| ?Background and objective?Hepatic fibrosis is a chronic liver injury characterized by the excess accumulation of extracellular matrix(ECM)components in liver,which can be regulated by multiple factors.Previous researches have shown that the injury induced by chronic liver fibrosis was irreversible once progressed to liver cirrhosis.However,in recent years,several studies have suggested that hepatic fibrosis is a dynamic and reversible pathological process during the early stage.In other word,it can be inhibited and even reversed.Many studies have demonstrated that the activation and the proliferation of hepatic stellate cells(HSCs)play critical role in hepatic fibrosis.It has been widely confirmed that HSC is in a quiescent condition in healthy liver tissue.Nevertheless,once the liver is infected by viruses,parasites or injured physicochemically,HSCs will be activated and transdifferentiate into myofibroblast(MFS)gradually.Moreover,the activated HSCs have the leading role in ECM production.Therefore,a further understanding about the mechanism of HSC activation and searching for gene targets to regulate HSC activation may provide a novel strategy to treat hepatic fibrosis.Long non-coding RNA(lnc RNA)is commonly defined as an RNA molecule with the length of 200bp-100 kbp that lacks protein-coding function.Lnc RNAs can interact with transcription factors,chromatin histone and other transcriptional regulatory proteins to regulate gene expression and chromosomal activity at the transcriptional level directly.Additionally,it can participate in RNA translation and shearing,degradation and phosphorylation modification of proteins,subcellular localization of RNA binding proteins and even cytoskeleton composition.Lnc RNAs are also considered as a competing endogenous RNA(ce RNA)to sponge other endogenous RNAs to regulate the expression of related downstream genes at posttranscriptional level.Autophagy is a lysosomal-dependent signal pathway that widely exists in eukaryotic cells,which provides energy and maintains metabolization by digesting damaged or redundant organelles.Autophagy includes macroautophagy,microautophagy and chaperone-mediated autophagy,and the autophagy we studied is macroautophagy.Some studies have found that the activating autophagy of HSCs exacerbate fibroblast transition and lead to hepatic fibrogenesis.Furthermore,the inhibition of autophagy down-regulates pre-type I collagen m RNA expression.There are many genes havingbeen confirmed to be associated with autophagy regulation,and autophagy-related gene 5(ATG5)is the one of them.With the development of hepatic fibrosis,ATG5 expression is up-regulated in liver septa and peri-vascular areas,which are consistent with the distribution of HSC.Therefore,inhibiting the expression of ATG5 and its downstream autophagy-related factors may provide a new therapy for treating hepatic fibrosis.In our previous study,we analyzed the differential expression of lnc RNAs in the liver tissue from 5 healthy volunteers and 4 liver cirrhosis patients by Arraystar Human lnc RNA Array v2.0 8*60k chips(covering 33,045 lnc RNAs and 30,215 m RNAs).Based on the data,we found the significantly upregulated lnc RNA,uc.420-,in liver cirrhosis tissues and confirmed the differential expression in hepatic fibrosis rat models.Meanwhile,we also found that the upregulation of uc.420-expression is accompanied by the downregulation of mi R-194-5p expression and upregulation of ATG5 expression,and silencing uc.420-expression promotes mi R-194-5p upregulation in HSC-T6 cell line.In summary,we hope to find a signal pathway consisting of uc.420-,mi R-194-5p and ATG5 and prove its influence on HSC autophagy and activation in hepatic fibrogenesis,which may provide a potential strategy for the treatment of hepatic fibrosis.?Methods?1.The interactions of mi R-194-5p with uc.420-and ATG5,and differential expression of uc.420-,mi R-194-5p,ATG5,autophagy related genes and fibrosis related genes in human liver cirrhosis tissues,animal model liver tissues,quiescent and activated primary HSC(1)Bioinformatics analysis is performed through the websites,mi Rmap,to predict the binding sites between mi R-194-5p and its downstream target gene ATG5 3'-UTR.(2)Reporter gene plasmids including wild type uc.420-reporter plasmid(WT-uc.420-)and mutation type uc.420-reporter plasmid(MUT-uc.420-)were constructed,and mi R-194-5p oligonucleotides(mi R-194-5p mimic)was synthesized.WT-uc.420-and MUT-uc.420-was co-transfected with mi R-194-5p mimic respectively into HEK-293 cells.And WT-uc.420-and MUT-uc.420-was co-transfected with NC mimic respectively in the same way.The luciferase activity of report gene was detected to prove mi R-194-5p is the target gene of uc.420-.