected via the tail vein.At weeks 1(n=8),2(n=8),4(n=8),8(n=8),12(n=8)after adenoviral transfection,the mouse liver tissues stored for further analysis.H&E staining and Sirius red staining were used to detect histological changes and collagen fibrils of liver tissues.qRT-PCR was used to detect levels of IGFBPrPl,a-SMA,NEAT1,miR-29b,Atg9a,and LC3B RNAs.Western blot was used to detect levels of IGFBPrPl,a-SMA,collagen I,Atg9a,LC3B,and SQSTM1/p62 proteins.Results:(1)The Level of IGFBPrPl was detected by qRT-PCR after AdIGFBPrP1 was transfected into mouse liver tissues:IGFBPrPl mRNA and protein levels increased and peaked at 1 w and then gradually decreased from 2 w to 12 w,but levels remained higher than those of the control group(P<0.05).(2)Histopathological changes of mouse liver tissues:H&E staining and Sirius red staining showed degeneration and necrosis of hepatocytes,infiltration of inflammatory cells,proliferation of bile ducts and the increasing of collagen fibers after AdIGFBPrP1 was transfected into mouse liver tissues.It suggests the occurence of mouse liver fibrosis.(3)Levels of a-SMA,and collagen I were detected by qRT-PCR and Western blot after IGFBPrPl was overexpressed:a-SMA mRNA and protein,and collagen I protein levels gradually increased over time(P<0.05).This indicated that HSCs were activated and ECM was increased after IGFBPrPl was overexpressed,which further indicated the occurence of liver fibrosis in mice(4)Levels of NEAT1 and miR-29b were detected by qRT-PCR after IGFBPrPl was overexpressed:NEAT1 RNA increased and peaked at 1 w and then gradually decreased from 2 w to 12 w,but levels remained higher than those of the control group(P<0.05)The expression trend of miR-29b was contrary to that of NEAT1.miR-29b RNA decreased and reached the lowest point at 1 w and then gradually increased from 2 w to 12 w,but levels remained lower than those of the control group(P<0.05)(5)The Level of Atg9a was detected by qRT-PCR and Western blot after IGFBPrPl was overexpressed:Atg9a mRNA and protein increased and peaked at 1 w and then gradually decreased from 2 w to 12 w,but levels remained higher than those of the control group(P<0.05)(6)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after IGFBPrPl was overexpressed:LC3B mRNA and LC3B-II/LC3B-I protein levels increased and peaked at 1 w and then gradually decreased from 2 w to 12 w,but levels remained higher than those of the control group(P<0.05).SQSTM1/p62 had the opposite trend(P<0.05)Conclusion:During IGFBPrPl-induced liver fibrosis,levels of NEAT1,Atg9a,and autophagy were increased in liver tissues.MiR-29b was decreased.HSC activation and ECM synthesis were increased.It is suggested that IGFBPrPl may act as an upstream factor to upregulate NEAT1,Atg9a and autophagy,and downregulate the expression of miR-29b.Then the activation of HSCs and the expression of ECM were promoted,and mouse liver fibrosis was occuredPart 2 Effects of upregulation or downregulation of IGFBPrP1 on IncRNA NEAT1,miR-29b,Atg9a, and autophagy of mouse hepatic stellate cell line JS1Experiment 2 Effects of upregulation of IGFBPrPl on lncRNA NEAT1,miR-29b,Atg9a,and autophagy of JS1 cellsObjective:To determine whether upregulation of IGFBPrPl affects levels of NEAT1,miR-29b,Atg9a,and autophagy in JS1 cells.Methods:Taking the mouse hepatic stellate cell line JS1 as the