| Background:Hepatic stellate cells(HSCs)are non-parenchymal cell.HSCs activation is a key for promoting liver fibrosis,and various cytokines participate in this process.Transforming growth factor beta 1(TGFβ1)plays an important role in activation of HSCs,which continuously activates HSCs via directly or indirectly ways in the early and late stages.HSCs activation leads to unbalance of synthesis and degradation of extracellular matrix(ECM)and liver fibrosis.Autophagy is a biological process that cells degrade the damaged organelles,misfolded proteins and invasive pathogens.Autophagy has been proved to play an important role in liver fibrosis.Long-noncoding RNA(lncRNA)is a nucleotides whose transcription length is more than 200nt.Materally expressed gene 3(MEG3)was first discovered lncRNA which is an anti-tumor gene.Its expression in hepatocellular carcinoma was lower around 200 times than that of normal tissue.Meanwhile,it also plays an important role in hepatic fibrosis.Insulin-like growth factor binding protein related protein 1(IGFBPrP1),is a new fibrotic factor found by our group under the support of the National Natural Science Foundation of China.IGFBPrP1 can activate HSCs,increase ECM production and regulate each other with TGFβ1.TGFβ1 is the strongest effector in liver fibrosis,which can induce autophagy and affect the expression of lncRNA MEG3 in liver fibrosis.Since our group found that IGFBPrP1 and TGFβ1 regulate each other,Whether IGFBPrP1 can also induce autophagy and affect the level of lncRNA MEG3.It is currently unknown.Autophagy is regulated by multiple signaling pathways.PI3K/Akt/mTOR signaling pathway is particularly critical.HSCs activation is regulated by various cytokines and signaling pathways.Our group used Singnal Transduction Pathway Finder PCR Array technology to screen genes,and found that IGFBPrP1 might play a role in liver fibrosis through PI3K/Akt signaling pathway.It is not clear whether IGFBPrP1 can activate HSCs and regulate autophagy by phosphorylated Akt,m TOR.P53 is an anti-oncogene.LncRNA MEG3 can interact with P53 to play a role in inhibiting cancer.It is currently unknown the relationship among IGFBPrP1,lncRNA MEG3 and P53.This study is divided into six parts,from part one to part three,we adopted the primary rat HSCs,because of that primary HSCs has a stable biological character and can reflect the state of body.We used adenovirus vector carrying IGFBPrP1(AdIGFBPrP1).In addition,autophagy promoter rapamycin and autophagy inhibitor3MA was adopted to treat HSCs,we aimed to investigate the interactions among IGFBPrP1,autophagy,and activation of primary rat HSCs.From part four to part six,we adopted JS-1(mouse hepatic stellate cell line),because of that we used JASPAR database to predict the binding sites between MEG3 and P53,it was found that they had binding sites in mice and humans,but no binding sites were found in rats.Meanwhile,pcDNA3.1-MEG3 and shRNA-MEG3 was used to overexpress and silence MEG3 to clarify the relationship among IGFBPrP1,lncRNA MEG3 and P53.We aimed to investigate the effects of autophagy and long noncoding RNA MEG3 on hepatic stellate cells activated by IGFBPrP1 and then provide an experimental and theoretical basis for the early diagnosis and prevention of liver fibrosis.Part 1 The effect of AdIGFBPrP1 on primary rat hepatic stellate cells activationand autophagyObjective:After successfully isolating,identifing and culturing the primary rats HSCs,investigating whether AdIGFBPrP1 can induce autophagy in the activation of primary HSCs.Methods:HSCs were isolated from the liver of Sprague-Dawley rats,and were identified and cultured for subsequent experiments.First,AdIGFBPrP1 was transfected into rat primary HSCs with