| Backgroud and ObjectiveLiver fibrosis is a pathological process in which the normal physiological structure and function of the liver are destroyed due to the increase and deposition of extracellular matrix(ECM)when liver is stimulated by a variety of injury factors.It's a common process of several chronic hepatic disease that could progress to cirrhosis or liver cancer with a poor prognosis if not treated timely.However,there's no effective non-invasive methods for the diagnosis and treatment of this disease yet,so it's necessary to explore the molecular mechanism involed in the occurrence and development of liver fibrosis.HSC(Hepatic stellated cell)is the key cell that synthesize and excrete the main source of ECM during liver fibrogenesis,so researchers always focus on its transition from static state to activated state.It has been reported that a variety of non-coding RNAs including miRNA and lnc RNA are involved in the regulation of HSC activation.circRNAs,as a member of non-coding RNAs,are characterized by it's circular structure without 5'-terminal cap and 3'-terminal poly A tail.They are stablely exist in eukaryotic cells and play an important biological function by acting as miRNA sponge,gene transcription regulation,encoding peptides.Existing studies have found that circ RNA plays an important role in the occurrence of tumor,myocardial fibrosis,pulmonary fibrosis,et al,and most of the circ RNAs contains miRNA response elements(MRE),which can bind to miRNAs as endogenous competitive RNAs(ce RNA mechanisms)to weaken their inhibitory effect on target genes.However,in the field of liver fibrosis,several circ RNAs have been found to be involved in the development of this disease by affecting HSC activation,which suggest that the effects of circ RNA on the activation of HSC/ liver fibrogenesis should not be ignored.Based on the results of RNAseq in liver fibrosis tissues,we analyzed the differently expressed circ RNAs and selected circ RNA?1989 for further reseach,The Objective of this study was to investigate the role of circ RNA?1989 in the activation of HSC and the occurrence of liver fibrosis,and preliminarily explore the ce RNA mechanism of circ RNA?1989 targeting miR-185-3p /Col1a1,so as to provide a new molecular target for the study of liver fibrosis.The research mainly consists of three parts:(1)Screening and validation of differentially expressed cir RNAs in the process of liver fibrosis;(2)To investigate the effect of circ RNA?1989 on HSC activation/liver fibrogenesis in vitro;(3)Explore the ce RNA mechanism of circ RNA?1989 on regulating HSC activation/liver fibrogenesis.Part 1 Screening and validation of differentially expressed cir RNAs in the process of liver fibrosis Methods1.Clinical liver fibrosis samples were collected and stained with HE,RNAseq was performed after pathological observation according to Metavir semi-quantitative assessment system.The differentially expressed circ RNAs were analyzed by bioinformatics,and the interested circ RNAs were selected for further study.2.Stimulated LX-2 cell lines by human recombinant TGF-?1,liver fibrosis indicators ?-sma,Col11a1 and the selected circ RNAs were detected by q RT-PCR.3.Selected circ RNA?1989 for further research,found its homologous circ RNA in mouse was mmu?circ?0001283 by searching data website such as circ Base,circbank.And the mouse liver fibrosis model was constructed with DMN,after HE staining and pathological identification of liver tissue,the expression of mmu?circ?0001283 in liver tissues were detected by q RT-PCR.Results1.RNAseq analysis was performed in 6 liver fibrosis tissue samples(F1 2,F2 2,F3 1,F4 1),compared to normal tissues,2630 up-regulated and 1734 down-regulated circ RNAs were selected for clustering heat map,among them,6 Ecirc RNAs and 10 ci RNAs were selected for validation.2.After TGF-?1 stimulation,the expression of liver fibrosis indicators ?