research object.Firstly,AdIGFBPrP1 of different multiplicity of infection(MOI)(10,50,100,and 200)was transfected into JS1,and the number of green fluorescent cells was observed by using inverted fluorescence microscope,and the most suitable MOI was screened out and used in subsequent experiments.Then the cells were divided into the AdIGFBPrP1 group by transfection with AdIGFBPrP1,the CAd group by transfection with control adenovirus,or the Control group by culturing with only DMEM containing 10%FBS.Cells were then collected at 6 h(n=3),12 h(n=3),24 h(n=3),48 h(n=3),and 72 h(n=3)after adenoviral transfection.qRT-PCR was used to detect levels of IGFBPrPl,a-SMA,NEAT1,miR-29b,Atg9a,and LC3B RNAs.Western blot was used to detect levels of IGFBPrPl,a-SMA,collagen I,Atg9a,LC3B,and SQSTM1/p62 proteins.Results:(1)The most suitable MOI was detected after AdIGFBPrP1 was transfected into JS1 cells:In MOI 200 group,the cell death rate was higher than 85%.The transfection efficiency in MOI 100 group was higher than that of MOI 10 and MOI 50 group(P<0.05).Therefore,MOI 100 was the most suitable MOI and used for subsequent experiments.(2)Levels of IGFBPrPl,a-SMA,and collagen I were detected by qRT-PCR and Western blot after AdIGFBPrP1 was transfected into JS1 cells:Levels of IGFBPrPl mRNA and protein increased from 6 h,peaking at 48 h,then decreasing at 72 h but remaining higher than the control group(P<0.05).Levels of a-SMA mRNA and protein increased from 6 h and gradually increased over time.The level of collagen I protein increased from 12 h and gradually increased over time(P<0.05).(3)Levels of NEAT1 and miR-29b RNA were detected by qRT-PCR after IGFBPrPl was overexpressed:NEAT1 RNA increased from 6 h,peaking at 48 h,then decreasing at 72 h but remaining higher than the control group(P<0.05).miR-29b RNA decreased from 6 h,reaching the lowest point at 48 h,then increasing at 72 h,but levels remained lower than those of the control group(P<0.05).(4)The Level of Atg9a was detected by qRT-PCR and Western blot after IGFBPrPl was overexpressed:Atg9a mRNA and protein increased from 6 h,peaking at 48 h,then decreasing at 72 h but remaining higher than the control group(P<0.05)(5)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after IGFBPrPl was overexpressed:LC3B mRNA and LC3B-II/LC3B-I protein increased from 6 h,peaking at 48 h,then decreasing at 72 h but remaining higher than the control group(P<0.05).SQSTM1/p62 had the opposite trend(P<0.05)Conclusion:After IGFBPrPl was upregulated in JS1 in vitro,levels of NEAT1,Atg9a and autophagy increased and the expression of miR-29b decreased.The activation of HSCs increased,and then the expression of ECM increased.It is suggested that IGFBPrPl,as an upstream factor,upregulates the expression of NEAT1,Atg9a and autophagy in HSCs,and downregulates the expression of miR-29b,thus promoting the activation and ECM expression of mouse HSCsExperiment 3 Effects of downregulating IGFBPrP1 on lncRNA NEAT1,miR-29b,Atg9a,and autophagy of JS1 cellsObjective:To determine whether downregulation of IGFBPrPl affects levels of NEAT1,miR-29b,Atg9a,and autophagy in JS1 cellsMethods:Firstly,qRT-PCR was used to select the highest silencing efficiency one from the three siIGFBPrPls and the selected one was used for subsequent experiments.Then groups were invided into the following groups:the