| Background:Cholestasis is a kind of digestive system disease caused by a variety of causes,such as bile acid,bilirubin and other excretions,which can't be normally discharged from the liver,leading to a series of symptoms caused by the elevated liver bile acid levels.At the early stage of cholestasis,the liver will produce a series of adaptive responses to protect the hepatocytes,such as:reducing the synthesis of bile acids in the liver,reducing the uptake of bile acids,promoting the efflux of bile acids,and increasing the activity of liver detoxification enzymes.If the condition runs out of the liver adaptive responses,it will lead to liver injuries,liver fibrosis,and even cirrhosis.The transportation of bile acids mainly relies on transport proteins on the hepatocyte membrane,including NTCP(Na~+-taurocholate cotransporting polypeptide),MRPs(multidrug resistance-associated proteins)and OATPs(organic-anion-transporting polypeptides).Studies have found that there are 11 kinds of OATPs transporters in human,especially OATP1A2,OATP1B1,OATP1B3 and OATP2B1.Studies have found that in human cholestasis liver,OATP1B1 and OATP1B3 are significantly reduced,which can significantly reduce the uptake of bile acids by liver cells and reduce the accumulation of bile acids in liver cells,thereby protecting the liver.OATP3A1 is located on the basolateral membrane of hepatocytes,which is a transporter of bile acids,bilirubin and other excretions.Studies have shown that OATP3A1 is upregulated in human obstructive cholestasis,which has the function of promoting intrahepatic bile acid excretion and protecting the liver.Cholesterol 7 alpha-hydroxylase(CYP7A1)is the key rate-limiting enzyme in the synthesis of bile acids,which converts cholesterol into bile acids.Studies have reported that CYP7A1 is significantly repressed in patients with alcoholic liver disease and cholestasis,which in turn reduces the synthesis of intrahepatic bile acids,thereby reducing the liver injury of bile acid.Liver receptor homolog-1(LRH-1)is a nuclear receptor,which is encoded by the NR5A2 gene and expressed mainly in the liver,intestine and pancreas.LRH-1 could promote CYP7A1 and CYP8B1 transcription and is involved in bile acid homeostasis and cholesterol transportation.MicroRNAs(miRNAs)are a kind of small non-coding RNAs that bind to the 3'UTR region of a downstream target gene to degrade the target gene and regulate biological functions.The miR-200 family is widely distributed in various tissues of human body,which mainly includes miR-200a,miR-200b,miR-200c,miR-141 and miR-429.It is widely involved in various biological activities,miR-200b decomposes into miR-200b-3p and miR-200b-5p in the cell,thereby regulating the expressions of target genes.There have been few studies on miR-200b-3p before,our previous studies have shown that miR-200b-3p is significantly elevated in cholestasis miRNA microarray in human liver tissue,which has not been verified in human liver tissue.The relationships between miR-200b-3p and OATPs and bile acid synthase remain still unclear.In this study,35human tissue samples were collected to investigate the relationship between miR-200b-3p and bile acid-synthetic enzymes and OATPs in cholestatic liver diseases.Methods:1.35 liver tissue samples were collected from patients undergoing liver surgery in the department of hepatobiliary surgery,including 24 obstructive cholestatic liver samples and11 control liver samples.All liver tissues were immediately cut into small pieces,which weight about 30mg and then stored in liquid nitrogen.2.The liver functions of all enrolled patients were detected by enzyme labeling,especially including AST,ALT,ALP,GGT,TBIL,DBIL,IBIL and TBA.3.MiRNA microarray was performed with cholestasis patients and normal control patients,and the several differentially related miRNAs were found.The mRNA levels of miR-200b-3p,miR-4665-5P,miR-363-5p,miR-3609 and OATP3A1 were detected in obstructive cholestasis and normal control group.4.Correlation analysis between miR-200b-3p with liver function indexes and OATP3A1 was performed.5.50 ng/mL FGF19,TNF-?,IL-1?,IL-8,IP-10,HGF and TWEAK were treated into HepG2 cells for 12 h,0-50ng/mL FGF19 were treated into HepG2 cells for 12 h and 50ng/mL FGF19 were treated into HepG2 cells for 0-24h.Then total RNA was extracted and the expression of miR-200b-3p was detected in each group.The regulators of miR-200b-3p were