MiR-185 Promote Hepatic Fibrosis Through Targeting SCARB1

BackgroundLiver fibrosis is a common pathological condition and is a pathological change because of chronic liver injury caused by various causes,which is represented by the accumulation of extracellular matrix(ECM).Liver fibrosis is a response to the healing of chronic liver injury and scarring.As a result of long-term damage,liver fibrosis may progress to cirrhosis,hepatic carcinoma and liver failure.However,there is no effective method to control liver fibrosis so far,so it is necessary to further study the specific molecular mechanism of liver fibrosis.Hepatic stellate cells(HSC),also called fat-storing cells,are the main cells that produce ECM during liver fibrogenesis.In recent years,the impact of epigenetic changes on the expression of liver fibrosis-related genes and the activation of HSC has been increasingly valued.Among them,the epigenetic regulatory mechanism of miRNAs plays an important role in many biological processes and metabolic homeostasis,including protein secretion and fatty acid metabolism.Studies have shown that micro RNA-30 a,micro RNA-9-5p,miR-142-3p and other micro RNAs can promote or inhibit HSC activation.Therefore,the study of the role of miRNAs in the activation of HSCs is of great significance in inhibiting or even reversing hepatic fibrosis.The Scavenger Receptor Class B Type I(SR-BI)is a member of the Class B family of scavenger receptor proteins and is a receptor for high-density lipoprotein(HDL).The gene encoding SR-BI has been named SCARB1.Selective lipid uptake(SLU)mediated via SR-BI plays a key role in reverse transport of cholesterol.And SR-BI is under the control of different nuclear receptors and transcription factors,including peroxisome proliferator-activated receptor(PPAR),liver X receptor(LXR),and liver receptor homolog-1(LRH-1)and sterol regulatory element binding proteins(SREBPs).These nuclear factors and related molecules participate in liver fibrosis by affecting inflammation and lipid metabolism.However,the mechanism of SCARB1 in liver fibrosis has not been reported.Our previous study found that miR-185 was up-regulated in peripheral blood of patients with hepatic fibrosis and could be used as a marker of peripheral blood circulation for the diagnosis of hepatic fibrosis,and related articles have been published.To further study the mechanism of miR-185 promoting hepatic fibrogenesis,we first used bioinformatics analysis and dual luciferase reporter assays to screen and verify that SCARB1 is the target gene for miR-185.Afterwards,we successfully constructed a stable cell line that interferes with SCARB1 expression,and confirmed that miR-185 targets SCARB1 in human HSCs to affect HSC activation and promote hepatic fibrosis formation,providing a new way for clinical prevention and treatment of liver fibrosis.Part 1: Screening and Validation of miR-185 Target GenesObjective Screening and Validating the Target Gene of miR-185Methods1.Predictive analysis of miR-185 by miRNA target gene prediction software such as Targetscan 、miRWalk andmiRDB.2.Detection of miR-185 and SCARB1 targeting in HEK293 instrumental cells: Construct wild-type or mutant plasmid carrying SCARB1 and co-transfect 293 T cells with miR-185.After transfection for 72 hours,use luciferase reporter system to detect the difference in luciferase expression and screen out the direct target SCARB1 of miR-185.3.Detection of miR-185 and SCARB1 targeting relationships in HSC: HSCs were transfected with miR-185 mimic and miR-185 inhibitor,and the expression level of SCARB1 in cells was detected by qRT-PCR and Western blot,And the changes of hepatic stellate cell activation markers col? and α-SMA.Results1.The miRNA target prediction software predicts that SCARB1 and miR-185 have binding sites in the 3’-UTR region and are potential targets for miR-185.2.Target gene identification results: The ratio of dual luciferase in cells transfected with the SCARB1 wild type plasmid and the miR-185 overexpression plasmid was reduced,and the difference was statistically significant.MiR-185 showed direct regulation of SCARB1 and inhibited its expression.3.Verification results of the targeting relationship between miR-185 and SCARB1 in HSC: Compared with the blank group and the negative control group,the expression of SCARB1 was decreased and the expression of col I and α-SMA increased after transfection of miR-185 mimic in HSC.However,there was no significant difference in the expression of SCARB1,col I and α-SMA between the blank group and the NC group;Compared with the blank group and the negative control group,the expression of SCARB1 increased after miR-185 inhibitor,and the expression of col I and α-SMA decreased.There was no significant difference in the expression of SCARB1,col I,and α-SMA between the blank group and the NC group.Conclusions1.Biological information predicts that SCARB1 is a target gene for miR-185.2.Luciferase report that SCARB1 is a direct target of miR-185.3.Overexpression of miR-185 can promote HSC activation,down-regulation of miR-185 can inhibit HSC activation.Part 2 Study on Mechanism of miR-185 Targeting SCARB1 to Activate HSCObjective Elucidate the biological mechanism by which miR-185 targets SCARB1 for HSC activation/hepatic fibrosis progression.Methods1.Construct three sh RNA-stable cell lines sh1,sh2,sh3 that interfere with SCARB1 expression.The qRT-PCR was used to detect the interference efficiency of the three stable strains.Select the highest interference efficiency for subsequent experiments.2.Rescue assay:qRT-PCR and Western blot were used to detect the expression of SCARB1 and the expression of HSC activation markers col? and α-SMA in HSCs transfected with miR-185 inhibitor group in the SCARB1 interference stable group and the SCARB1 interference stable group.3.CCK8 proliferation experiments demonstrated whether miR-185 targets SCARB1 affects HSC activation.4.Apoptosis experiments confirmed whether miR-185 targeting SCARB1 affects HSC apoptosis.Results1.qRT-PCR was used to detect the interference efficiency of SCARB1 in sh1,sh2,sh3 in three different interference stable cell lines.The sh1 interference-stable cell line SCARB1 had the highest interference efficiency,so it was selected as the SCARB1 interference stable cell line during the next experiment.2.The results of rescue assay showed that compared with the SCARB1 interference control group,the expression of SCARB1 in the SCARB1 interference-stable group was significantly decreased,and the expression of col? and α-SMA was significantly increased;After transfection of miR-185 inhibitor in the SCARB1 interference-stable group,compared with the SCARB1 interference-stable group,SCARB1 expression increased while col? and α-SMA decreased.It was further verified that miR-185 targets SCARB1 to promote HSC activation/liver fibrosis.3.CCK8 proliferation test results:(1)Compared with the blank control group and the SCARB1 interference control group,transfection with SCARB1 interfered with lentivirus-stable cell significantly proliferated at 24 h,48 h,72 h and 96 h after transfection.(2)After transfected with miR-185 inhibitor in the SCARB1 interference-stable group,cell proliferation was inhibited at 24 h,48 h,72 h,and 96 h after transfection compared with the SCARB1 interference-stable group,with statistical significance.4.Apoptosis experimental results:(1)Compared with the blank control group and the SCARB1 interference control group,cell apoptosis was decreased 48 h after transfection of the SCARB1 lentivirus-stable stable strain,and there was a statistical difference.(2)48h after transfection of miR-185 inhibitor in the SCARB1 interference-stable group,apoptosis was increased compared to the SCARB1 interference-stable group.Conclusions1.Successfully constructed SCARB1 interference stable cell lines.2.miR-185 promotes HSC activation by targeting SCARB1,which in turn promotes the formation of hepatic fibrosis.3.Interference with SCARB1 gene expression can promote HSC proliferation.4.Interfering with SCARB1 gene expression can inhibit HSC apoptosis.