control group was cultured for a total of 48 h in DMEM containing 10%FBS.The EBSS group was cultured in DMEM containing 10%FBS for 44 h then in earle’s balanced salt solution(EBSS)for 4 h.The EBSS+siRNA-NC group was cultured with control siRNA for 44 h followed by incubation in EBSS for 4 h.The EBSS+siIGFBPrPl group was incubated with siIGFBPrPl for 44 h followed by incubation in EBSS for 4 hResults:(1)Screening for the most effective silGFBPrPl:The efficiency of siIGFBPrP1-953 was higher than that of siIGFBPrPl-857 and silGFBPrPl-647(P<0.05)Hence,siIGFBPrPl-953 was selected for the follow-up experiment(2)Levels of IGFBPrPl,a-SMA,and collagen I were detected by qRT-PCR and Western blot after silGFBPrPl was transfected into JS1 cells:IGFBPrPl mRNA and protein were decreased in EBSS+siIGBPrP1 group compared to levels in the EBSS-only treated group(P<0.05).α-SMA mRNA and protein,and collagen Ⅰ protein were reduced in EBSS+siIGBPrP1 group compared to EBSS group(P<0.05).(3)Levels of NEAT1 and miR-29b RNA were detected by qRT-PCR after siIGFBPrP1 was transfected into JS1 cells:NEAT1 RNA was decreased in EBSS+siIGBPrP1 group compared to levels in the EBSS-only treated group(P<0.05).miR-29b RNA was increased in EBSS+siIGBPrPl group compared to levels in the EBSS-only treated group(P<0.05).(4)The Level of Atg9a was detected by qRT-PCR and Western blot after siIGFBPrPl was transfected into JS1 cells:Atg9a mRNA and protein were decreased in EBSS+siIGBPrPl group compared to levels in the EBSS-only treated group(P<0.05).(5)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after siIGFBPrPl was transfected into JS1 cells:LC3B mRNA and LC3B-Ⅱ/LC3B-Ⅰ protein were decreased in EBSS+siIGBPrPl group compared to levels in the EBSS-only treated group(P<0.05),while SQSTM1/p62 in EBSS+siIGBPrPl group was higher than levels in the EBSS-only treated group(P<0.05).Conclusion:After downregulating IGFBPrPl in JS1 cells treated with EBSS in vitro,levels of NEAT1,Atg9a and autophagy decreased,and the expression of miR-29b increased compared with the EBSS group.JS1 cell activation and ECM expression decreased.It is suggested that IGFBPrPl,as an upstream factor,regulates levels of NEAT1,miR-29b,Atg9a and autophagy in mouse HSCs,thus affecting mouse liver fibrosis.Part 3 Effects of changing levels of lncRNA NEAT1,miR-29b,or Atg9a on IGFBPrP1-induced autophagy,activation,and ECM expression in JS1 cellsExperiment 4 Effects of upregulation or downregulation of lncRNA NEAT1 on IGFBPrP1-induced autophagy, activation,and ECM expression in JS1 cellsObjective:To explore whether IGFBPrPl-induced JS1 autophagy,activation,and ECM expression changed after upregulating or downregulating NEAT1.Methods:Firstly,qRT-PCR was used to select the best silencing effect siNEATl for subsequent experiments.Then JS1 cells were divided into the following group:control group was cultured for a total of 48 h in DMEM containing 10%FBS.AdIGFBPrP1 group was transfected with AdIGFBPrP1 for 48 h.The AdIGFBPrPl+vector-NC group was cultured with control vector for 6 h followed by incubation with AdIGFBPrP1 for 42 h.AdIGFBPrP 1+NEAT1 group was incubated with 4 μg per well NEAT 1 for 6 h followed by incubation in AdIGFBPrP1 for 42 h.AdIGFBPrPl+siRNA-NC group was cultured with control siRNA-NC for 6 h followed by incubation with AdIGFBPrP1 for 42 h.AdIGFBPrPl+siNEAT1 group was cultured with 50 nM siIGFBPrPl for 6 h followed by incubation with AdIGFBPrP1 for 42 h.qRT-PCR was used to detect levels of NEAT1,LC3B,and a-SMA RNAs.Western blot was used to detect levels of LC3B,SQSTM1/p62,a-SMA and collagen I proteins.Results:(1)Screening the most effective siNEATl:NEAT1 RNA was decreased in siNEAT1-1654 group compared to the level in siNEATl-183 and siNEATl-2644 group(P<0.05).Therefore,siNEAT1-1654 was selected for subsequent experiments.