Screening And Functional Study Of Differentially Expressed LncRNA Associated With Cholesterol Gallstone

Part Ⅰ.Screening and functional study of differentially expressed lncRNA associated with cholesterol gallstone.Background and objective:Cholelithiasis is one of the most common diseases in general surgery,with an incidence of 5%to 25%in adults in western countries.In China,with the development of economy and the westernization of life style,its incidence is increasing year by year.Epidemiological study shows that the overall prevalence rate of cholelithiasis in Shanghai is 7.02%,and the cholecystectomy rate is 2.48%.Although 75%of gallstone patients are asymptomatic,once symptoms or complications occur,it will seriously affect the health and quality of life of residents,and bring a heavy burden of economic and health resources.There are many risk factors for gallstone formation,including age,female,race and stone-related genes,while metabolic syndrome,dietary factors,gallbladder dysfunction and drug factors also play an important role in gallstone formation.However,the cause of gallstone has not been fully clarified.There is still much space for improvement in its prevention and treatment.Therefore,it is still important to explore the pathogenesis of gallstone and seek effective stone prevention measures.Although the mechanism of gallstone formation has not been fully elucidated,great progress has been made in recent years.Cholesterol gallstone formation mainly goes through three stages:cholesterol supersaturation in bile,abnormal nucleation process and abnormal gallbladder function.The final causes of gallstone mainly include the following aspects:stone-related genes,including LITH gene;excessive secretion of cholesterol in liver;formation of supersaturated gallbladder bile impaired gallbladder contractile function;and intestinal absorption function.Long non-coding RNAs(lncRNAs,lncRNAs)are non-coding RNAs with a length of more than 200 nucleotides,which have been proven to be associated with a variety of human diseases.Current reports suggest that lncRNAs can regulate cellular cholesterol metabolism.For example,lnc-HC can regulate the expression of cholesterol metabolism-related genes CYP7A1 and ABCA1 by binding to HNPA2B1 at the post-transcriptional level.At present,there are few reports on the regulation of cholesterol gallstone by long-chain non-coding RNA(long non-coding RNA,lncRNA).In this study,a mouse cholesterol gallstone model was established.The differentially expressed lncRNA in mouse liver was analyzed and bioinformatics methods were used to explore the biological function of differential genesMethods:1.24 C57BL/6 mice were categorized into two groups:16 mice were fed lithogenic diet as cholesterol gallstone group,and another eight mice were fed standard diet as a control group.Two groups of mice were fed in five weeks to form the animal model2.The liver tissue,gallbladder,and bile in two groups of mice were collected.The volume of gallbladder samples was noted and photographed.Lipid analysis was taken in mice bile samples.The contents of total cholesterol,phospholipid,and total bile acid were collected and calculated as cholesterol stone index(CSI)3.The fresh mice liver tissues were prepared to analyze total IncRNA and discussing the variation between two groups.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were exploited to find out potential cis-and trans-differentially expressed genes(DEGs)4.After setting the RNA-sequence library,the differential mRNAs(DE-mRNAs)and the differential lncRNAs(DE-lncRNAs)in CSG and NG were analyzed through the"DESeq2" package of R software.The "cluster Profiler" package was used in the enrichment analysis of DE-mRNAs.Based on the Cytoscape software,PPI networks and competing endogenous RNA(ceRNA)were constructed.The potential hub RNAs were validated through quantitative real-time polymerase chain reaction(qRT-PCR).The immunohistochemical method was exploited to testify the protein expression of two mice groups.Results:1.A total of 16 mice model with cholesterol gallstone was set up successfully.The bile of the gallbladder of the mice in the control group was transparent and yellow,and the gallbladder of the mice in the cholesterol gallstone group was larger,and the bile was turbid and dark in color.Observed under the microscope,There was no obvious stone formation in the gallbladder bile of the control group,and the gallbladder bile was fused into clusters and formed with cholesterol stones in the cholesterol gallstone group Compared with the control group,the liver weight of mice in the model group increased significantly,and there was a statistical difference between the two groups.The results of the analysis of bile lipids in the gallbladder of the two groups of mice indicated that the total cholesterol,total cholesterol/total bile acid,total cholesterol/phospholipid and CSI in the model increased,and the total bile acid in the cholesterol gallstone group decreased The results of gallbladder bile lipid analysis suggest that changes in bile cholesterol content play an important role in the formation of gallstones.The results of gallbladder bile lipid analysis suggest that changes in bile cholesterol content induced by lithogenic diet fed mice play an important role in the formation of cholesterol gallstone2.It illustrates that 33 lncRNA were varied through the differential expression analyses of lncRNA in the liver tissue of two mice groups,including 17 enhanced genes were 16 declining genes in the cholesterol gallstone group.In mRNA analyses,181 differentially expressed genes were screened out,including 104 uprising genes and 77 decreasing genes in the cholesterol gallstone group.Those DEGs were analyzed under Bidirectional hierarchical clustering analysis and performed as a heatmap plot3.We applied Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)functional enrichment analyses to analyze the function of DEGs.There are 419 biological processes,86 cellular components,134 molecular functions in the GO subset,and eight pathways in KEGG subset that were