(3)Reporter gene plasmids including wild type ATG5 3'-UTR reporter plasmid(WT-ATG5 3'-UTR)and mutation type ATG5 3'-UTR reporter plasmid(MUT-ATG53'-UTR)were constructed.The two different mimics mentioned above and the two kinds of reporter gene plasmids were co-transfected into HEK-293 cells as following groups:WT-ATG5 3'-UTR + mi R-194-5p mimic group,WT-ATG5 3'-UTR + NC mimic group,MUT-ATG5 3'-UTR + mi R-194-5p mimic group and MUT-ATG5 3'-UTR + NC mimic group.The luciferase activity of report gene was detected to prove ATG5 is the target gene of mi R-194-5p.(4)After obtaining the informed consent of the patients and signing the relevant letters of authorization,5 specimens of normal liver tissue and 5 specimens of liver tissue diagnosed as cirrhosis were retained.(5)Construction of animal model of hepatic fibrosisIn order to clarify the effect of uc.420-on hepatic fibrosis,TAA was used to induce hepatic fibrosis in rats.TAA liver fibrosis model: Male SD rats were divided into 2 groups randomly(10 rats in each group).The liver fibrosis model was induced by intraperitoneal injection with 20mg/ml TAA solution at a dose of 1 ml/100 g,three times a week,and sacrificed them to save the liver tissues after 16 weeks.Intraperitoneal injection with 0.9% saline at a dose of 1ml/100 g was performed to the control group three times a week,and them to save the liver tissues after 16 weeks.(6)Collagenase-modified in situ circular perfusion method was used to separate primary SD rat HSC.The primary HSCs mentioned above were divided into 3 groups:group 1 quiescent primary HSCs,group 2 and group 3 with TGF-?1(2ng/ml)stimulated3d and 7d respectively.(7)Detection of the expression of uc.420-,mi R-194-5p,ATG5,autophagy pathway genes and fibrosis related genesReal time RT-PCR was used to detect the differential expression of uc.420-and mi R-194-5p in liver tissue of patient with hepatic cirrhosis,liver tissue of hepatic fibrosis models and activated primary HSCs of rats in different period.Real time RT-PCR and western-blot were used to determine expression of ATG5 and autophagy related genes,including ATG5,ATG7,LC3,Beclin 1 and p62,and fibrosis related genes including TIMP2,MMP2,MMP9,COL?A1,Col-? and ?-SMA in patients with liver cirrhosis,animal model of liver fibrosis,quiescent primary HSCs,and activated primary HSCs in different period.2.Inhibition of uc.420-regulated mi R-194-5p,autophagy related genes and fibrosis related genes in vitro(1)The uc.420-small interfering RNA(si-uc.420-)has been designed and synthesized to down-regulate uc.420-expression in our previous study.In vitro,the uc.420-expression of the si-uc.420--transfected HSC-T6 is downregulated,and the expression of mi R-194-5p,ATG5,autophagy related genes and fibrosis related genes are detected at RNA level.(2)Mi RZip-shuc.420-lentiviral recombinant plasmid and its negative control,mi RZip-sh NC lentiviral recombinant plasmid,has been designed and synthesized,which all have puromycin(PURO)resistance.And then the plasmids were packaged in HEK-293 cells to obtain the lentiviruses lenti-shlnc.420-and lenti-sh NC.(3)The concentration of PURO was detected by killing curve.HSC-T6 was infected by lenti-shuc.420-and lenti-sh NC respectively for 48 h.After that,changing the medium with PURO(2ng/ml)to obtain the stable cell strain silencing uc.420-,lenti-shuc.420--HSC-T6 and its negative control stable cell strain,lenti-sh NC-HSC-T6.The expression of uc.420-and mi R-194-5p was detected at RNA level.The expression of ATG5,autophagy related genes and fibrotic genes were detected by Real time RT-PCR and western-blot.(4)The primary SD rat HSCs were obtained by the above-mentioned protocol and change the culture medium every 2 days.After being activated by TGF-?1(2ng/ml)for7d,the primary HSCs are infected by lenti-shlnc.420-or lenti-sh NC.And The expression of ATG5,autophagy related genes and fibrotic genes were detected by Real time RT-PCR.3.Inhibition of uc.420-regulated cell biological activity and autophagy activity of activated primary HSC and HSC-T6 in vitro(1)The cell biological activity of lenti-shuc.420--HSC-T6 and lenti-sh NC-HSC-T6 is tested as following methods: transwell method is used to detect the transfer ability,CCK-8 assay is used to observe the cell strains' proliferation,and apoptosis is detected by flow cytometry according to manufacturer's instructions.(2)The primary HSCs in rats are obtained as the protocol mentioned above and are activated by TGF-?1(2ng/ml)for 7d.And then,the activated primary HSCs are divided into 2 groups to be infected by lenti-shuc.420-and lenti-sh NC,respectively.The cell biological activity of the 2 groups are tested by transwell method,CCK-8 assay and flowcytometry according to manufacturers' instructions.(3)The autophagosomes in the cells of lenti-shuc.420--HSC-T6 and lenti-sh NC-HSC-T6 are counted through transmission electron microscope.To test the change of autophagy flux,lenti-shuc.420--HSC-T6 and lenti-sh NC-HSC-T6 are infected by m RFP-GFP-LC3 adenovirus according to the manufacturer's protocol,and the luciferase activity of autophagy flux in the stable cell strains are examined under confocal microscope.