(2)The expression of NEAT1 RNA was detected by qRT-PCR after NEAT1 or siNEAT1 was transfected into AdIGFBPrP1-treated JS1 cells:NEAT1 RNA in AdIGFBPrP1+NEAT1 group was higher than the level in AdIGFBPrP1 group(P<0.05).NEAT1 RNA in AdIGFBPrP1+siNEATl group was lower than the level in AdIGFBPrP1 group(P<0.05).(3)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after NEAT1 or siNEATl was transfected into AdIGFBPrP1-treated JS1 cells:Increased levels of LC3B mRNA and LC3B-II/LC3B-I ratio,and decreased expression of SQSTM1/p62 protein were observed in AdIGFBPrP1+NEAT1 group compared to the AdIGFBPrP1-only treated group(P<0.05).Decreased levels of LC3B mRNA and LC3B-II/LC3B-I ratio,and increased expression of SQSTM1/p62 protein were observed in AdIGFBPrP1+siNEATl group compared to the AdIGFBPrP1-only treated group(P<0.05).(4)Levels of a-SMA and collagen I were detected by qRT-PCR and Western blot after NEAT1 or siNEATl was transfected into AdIGFBPrP1-treated JS1 cells:Increased a-SMA mRNA and protein,and collagen I protein were observed in AdIGFBPrP1+NEAT1 group compared to the AdIGFBPrPl-only treated group(P<0.05).Decreased a-SMA mRNA and protein,and collagen I protein were observed in AdIGFBPrP1+siNEAT1 group compared to the AdIGFBPrP1-only treated group(P><0.05)Conclusion:Upregulation of NEAT1 promotes IGFBPrP1-induced mouse HSC autophagy,activation,and ECM expression in vitro.Downregulation of NEAT1 inhibits IGFBPrPl-induced mouse HSC autophagy,activation,and ECM expression in vitro.These results demonstrate that IGFBPrPl can upregulate NEAT1,thus promoting HSC autophagy,activation and ECM expressionExperiment 5 Effects of upregulation or downregulation of miR-29b on IGFBPrPl-induced autophagy, activation,and ECM expression in JS1 cellsObjective:To explore whether upregulation or downregulation of miR-29b affects IGFBPrPl-induced JS1 autophagy,activation,and ECM expressionMethods:JS1 cells were divided into the following group:control group was cultured for a total of 48 h in DMEM containing 10%FBS.AdIGFBPrP1 group was transfected with AdIGFBPrP1 for 48 h.The AdIGFBPrP1+mimics-NC group was cultured with control mimics for 6 h followed by incubation with AdIGFBPrP1 for 42 h.AdIGFBPrP1+miR-29b mimics group was incubated with 50nM miR-29b mimics for 6 h followed by incubation in AdIGFBPrP1 for 42 h.AdIGFBPrP1+inhibitors-NC group was cultured with control inhibitors for 6 h followed by incubation with AdIGFBPrP1 for 42 h AdIGFBPrP1+miR-29b inhibitors group was cultured with 100 nM miR-29b inhibitors for 6 h followed by incubation with AdIGFBPrP1 for 42 h.qRT-PCR was used to detect levels of miR-29b,LC3B,and a-SMA RNAs.Western blot was used to detect levels of LC3B,SQSTM1/p62,a-SMA and collagen I proteinsResults:(1)The expression of miR-29b RNA was detected by qRT-PCR after miR-29b mimics or inhibitors were transfected into AdIGFBPrP1-treated