-sma and Col11a1 in LX-2 were increased 3.83 fold and 3.29 fold separately,p<0.05.Meanwhile,13 circ RNAs,including circ RNA?1989 were successfully verified during LX-2 activation.3.HE staining indicated that the liver fibrosis mice model was constructed by DMN,q RT-PCR results suggested that:compared with the Blank group,the expressions of ?-SMA and Col11a1 in DMN-liver fibrosis tissues was increased 8.12 fold and 4.73 fold,separately,p<0.05,meanwhile,the expression of mmu?circ?0001283 was increased 2.52 fold,p<0.05.Conclusions1.There are various circ RNAs differently expressd in the process of HSC activation and liver fibrogenesis;2.Circ RNA?1989 was increased in the activation of HSC and the development of liver fibrosis.Part 2 To investigate the effect of circ RNA?1989 on HSC activation/liver fibrogenesis in vitro Methods1.According to circ RNA?1989 gene sequence information,3 si RNA targets were designed for plasmid construction and lentivirus packaging,grouped as NC,KD1,KD2,KD3;2.Infected LX-2 with circ RNA?1989 interfered lentivirus,q RT-PCR was conducted to detect the interference efficiency of 3 si RNA targets,and the best one was selected;3.Knockdown circ RNA?1989 expression in LX-2,CCK-8 assay was conducted to test the cell OD450 value in 5 days continuously,for detecting the changes in cell proliferation and activation ability;Annexin V-APD single staining with flow cytometry were used to determine whether the cells were apoptotic;q RT-PCR and Western blot were conducted to detect the changes of liver fibrosis indicators,?-SMA and Col11a1.Results1.Circ RNA?1989 interfering lentivirus was successfully constructed,the infected LX-2 emit green fluorescence witih the rate of 80%.Compared to NC,the interference efficient of KD1 is 25.07%,KD is 24.10%,KD3 is 72.27%,p<0.05,so we choose KD3 for cellular function experiments.2.Down-regulated circ RNA?1989 in LX-2,CCK-8 assay showed that,on day 2,there was no significant difference in the OD450 values between group NC and group KD,p = 0.23;but on day 3?5,OD450 value in group KD were lower than that in NC group,p<0.05;compared with day1,OD450 fold of the KD group on day 2?5 were lower than that of the NC group,p<0.05;meanwhile,apoptosis ratio in group NC is(2.63±0.1)%,in group KD is(13.27±0.90)%,so it's increased 5.04 fold in group KD,p<0.05;3.The m RNA expression of ?-SMA and Col11a1 in cric RNA?1989 knockdown LX-2 were decreased by 81.87% and 30.88%,separately,p<0.05;Western blot results indicated that the protein expression of ?-SMA and Col11a1 were both significantly decreased.ConclusionsThree si RNA targets were designed for plasmid construction and lentivirus packaging,circ RNA?1989 were down-regulated in LX-2 stablely.Subsequently,the cell proliferation and activation ability were inhibited,and apoptosis rate was increased,meanwhile,the expression of ?-SMA and Col11a1 were decreased in m RNA and protein level.These results indicated that circ RNA?1989 may be involved in the development of liver fibrosis by promoting the activation of HSC and inhibiting its apoptosis.Part3 Explore the ce RNA mechanism of circ RNA?1989 on regulating HSC activation/ liver fibrogenesis Methods1.By bioinformatics analysis,the miRNAs that may have binding sites with circ RNA?1989 were obtained and a reticulation analysis map were drawed;miRNAs that differentially expressed in liver fibrosis tissues were screened by RNAseq,and the heat map was obtained.The intersection of the two results indicates that miR-185-3p may be the targeted miRNA of circ RNA?1989;2.miR-185-3p was detected in TGF-?1 stimulated LX-2 and DMN induced liver fibrosis mice by q RT-PCR.On Target Scan database,the target gene of miR-185-3p were selected,and verified by double luciferase reporter assay;transfected LX-2 with miR-185-3p mimics/inhibitor,the liver fibrosis indicators were detected by q RT-PCR and Western blot;3.According to the bingding sites between circ RNA?1989 