(4)The autophagosomes in the activated primary HSCs infected by lenti-shuc.420-or lenti-sh NC are observed via transmission electron microscope and counted.To test the change of autophagy flux,2 groups of infected activated primary HSCs were infected by m RFP-GFP-LC3 adenovirus according to manufacturer's protocol,then the luciferase activity of autophagy flux in the 2 groups were observed through confocal microscope.4.Statistical analysisThe analysis of variance(ANOVA)and the Student t test were used for comparison of normally distributed data among the groups and between paired data respectively.Data not normally distributed are compared using the Manne Whitney tests.All results are presented at least three independent experiments and values are reported as mean ąSD and a P value <0.05 was considered significant.?Result?1.The target interactions of mi R-194-5p with uc.420-and ATG5(1)Cluster heatmap and bioinformatics analysis demonstrated that mi R-194-5p is the target gene of uc.420-,and ATG5 is the target gene of mi R-194-5p.(2)The result revealed that,compared with the group WT-uc.420-+ NC mimic,the luciferase activity of the group WT-uc.420-+ mi R-194-5p mimic reduced 44.5%(P<0.01).Compared with the group MUT-uc.420-+ NC mimic,there was no significant change in luciferase activity in group MUT-uc.420-+ mi R-194-5p mimic.(3)The result suggested that compared with the group WT-ATG5 3?-UTR + NC mimic,the luciferase activity of the group WT-ATG5 3?-UTR + mi R-194-5p mimic was reduced 37.6%(P<0.01).And compared with the group MUT-ATG5 3?-UTR + NC mimic,there was no significant change in luciferase activity in group MUT-ATG53?-UTR + mi R-194-5p mimic.2.Differential expression of uc.420-,mi R-194-5p,autophagy related genes andfibrosis related genes in human liver cirrhosis tissues,animal model liver tissues,quiescent and activated HSCReal time RT-PCR analysis showed that the expression of uc.420-,ATG5,ATG7,LC3,Beclin 1,Col-?,?-SMA,MMP2 and MMP9 was up-regulated,but the expression of mi R-194-5p and TIMP2 was down-regulated in liver tissues of human liver cirrhosis and liver fibrosis model,as well as in activated HSC.At protein level,the expression of ATG5,ATG7,LC3A/B,Beclin 1,COL?A1 and ?-SMA was up-regulated,but the expression of p62 was down-regulated.3.Down-regulation of uc.420-inhibits the expression of ATG5,autophagy pathway genes and fibrosis-related genes in vitro(1)After transfected by si-uc.420-,the expression of mi R-194-5p and TIMP2 significantly promoted and the expression of ATG5,ATG7,LC3,Beclin 1,Col-?,?-SMA,MMP2 and MMP9 are all inhibited at RNA level.(2)After infected by lenti-shuc.420-or lenti-sh NC,compared with the lenti-sh NC group,the lenti-shuc.420-group expression of mi R-194-5p and TIMP2 significantly promoted and the expression of ATG5,ATG7,LC3,Beclin 1,Col-?,?-SMA,MMP2 and MMP9 are all inhibited at RNA level and protein level.4.Inhibition of uc.420-regulated cell biological activity and autophagy activity of activated primary HSC and HSC-T6 in vitro(1)Compared with the group lenti-sh NC-HSC-T6,lenti-shuc.420-exerted a vital role on inhibiting proliferation of group lenti-shuc.420--HSC-T6 and inducing the cell apoptosis.Meanwhile,transwell assay results showed that the migration of lenti-shuc.420--HSC-T6 was inhibited significantly by lenti-shuc.420-.(2)After infected by lenti-shuc.420-or lenti-sh NC,compared with the group lenti-sh NC,the uc.420-downregulation in group lenti-shuc.420-exerts a critical role on inhibiting proliferation of activated hepatic fibroblast and inducing the cell apoptosis.(3)Through transmission electron microscope,autophagosomes in the cells of group lenti-shuc.420--HSC-T6 are less than in group lenti-sh NC-HSC-T6.Furthermore,compared with the group lenti-sh NC-HSC-T6,the luciferase activity of autophagy flux in group lenti-shuc.420--HSC-T6 is significantly inhibited after infection of m RFP-GFP-LC3 adenovirus.(4)Through transmission electron microscope,autophagosomes in the primaryHSCs in rats of group lenti-shuc.420-are less than in group lenti-sh NC-.Furthermore,compared with the group lenti-sh NC-,the luciferase activity of autophagy flux in group lenti-shuc.420--HSC-T6 is significantly inhibited after infection of m RFP-GFP-LC3 adenovirus.?Conclusion?1.uc.420-and ATG5 are target genes of mi R-194-5p.2.The expression of uc.420-is up-regulated and the expression of mi R-194-5p is down-regulated in human cirrhotic liver tissues,animal model liver tissues and activated primary HSCs in rats.Meanwhile,the expression of autophagy related genes and fibrosis related genes are activated significantly in human cirrhotic liver tissues,animal model liver tissues and activated primary HSCs in rats.3.Silencing uc.420-can promote the upregulation of mi R-194-5p and inhibit the expression of autophagy related genes and fibrosis related genes.4.Silencing uc.420-can lead to a significant attenuation on the biological characteristics of HSC.5.Silencing uc.420-can lead to a significant attenuation autophagy in HSC. |
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