JS1 cells:miR-29b RNA in AdIGFBPrP1+miR-29b mimics group was higher than the level in AdIGFBPrP1 group(P<0.05).miR-29b RNA in AdIGFBPrP1+miR-29b inhibitors group was lower than the level in AdIGFBPrP1 group(P<0.05)(2)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after miR-29b mimics or inhibitors were transfected into AdIGFBPrP1-treated JS1 cells:Decreased levels of LC3B mRNA and LC3B-II/LC3B-I ratio,and increased expression of SQSTM1/p62 protein were observed in AdIGFBPrP1+miR-29b mimics group compared to the AdIGFBPrP1-only treated group(P<0.05).Increased levels of LC3B mRNA and LC3B-II/LC3B-I ratio,and decreased expression of SQSTM1/p62 protein were observed in AdIGFBPrP1+miR-29b inhibitors group compared to the AdIGFBPrP1-only treated group(P<0.05).(3)Levels of a-SMA and collagen I were detected by qRT-PCR and Western blot after miR-29b mimics or inhibitors were transfected into AdIGFBPrP1-treated JS1 cells:Decreased a-SMA mRNA and protein,and collagen I protein were observed in AdIGFBPrP1+miR-29b mimics group compared to the AdIGFBPrP1-only treated group(P<0.05).Increased a-SMA mRNA and protein,and collagen I protein were observed in AdIGFBPrP1+miR-29b inhibitors group compared to the AdIGFBPrP1-only treated group(P<0.05).Conclusion:Upregulation of miR-29b inhibits IGFBPrPl-induced mouse HSC autophagy,activation,and ECM expression in vitro.Downregulation of miR-29b promotes IGFBPrPl-induced mouse HSC autophagy,activation,and ECM expression in vitro.These results suggest that IGFBPrPl can downregulate miR-29b,thus promoting HSC autophagy,activation and ECM expressionExperiment 6 Effects of downregulation of Atg9a on IGFBPrP1-induced autophagy,activation, and ECM expression in JS1 cellsObjective:To explore whether downregulation of Atg9a affects IGFBPrPl-induced JS1 autophagy,activation,and ECM expressionMethods:Firstly,selecting the most effective siAtg9a for subsequent experiments.Then JS1 cells were divided into the following group:The control group was cultured for a total of 48 h in DMEM containing 10%FBS.The AdIGFBPrP1 group was transfected with AdIGFBPrP1 for 48 h.The AdIGFBPrP1+siRNA-NC group was cultured with control siRNA for 6 h followed by incubation with AdIGFBPrP1 for 42 h.The AdIGFBPrP1+siAtg9a group was incubated with 50 nM siAtg9a for 6 h followed by incubation with AdIGFBPrP1 for 42 h.qRT-PCR was used to detect levels of Atg9a,LC3B,and a-SMA mRNAs.Western blot was used to detect levels of Atg9a,LC3B,SQSTM1/p62,a-SMA and collagen I proteins.Results:(1)Screening for the most effective siAtg9a:Atg9a mRNA in the siAtg9a-1078 group was lower than the level in the siAtg9a-702 and siAtg9a-1404 group(P<0.05).Hence,siAtg9a-1078 was used for the subsequent experiments.(2)Levels of Atg9a was detected by qRT-PCR and Western blot after downregulation of Atg9a in AdIGFBPrP1-treated JS1 cells:Atg9a mRNA was decreased in AdIGFBPrPl+siAtg9a group compared to the AdIGFBPrP1-only treated group(P<0.05).(3)Levels of LC3B and SQSTM1/p62 were detected by qRT-PCR and Western blot after downregulating Atg9a in AdIGFBPrP1-treated JS1 cells:LC3B mRNA and LC3B-II/LC3B-I protein were decreased while SQSTM1/p62 protein was increased in the AdIGFBPrP1+siAtg9a group compared to the AdIGFBPrPl-only treated group(P<0.05).