and miR-185-3p,3 plasim were conducted:binding sequence plasmid(GACCA),Complementary sequence plasmid(CTGGT),Mutant sequence plasmid(GCAAG);double luciferase reporter assay verified the bingding sites of circ RNA?1989 and miR-185-3p.miR-185-3p was detected by q RT-PCR when circ RNA?1989 were interfered;circ RNA?1989 was detected when miR-185-3p mimics/inhibitor were transfected.Results1.Bioinformatics analysis showed that circ RNA?1989 may binding with miR-185-3p,miR-1225,miR-484,et al;while RNAseq results suggested that miR-185-3p was down-regulated in liver fibrosis tissues,so it was selected as a targeted miRNA for circ RNA?1989;2.The results of q RT-PCR indicated that,compared with Blank group,the expression of hsa-miR-185-3p in TGF-?1 stimulated LX-2 was decreased by 60.17%,p<0.05;by searching on miRbase,we found the gene sequence of miR-185-3p was highly homologous in human and mouse populations,and the expression of mmu-miR-185-3p in DMN induced mice liver fibrosis tissues was down-regulated by 54.68%,p<0.05;3.Target Scan suggested that there are 7 ribonucleotides of hsa-miR-185-3p are complementary to the 642-648 sites of Col1a1 3'UTR.Col1a1 WT(wild type)plasmid and Col1a1 MUT(mutant type)plasmid were constructed,and the dual-fluorescence reporter gene experiment was conducted for double luciferase reporter assay,the results indicated that:compared with cells co-transfected with Col1a1 WT plasmid and miRNA inhibitor NC,the ratio of firefly luciferase to renilla luciferase in 293 T cells co-transfected with Col1a1 WT plasmid and miR-185-3p inhibitor was increased by 1.87 fold,p<0.05;however,compared with cells co-transfected with Col1a1 MUT plasmid and miRNA inhibitor NC,the ratio of firefly luciferase to renilla luciferase in 293 T cells co-transfected with Col1a1 MUT plasmid and mir-185-3p inhibitor was not statistically significant,p=0.993.4.The results of q RT-PCR suggested that after transfection with miR-185-3p inhibitor,the expressions of Col1a1 and ?-SMA in LX-2 were increased 2.12 fold and 2.97 fold,separately,p<0.05.After transfection with miR-185-3p mimics,the expression of Col1a1 and ?-SMA were decreased 70.89% and 87.77%,separately,p<0.05.Western blot results suggested that the protein expression of Col1a1 and ?-SMA in LX-2 transfected with miR-185-3p inhibitor was increased,whereas decreased when miR-185-3p mimics were transfected.5.Bioinformatics predicted that there are 5 ribonucleotides of miR-185-3p bind to circ RNA?1989 at 159-163 sites.The results of double fluorescence reporter assay suggested that: compared with the cells co-transfected with the original sequence plasmid and miR-185-3p mimics,the ratio of firefly luciferase to renilla luciferase in the cells co-transfected with the complementary sequence plasmid and mir-185-3p mimics was increased 4.38 fold,while the ratio of firefly luciferase to renilla luciferase in the cells co-transfected with the mutant sequence plasmid and mir-185-3p mimics was increased 4.46 fold,p< 0.05;6.The results of q RT-PCR showed that the expression of circ RNA?1989 in LX-2 transfected with mir-185-3p inhibitor was 1.96 fold higher than that of the NC group,p<0.05.The expression of circ RNA?1989 in LX-2 transfected with miR-185-3p was decreased 37.55%,p<0.05.After knocking down circ RNA?1989 in LX-2,the expression of miR-185-3p was increased 1.93 fold,p<0.05.Conclusions1.It seems that circ RNA?1989 may bind to a variety of miRNAs and may take biological functions by acting as miRNAs sponge;and miR-185-3p is one of its potential target miRNA;miR-185-3p may regulate the activation of HSC by targeting Col1a1,and inhibit HSC activation/liver fibrogenesis;2.miR-185-3p may target with circ RNA?1989,as a ce RNA,circ RNA?1989 may weaken the inhibition effects of miR-185-3p on Col1a1,and reverse its regulatory effect on HSC activation/liver fibrogenesis. |
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