different MOI(10,20,40,80,100).After 48hours,green fluorescence of retinol esters was observed with inverted fluorescence microscope.Then the best MOI was screened according to transfection efficiency for subsequent experiments.HSCs were grouped as follows:normal group:HSCs were treated with 10%fetal bovine serum;CAd group:HSCs were treated with adenovirus vector carrying no cDNA;starvation group:HSCs were cultured with 2%FBS;Ad IGFBPrP1 group:HSCs were treated 24 h with AdIGFBPrP1 and then cultured in2%FBS for 3 h,6 h,12 hours,24 hours and 48 hours.Ultrastructural changes of HSCs in autophagy was detected with transmission electron microscopy;Autophagy lysosome was detected with MDC staining;LC3B expression was detected by immunofluorescence staining;IGFBPrP1,TGFβ1,α-SMA,LC3B mRNA levels were examined by quantitative PCR;IGFBPrP1,TGFβ1,collagen I,α-SMA,LC3B,Beclin1,SQSTM1/p62 protein levels were examined by western blot.Results:(1)The yield of the cultured HSCs was 3.5-4×10~7 cells per rat liver.Trypan blue staining,Cell survival rate was more than 80%according to trypan blue staining.Under the inverted fluorescence microscope,the cells with green fluorescence were more than 80%observed by inverted fluorescence microscope.(2)Cell morphology:cells were adherent after 24 h and stretched into heterogeneous morphology after 72 h.Most of HSCs showed myofibroblast-like morphology after 7d.(3)Desmin andα-SMA were positive in HSCs and the rate of positive cell was more than 80%.(4)The best transfection efficiency was MOI 80.(5)The expressions of IGFBPrP1 and TGFβ1 after Ad IGFBPrP1 transfection in rat primary HSCs:IGFBPrP1 protein and m RNA gradually increased,with a peak at 48h(P<0.05),showed that Ad IGFBPrP1 was transfected successfully.TGFβ1 mRNA and protein increased with time(P<0.05).(6)The expressions ofα-SMA and collagen I after AdIGFBPrP1 transfection in rat primary HSCs:the expressions ofα-SMA and collagen I started from 3 h,and gradually increased with time(P<0.05).(7)The expressions of LC3B,Beclin1 and SQSTM1/p62 after Ad IGFBPrP1 after transfection in primary rat HSCs:LC3B,Beclin1 protein and mRNA increased,with a peak at 12 h(P<0.05).SQSTM1/p62 protein level was opposite to Beclin1 and LC3B(P<0.05).(8)MDC staining:the bright spot increased significantly,representing the increased autophagy lysosome.(9)Transmission electron microscope:there were many bilayer vesicles in the cells,and some vesicles encapsulated organelles,such as mitochondria,and some vesicles were combined with lysosomes to form autophagy lysosomes.Conclusion:The primary rat HSCs were successfully isolated and cultured;IGFBPrP1 can induce HSCs autophagy in the primary rat,promote the continuous expression of TGFβ1,activate HSCs,and promote the synthesis of ECM.Part 2 The effect of Autophagy promoter or inhibitor on autophagy andactivation in AdIGFBPrP1-treated primary rat HSCsObjective:To determine whether IGFBPrP1 affect the autophagy and thus affect the activation of HSCs and ECM deposition.Methods:(1)The best time and concentration of rapamycin was screened,groups were divided as follows:normal group:10%FBS;rapamycin low concentration group:cells were treated with 25nmol/L rapamycin and cultured in 2%FBS for 6 h,12 h,24 h;rapamycin medium concentration group:cells were treated with 50nmol/L rapamycin and cultured in 2%FBS for 6 h,12 h,24 h;high concentration of rapamycin group:cells were treated with 100nmol/L treated rapamycin and cultured in 2%FBS for 6 h and 12 h,24 h.(2)HSCs were treated with Rapamycin and AdIGFBPrP1,the groups were as follows:normal group:10%FBS;Ad IGFBPrP1 group:cells were transfected with MOI 80Ad IGFBPrP1 and cultured with 2%FBS for 24 hours;rapamycin group:cells were cultured with 100nmol/L rapamycin and 2%FBS for 24 hours;AdIGFBPrP1+rapamycin group:cells were transfected with MOI 80 AdIGFBPrP1 and cultured with100nmol/L rapamycin in 2%FBS for 24 h.