(4)Levels of a-SMA and collagen I were detected by qRT-PCR and Western blot after downregulating Atg9a in AdIGFBPrP1-treated JS1 cells:a-SMA mRNA and protein,and collagen I protein were decreased in the AdIGFBPrP1+siAtg9a group compared to the AdIGFBPrP1-only treated group(P<0.05).Conclusion:Downregulation of Atg9a inhibits IGFBPrPl-induced mouse HSC autophagy,activation,and ECM expression in vitro.These results suggest that IGFBPrPl can upregulate Atg9a,thus promoting HSC autophagy,activation and ECM expression.Part 4 The relationship among IncRNA NEAT1,miR-29b,and Atg9a in IGFBPrP1-induced autophagy,activation,and ECM expression in JS1 cells Experiment 7 The relationship between miR-29b and Atg9a in IGFBPrPl-induced autophagy,activation,and ECM expression in JS1 cellsObjective:To explore the relationship between miR-29b and Atg9a in IGFBPrPl-induced JS1 autophagy,activation,and ECM expressionMethods:(1)Whether Atg9a is the target gene of miR-29b was confirmed by dual-luciferase reporter assays(2)Groups in the experiment that miR-29b was upregulated or downregulated were the same as that in Experiment 5.The expression of Atg9a mRNA was detected by qRT-PCR The expression of Atg9a protein was detected by Western blot and immunofluorescence(3)The groups in the experiment that miR-29b and Atg9a were downregulated were as follows:Control group:JS1 cells were cultured for 48 h;AdIGFBPrP1 group:JS1 cells were transfected with AdIGFBPrP1 for 48 h;AdIGFBPrP1+miR-29b inhibitors group:JS1 cells were transfected with miR-29b inhibitors at a final concentration of 100 nM for 6 h,and then cultured with AdIGFBPrP1 for 42 h;AdIGFBPrP1+miR-29b inhibitors+siAtg9a group:JS1 cells transfected with miR-29b inhibitors(100 nM)+siAtg9a(50 nM)for 6 h,and then cultured with AdIGFBPrP1 for 42 h.The mRNA levels of LC3B and a-SMA were detected by qRT-PCR.The protein levels of LC3B,SQSTM1/p62,a-SMA and collagen I were detected by Western blotResults:(1)Atg9a binding to miR-29b was detected by dual-luciferase reporter assays:Relative luciferase activity in the miR-29b mimics+Atg9a wild type(Atg9a Wt)group was significantly lower than that in the miR-29b mimics+Atg9a mutant type(Atg9a Mut)group(P<0.05)(2)qRT-PCR,Western blot,and immunofluorescence were used to detect the expression of Atg9a in AdIGFBPrP1-treated JS1 cells transfected with miR-29b mimics or inhibitors:Atg9a mRNA and protein were decreased in the AdIGFBPrP1+miR-29b mimics group compared to the AdIGFBPrP1-only treated group(P<0.05).Atg9a mRNA and protein were increased in AdIGFBPrP1+miR-29b inhibitors group compared to the AdIGFBPrP1-only treated group(P<0.05)(3)qRT-PCR and Western blot were used to detect LC3B and SQSTM1/p62 levels in AdIGFBPrP1-treated JS1 cells transfected with miR-29b inhibitors+siAtg9a:Levels of LC3B mRNA and LC3B-II/LC3B-I protein in the AdIGFBPrP1+miR-29b inhibitors+siAtg9a group were lower than that in AdIGFBPrP1+miR-29b inhibitors group(P<0.05),while the level of SQSTM1/p62 protein was higher than that in the AdIGFBPrP1+miR-29b inhibitors group(P<0.05)(4)qRT-PCR and Western blot were used to detect a-SMA and collagen I levels in AdIGFBPrP1-treated JS1 cells transfected with miR-29b inhibitors+siAtg9a:Levels of a-SMA mRNA and protein,and collagen I protein in the AdIGFBPrP1+miR-29b inhibitors+siAtg9a group were lower than that in the AdIGFBPrP1+miR-29b inhibitors group(P<0.05)Conclusion:IGFBPrPl inhibits miR-29b expression thus promoting the Atg9a level,thus promoting mouse HSC activation and ECM expression in vitroExperiment 8 The relationship between lncRNA NEAT1 and miR-29b in IGFBPrPl-induced autophagy, activation,and ECM expression in JS1 cellsObjective:To explore the relationship between NEAT1 and miR-29b in IGFBPrPl-induced JS1 autophagy,activation,and ECM expressionMethods:(1)Whether NEAT1 binding to miR-29b was confirmed by dual-luciferase reporter assays.