explored.6.After miR-200b-3p mimics transfection into HepG2 cells for 24h,total RNA and total protein were extracted,and the expression of miR-200b-3p was detected by RT-PCR.Bile acid synthesis-related enzymes were detected,including CYP7A1,CYP7B1,CYP8B1,CYP27A1.Nuclear receptors were detected,including LRH-1,FXR,HNF4?,HNF1?,SHP.Liver detoxification enzymes were detected,including GSTM1-4,GSTA1-4,CYP3A4,UGT2B4,UGT2B7,SULT2A1.Membrane transporters were detected,including MRP2,MRP3,OST?,OST?,OATP3A1.And the protein levels of LRH-1,CYP7A1,CYP8B1,MRP3 and OATP3A1 were detected by WB.7.After miR-200b-3p mimics transfection into PLC5 cells for 24h,total RNA and total protein were extracted,and the expressions of miR-200b-3p and OATP3A1 was detected by RT-PCR.And OATP3A1 protein level was detected by WB.8.After miR-200b-3p inhibitor transfection into HepG2 and PLC5 cells for 24h,total RNA and total protein were extracted,and the expressions of miR-200b-3p and OATP3A1was detected by RT-PCR.And OATP3A1 protein level was detected by WB.9.The potential binding site of miR-200b-3p and LRH-1 was predicted and synthesized,the wild-type sequence(LRH-1-wt)and the mutated sequence(LRH-1-mut)of miR-200b-3p were synthesized into the pmirGLO plasmid,luciferase reporter gene assay was used to determine two luciferase relative activities.Results:1.The levels of AST,ALT,ALP,GGT,TBIL,DBIL,IBIL and TBA in human obstructive cholestasis patients were much higher than these in normal control patients(P<0.05).2.RT-PCR confirmed that the miRNA-200b-3p expression in human obstructive cholestasis livers were 1.8-fold higher than these in normal control livers(P<0.05).3.In miRNA-200b-3p high expression group,TBA level of patients was decreased,and it was negatively correlated with miRNA-200b-3p(P<0.05),and there was no significant correlation with other liver function indexes(P>0.05).4.In cholestatic liver tissue,LRH-1 and CYP8B1 were significantly up-regulated,while CYP7A1 was significantly down-regulated.In miRNA-200b-3p high expression group,levels of LRH-1 and CYP8B1 were significantly lower than those in miRNA-200b-3p low expression group(P<0.05);CYP7A1 was significantly lower than the control group(P<0.05);and miR-200b-3p was negatively correlated with LRH-1 and CYP8B1.5.FGF19 significantly increased the expression of miR-200b-3p for 2.6-fold,and with time-dependence and dose-dependence(P<0.01),while there was no significant difference with other factors,such as IL-1?,IL-8,IP-10,TNF-?,HGF,TWEAK(P>0.05).6.After miR-200b-3p mimics transfection into HepG2 cells for 24 hours,total RNA and total protein were extracted.The miR-200b-3p level was significantly upregulated(P<0.01),and the levels of LRH-1,CYP7A1 and CYP8B1 were significantly decreased(P<0.05),while OATP3A1 was significantly increased(P<0.05).7.After miR-200b-3p mimics transfection into PLC5 cells for 24 hours,total RNA and total protein were extracted.The expression of miR-200b-3p was significantly increased(P<0.01),and the expression of OATP3A1 was significantly increased(P<0.05).8.After miR-200b-3p inhibitor transfection into HepG2 and PLC5 cells for 24 hours,total RNA and total protein were extracted.The expression of miR-200b-3p was significantly decreased(P<0.01),and the expression of OATP3A1 was significantly decreased(P<0.05).9.Luciferase reporter gene assay showed that the relative luciferase activity in group of pmirGLO-LRH-1 wt and miR-200b-3p mimics co-transfection was significantly decreased(P<0.05),while it was recovered in pmirGLO-LRH-1 mut group(P<0.05),which suggesting that miR-200b-3p could bind with LRH-1.Conclusion:1.The expression of miR-200b-3p was significantly increased in human obstructive cholestatic liver tissues.Correlation analysis revealed that miR-200b-3p was negatively correlated with TBA,which might indicate that it might be involved in bile acid metabolism.2.The FGF19 might induce the expression of miR-200b-3p with time-dependent and dose-dependent.3.miR-200b-3p might inhibit the expression of nuclear receptor LHR-1,and then down-regulate the expressions of bile acid synthase CYP7A1 and CYP8B1,thereby reducing intrahepatic bile acid synthesis.Meanwhile,miR-200b-3p might indirectly up-regulate the expression of OATP3A1,which increased the bile acid efflux,thereby alleviating the accumulation of bile acids in the liver cells and protecting the liver. |
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