(3)The best time and concentration of 3MA was screened,groups were divided as follows:normal group:10%FBS;3MA low concentration group:the cell was used with 2.5mmol/L 3MA and were cultured in 2%FBS for 6 h,12 h,24 h;3MA medium concentration group:cells cultured with 5mmol/L 3MA and were cultured in 2%FBS for 6 h,12 h,24 h;high concentration of 3MA group:cells cultured with 10mmol/L rapamycin with 2%FBS for 6 h and 12 h,24 h.(4)HSCs were cultured with 3MA and AdIGFBPrP1,the groups were as follows:normal group:10%FBS;Ad IGFBPrP1 group:cells were transfected with MOI 80Ad IGFBPrP1 and cultured with 2%FBS for 24 hours;3MA group:cells were cultured with 10mmol/L 3MA and 2%FBS for 24 hours;AdIGFBPrP1+3MA group:cells were transfected with MOI 80 AdIGFBPrP1 and cultured with 10mmol/L 3MA in 2%FBS for 24 h.IGFBPrP1,TGFβ1,α-SMA,LC3B mRNA were examined by RT-qPCR;IGFBPrP1,TGFβ1,α-SMA,collagenI,LC3B,Beclin1,SQSTM1/p62 protein were examined by Western blot;autophagy lysosome was detected with MDC staining;LC3B expression was detected by Immunofluorescence staining.Results:(1)The best concentration and time of rapamycin were that 100nmol/L rapamycin was used to treat HSCs for 24 h.(2)HSCs were treated with rapamycin and AdIGFBPrP1,LC3B mRNA,protein level and green fluorescent spots were increased.Beclin1 protein level was increased.SQSTM1/p62 protein expression decreased(P<0.05).(3)MDC staining:bright particles in the group treated with rapamycin and Ad IGFBPrP1 were the most.(4)HSCs were treated with rapamycin and AdIGFBPrP1,the expression of IGFBPrP1and TGFβ1protein and m RNA increased significantly in the group(P<0.05).(5)α-SMA and collagen I level after rapamycin and AdIGFBPrP1 treated:the expression ofα-SMA and collagen I protein and mRNA levels increased(P<0.05).(6)The best concentration and time of 3MA were that 10mmol/L 3MA was used to treat HSCs for 24 h.(7)HSCs were treated with 3MA and AdIGFBPrP1,LC3B mRNA,protein level and green fluorescent spots were decreased.Beclin1 protein level was decreased.SQSTM1/p62 protein expression increased(P<0.05).(8)MDC staining:bright particles in the group treated with 3MA and Ad IGFBPrP1decreased.(9)HSCs were treated with rapamycin and AdIGFBPrP1,the expression of IGFBPrP1and TGFβ1 protein and mRNA levels decreased significantly in the group(P<0.05).(10)α-SMA and collagen I level after 3MA and Ad IGFBPrP1 treated:3MA inhibitedα-SMA and collagen I level which could be increased by IGFBPrP1(P<0.05).Conclusion:Increased autophagy can promote IGFBPrP1-stimulated induction of HSCs activation and ECM deposition;decrased autophagy can inhibit IGFBPrP1-stimulated induction of HSCs activation and ECM deposition.Part 3 Role of PI3K/Akt/mTOR signaling pathway in AdIGFBPrP1 inducedautophagy and activation in primary rat HSCsObjective:To clarify whether IGFBPrP1-induced HSCs autophagy via PI3K/Akt/m TOR signaling pathway.Methods:HSCs were treated with Ad IGFBPrP1,rapamycin and 3MA.The groups were divided as follows:normal group:HSCs were treated with 10%FBS;Ad IGFBPrP1 group:cells were transfected with AdIGFBPrP1 and cultured with 2%FBS for 12 hours;rapamycin group:cells were cultured with 100nmol/L rapamycin and 2%FBS for 24 hours;AdIGFBPrP1+rapamycin group:cells were transfected with AdIGFBPrP1 and cultured with 100nmol/L rapamycin in 2%FBS for 24 h.3MA group:cells were cultured with 10mmol/L 3MA and 2%FBS for 6 hours;Ad IGFBPrP1+3MA group:cells were transfected with AdIGFBPrP1 and cultured with 10mmol/L 3MA in 2%FBS for 6 h.Akt、p Akt、mTOR、pmTOR protein were examined by Western blot.Results:Ad