(2)Groups in the experiment that NEAT1 was upregulated or downregulated were the same as that in Experiment 4.The expression of miR-29b RNA was detected by qRT-PCR(3)The groups in the experiment that NEAT1 and miR-29b mimics was co-transfected were as follows:Control group:JS1 cells were cultured for 48 h;AdIGFBPrP1 group:JS1 cells were transfected with AdIGFBPrP1 for 48 h;AdIGFBPrP1+NEAT1 group:JS1 cells were transfected with NEAT1(4 μg per well)for 6 h,and then cultured with AdIGFBPrP1 for 42 h;AdIGFBPrP1+NEAT1+miR-29b mimics group:JS1 cells transfected with NEAT1(4 μg per well)+miR-29b mimics(50 nM)for 6 h,and then cultured with AdIGFBPrP1 for 42 h.The mRNA levels of Atg9a,LC3B and a-SMA were detected by qRT-PCR.The protein levels of Atg9a,LC3B,SQSTM1/p62,a-SMA and collagen I were detected by Western blot.The expression of Atg9a protein was detected by immunofluorescenceResults:(1)NEAT1 binding to miR-29b was detected by dual-luciferase reporter assays:Relative luciferase activity in miR-29b+NEAT1 Wt group was significantly lower than that in miR-29b+NEAT1 Mut group(P<0.05).(2)Localization of NEAT 1 was detected by RNA nuclear-cytoplasmic fractionation:NEAT1 is located both in the nucleus and the cytoplasm.(3)qRT-PCR was used to detect the expression of miR-29b RNA in AdIGFBPrP1-treated JS1 cells transfected with NEAT1 or siNEATl:The expression of miR-29b RNA in the AdIGFBPrP1+NEAT1 group was lower than the level in the AdIGFBPrP1 group(P<0.05).The expression of miR-29b RNA in the AdIGFBPrP1+siNEAT1 group was higher than the level in the AdIGFBPrP1 group(P<0.05).(4)qRT-PCR,Western blot,and immunofluorescence were used to detect Atg9a,LC3B,and SQSTM1/p62 levels in AdIGFBPrP1-treated JS1 cells transfected with NEAT1+miR-29b mimics:Levels of Atg9a mRNA and protein,and LC3B mRNA and LC3B-II/LC3B-I protein in the AdIGFBPrP1+NEAT1+miR-29b mimics group were lower than that in AdIGFBPrP1+NEAT1 group(P<0.05),while levels of SQSTM1/p62 protein was higher than that in AdIGFBPrP1+NEAT1 group(P<0.05).(5)qRT-PCR and Western blot were used to detect a-SMA and collagen I levels in AdIGFBPrP1-treated JS1 cells transfected with NEAT1+miR-29b mimics:Levels of a-SMA mRNA and protein,and collagen I protein in the AdIGFBPrP1+NEAT1+miR-29b mimics group were lower than that in the AdIGFBPrP1+NEAT1 group(P<0.05).Conclusion:IGFBPrPl promotes the NEAT1 expression,and then inhibits the expression of miR-29b thus increasing the Atg9a level to promoting mouse HSC activation and ECM expression in vitro.ConclusionIGFBPrPl can upregulate NEAT1 expression in mouse HSCs,and NEAT 1 competitively binds to miR-29b,thus reducing miR-29b inhibition on Atg9a and upregulating the expression of Atg9a.In other words,IGFBPrPl regulates the NEAT1/miR-29b/Atg9a pathway.Furthermore,Atg9a regulates HSC autophagy,thus promoting HSC activation and ECM increase,thus leading to mouse liver fibrosis.