IGFBPrP1 could inhibit the phosphorylation level of Akt and mTOR.Rapamycin combined with 3MA could increase the effect of Ad IGFBPrP1(P<0.05).Conclusion:IGFBPrP1 can promote autophagy via downregulating the phosphorylation levels of Akt and mTOR in hepatic stellate cells.Part 4 The expression of LncRNA MEG3 in JS-1 transfected with AdIGFBPrP1Objective:To identify the expression of lncRNA MEG3 in JS-1 transfected with Ad IGFBPrP1.Methods:Firstly,HSCs were transfected with different MOI(10,50,100,200)of Ad IGFBPrP1 and the intensity of green fluorescence was observed with inverted fluorescence microscope after 48 h.Transfection efficacy was assessed and the optimization of MOI was used for next experiments.The groups were divided as follows:normal group:cells were treated with 10%FBS;CAd group:HSCs were treated with adenovirus vector carrying no cDNA;AdIGFBPrP1 group:cells were transfected with MOI 100 AdIGFBPrP1 and cultured with 2%FBS for 3,6,12,24 h.The IGFBPrP1 and MEG3 RNA were detected in JS-1.Results:(1)The best transfection efficiency was MOI 100.(2)IGFBPrP1 mRNA gradually increased with time,indicating that the Ad IGFBPrP1gene was successfully transferred to JS-1.MEG3 RNA decreased with time(P<0.05).These results suggested that the decrease of MEG3 may be related to IGFBPrP1.Conclusion:In the hepatic stellate cells treated with AdIGFBPrP1,the expression of MEG3 RNA decreased following IGFBPrP1 mRNA was up-regulated.Part 5 The effect of lncRNA MEG3 up-regulation or down-regulation in JS-1autophagy induced by AdIGFBPr P1Objective:To determine the effect of lncRNA MEG3 up-regulation or down-regulation in JS-1 autophagy induced by IGFBPrP1.Methods:(1)AdIGFBPrP1 and pcDNA3.1-MEG3 were transfected into JS-1,the groups were divided as follows:normal group:cells were treated with 10%FBS;pcDNA3.1-MEG3+CAd group:cells were transfected with pcDNA3.1-MEG3 for 6 h and then cultured with MOI 100 CAd in 2%FBS for 24 h.pcDNA3.1-control+Ad IGFBPrP1group:cellsweretransfectedwith pcDNA3.1-control for 6 h and then cultured with MOI 100 AdIGFBPrP1 in 2%FBS for 24 h.pcDNA3.1-MEG3+AdIGFBPrP1:cells were transfected with pcDNA3.1-MEG3 for 6 h then cultured with MOI 100 Ad IGFBPrP1 in 2%FBS for24 h.(2)Ad IGFBPrP1 and shRNA-MEG3 were transfected into JS-1,the groups were divided as follows:normal group:cells were treated with 10%FBS;shRNA-MEG3+CAd group:cells were transfected with shRNA-MEG3 for 6 h then cultured withMOI 100 CAd in 2%FBS for 24 h.shRNA-NC+AdIGFBPrP1 group:cells were transfected with shRNA-NC for 6 h then cultured with MOI 100Ad IGFBPrP1 in 2%FBS for 24 h.shRNA-MEG3+Ad IGFBPrP1:cells were transfected with shRNA-MEG3 for 6 h then cultured with MOI 100 AdIGFBPrP1 in 2%FBS for 24 h.MEG3,IGFBPrP1,TGFβ1,LC3B,α-SMA mRNA were detected by RT-qPCR;IGFBPrP1、TGFβ1,LC3B,Beclin1,SQSTM1/p62,α-SMA,collagen I protein were examined by Western blot;the expression of LC3B was detected by fluorescence;autophagy lysosome was detected by MDC staining.Results:(1)LncRNA MEG3 increased after pcDNA3.1-MEG3 and AdIGFBPrP1 co-transfected in JS-1:but it was lower than that of pcDNA3.1-MEG3 group(P<0.05).(2)LC3B,Beclin1 and SQSTM1/p62 level after transfection of pcDNA3.1-MEG3and AdIGFBPrP1:pcDNA3.1-MEG3 and AdIGFBPrP1 transfected group significantly inhibited LC3B induced by AdIGFBPrP1,the green fluorescence decreased,Beclin1 level decreased,SQSTM1/p62 protein increased(P<0.05).(3)MDC staining:autophagy lysosome decreased in pcDNA3.1-MEG3 and Ad IGFBPrP1 transfected group.(4)IGFBPrP1 and TGFβ1 level decreased in pcDNA3.1-MEG3 and Ad IGFBPrP1transfected group(P<0.05).(5)α-SMA and collagen I level decreased in pcDNA3.1-MEG3 and Ad IGFBPrP1transfected group(P<0.05).(6)shRNA-MEG3 interference efficiency screening:constructing three lncRNA MEG3 interference sequences to transfect JS-1(小鼠肝星状细胞株).After 48 hours,RT-qPCR was used to detect MEG3 expression,and the most efficient interference sequence was selected.(7)MEG3 RNA level in shRNA-MEG3+AdIGFBPrP1 group is lower than that in shRNA-MEG3(P<0.05).(8)LC3B,Beclin1 and SQSTM1/p62 level after transfection of shRNA-MEG3 and Ad IGFBPrP1:shRNA-MEG3+Ad IGFBPrP1 significantly promoted LC3B induced by AdIGFBPrP1,the green fluorescence increased,Beclin1 level increased,SQSTM1/p62 protein decreased(P<0.05).(9)MDC staining:autophagy lysosome increased in shRNA-MEG3+Ad IGFBPrP1group.(10)IGFBPrP1 and TGFβ1 level increased in shRNA-MEG3+AdIGFBPrP1 group(P<0.05).(11)α-SMA and collagen I level increased in pcDNA3.1-MEG3+AdIGFBPrP1 group(P<0.05).Conclusion:LncRNA MEG3 inhibits autophagy in hepatic stellate cells induced by IGFBPrP1.Part 6 The effect of lncRNA MEG3 on transcription factor P53 in JS-1transfected with AdIGFBPrP1Objective:To explore whether lncRNA MEG3 affect autophagy via P53 after Ad IGFBPrP1 transfected with JS-1.Methods:(1)AdIGFBPrP1 and pcDNA3.1-MEG3 were transfected into JS-1,the groups were divided as follows:normal group:cells were treated with 10%FBS;pcDNA3.1-MEG3+CAd group:cells were transfected with pcDNA3.1-MEG3 for 6 h thenculturedwithMOI100CAdin2%FBSfor24h.pcDNA3.1-control+Ad IGFBPrP1group:cellsweretransfectedwith pcDNA3.1-control for 6 h then cultured with MOI 100 AdIGFBPrP1 in 2%FBS for24 h.pcDNA3.1-MEG3+AdIGFBPrP1:cells were transfected with pcDNA3.1-MEG3for 6 h then cultured with MOI 100 Ad IGFBPrP1 in 2%FBS for 24 h.(2)Ad IGFBPrP1 and shRNA-MEG3 were transfected into JS-1,the groups were divided as follows:normal group:cells were treated with 10%FBS;shRNA-MEG3+CAd group:cells were transfected with shRNA-MEG3 for 6 h then cultured withMOI 100 CAd in 2%FBS for 24 h.shRNA-NC+AdIGFBPrP1 group:cells were transfected with shRNA-NC for 6 h then cultured with MOI 100Ad IGFBPrP1 in 2%FBS for 24 h.shRNA-MEG3+Ad IGFBPrP1:cells were transfected with shRNA-MEG3 for 6 h then cultured with MOI 100 AdIGFBPrP1 in 2%FBS for 24 h.P53 mRNA were detected by RT-qPCR;P53 protein were examined by Western blot;the expression and location of P53 was detected by fluorescence;lncRNA MEG3 and transcription factor P53 interaction was detected by luciferase activity assay.Results:(1)P53 level decreased after pcDNA3.1-MEG3+AdIGFBPrP1 group than pcDNA3.1-MEG3 group,but it was more than that of Ad IGFBPrP1 group.These indicated overexpression of MEG3 can increase P53 after treated with IGFBPrP1(P<0.05).(2)Immunofluorescence was used to detect the location and expression of P53 protein after co-transfection of pcDNA3.1-MEG3 and AdIGFBPrP1:the red fluorescence was significantly increased,and a large amount of red fluorescence was distributed in cytoplasm.(3)Luciferase activity assay was used to detect P53 transcriptional activity after co-transfection of of pcDNA3.1-MEG3 and Ad IGFBPrP1:the activity of luciferase in the co-transfection group increased significantly,indicating the interaction between lncRNA MEG3 and P53.(4)The expression of P53 after shRNA-MEG3 transfection:the expression of P53 in co-transfection was reduced than Ad IGFBPrP1 group,indicating silencing of MEG3can reduce P53 activity after treated with IGFBPr P1(P<0.05).(5)The location and expression of P53 protein was detected by immunofluorescence after shRNA-MEG3 and AdIGFBPrP1 co-transfection:After gene silencing MEG3,the fluorescence intensity of P53 decreased.Conclusion:After treated with IGFBPrP1 in hepatic stellate cells,lncRNA MEG3interacted with P53 and increased P53 expression,thereby regulating the autophagy in hepatic stellate cells. |
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