discovered in enhancing the DEGs group in the cholesterol gallstone group.A total of 229 biological processes(GO_BP),44 cellular components(GO_CC),54 molecular functions(GO_MF)in the GO subset,and seven pathways in the KEGG subset were seen in decreasing DEGs in the cholesterol gallstone group4.The intersection of 117 proteins was provided in the PPI network,and the relationships were depicted through Cytoscape software.It illustrated about 101 hub points 65 uprising DEGs and 36 decreasing DEGs were paired proteins.Ptpre,Kdm4a,Sptanl,ranked top 15,might be the hub proteins in this network5.After co-analyzing the differentially expressed mRNAs and IncRNAs,173 co-expression relation pairs were screened out,including 96 mRNA and 24 lncRNA.Each lncRNA was analyzed through GO functions and KEGG enrichment pathways.It demonstrates 457 GO_BP,80 GO_CC,137 GO MF,and 11 KEGG pathways were statistically enriched6.Paired relationship of 86 mRNA-mRNA was screened out through miRanda software,including 49 miRNAs and ten mRNA.9320 paired miRNA-lncRNA were included,including 1754 miRNAs and 19 lncRNAs7.A total of 42 miRNA-mRNA relationship pairs,127 miRNA-lncRNA relationship pairs,and 115 mRNA-lncRNA relationship pairs were found in the ceRNA network,including 24 up-regulated mRNAs,11 lncRNAs,and 53 down-regulated mRNAs,47miRNAs8.The hub genes from two groups were validated through qRT-PCR.Compared to control group,the expression of Kdm4a elevated whereas Meg3,Pabpc4,Cep131,and Numb1 declined,which proceeded to support former genetic analysis9.The result of IHC proved Kdm4a present higher protein expression in liver tissue of cholesterol gallstone group compared to control groupConclusions:1.The cholesterol gallstone mice model was successfully set up.A total of 181 differential mRNAs and 33 differential lncRNAs were detected in cholesterol gallstone group and control group2.Kdm4a was selected to define a hub point in the PPI network,and lncRNA MEG3 was discovered as a potential hub gene3.In the qRT-PCR analysis,Kdm4a was enhanced,whereas Meg3,Pabpc4,Cep131,and Numbl were decreased in cholesterol gallstone group,compared to control group.The protein expression of Kdm4a was validated through IHCPart Ⅱ.LncRNA 4732465J04Rik promotes the formation of cholesterol gallstones in mice via regulating the miR-716/Scarb1 axisBackground and objectives:The supersaturation of cholesterol in bile synthesized by hepatic cells and nucleation of cholesterol crystals promoted by bile proteins contribute to the formation of cholesterol gallstones.Moreover,impaired gallbladder motor function leading to cholestasis is also another factor leading to the formation of cholesterol gallstones.Nevertheless,some researchers have demonstrated that changes in the synthesis of prostaglandins in the gallbladder and excessive calcium concentration in the bile are also factors promoting the formation of gallstones.In some patients,the prerequisite condition for the formation of gallstones is considered to be cholestasis,where cholesterol crystals composed of viscous glycoproteins,and it shall be the only abnormal manifestation during biliary colic,pancreatitis or cholangitis detected by ultrasound.Lots of studies have indicated that gallstone associated-genes such as CCKAR,CYP 7A1,FXR2,SREBF2,HMGCR,SCARB1 and ABCB11 play vital roles in the formation of gallstone.Cholesterol is an essential component for maintaining the fluidity and integrity of cell membranes and cell functions,and it can be obtained through selective high-density cholesteryl ester(CE)pathway.The scavenger receptor B1 gene(Scarb1)is a high-density lipoprotein receptor on the cell surface that mediates the uptake of high-density lipoprotein-cholesterol esters.It is most abundantly expressed in the liver,where it provides cholesterol for the synthesis of bile acids and cholesterol esters for storage in steroid tissues,or participates in the steroid synthesis in rodent.The transcription of SCARB1 is regulated by hormones in the adrenal gland,ovary and testis,and it is also regulated post-transcriptionally and post-translationally in the liver and other organs.Furthermore,it has been demonstrated that Scarb1 is involved in a variety of disease biological processes and associated with the pathogenesis of atherosclerosis,stone formation,inflammation,hepatitis C virus infection and other diseases.Scarb1 plays an important role in the process of hepatic cells obtaining high-density lipoprotein cholesterol from peripheral cells.As high-density lipoprotein cholesterol is an indispensable component of cholesterol in the gallbladder bile,Scarb1 exerts important function in the formation of cholesterol gallstonesA better understanding of the mechanism underlying the formation of cholesterol gallstones could provide more new molecular targets for treatment.With the rapid development of high-throughput sequencing technology,transcriptome sequencing(RNA-seq)technology has been widely used in various fields.In this study,we constructed a gallbladder cholesterol gallstone mice model and obtained liver tissue for RNA-seq Furthermore,bioinformatics analysis methods were used to construct a ceRNA network associated with the gallstone genes,especially lncRNAs with significant difference Consequently,we showed that lncRNA 4732465J04RIK may up-regulate the expression of the lithogenic gene Scarb1 by sponging miR-761 in vivo and in vitro experiments,thus affecting the formation of cholesterol stones.Methods:1.After retrieving the lncRNA expression profile and genes associated with gallstone formation,bioinformatics methods were used to analyze the relationship between lncRNA and mRNA,thus constructing the ceRNA network2.42 C57BL/6 male mice with 8-week-old were randomly divided into 6 groups Group 1(G1):seven mice were injected with the negative control virus and fed with standard diet;Group 2(G2):seven mice were injected with the negative control virus and fed with lithogenic diet;Group 3(G3):seven mice were injected with the shRNA-lncRNA 4732465J04Rik virus and fed with standard diet;Group 4(G4):seven mice were injected with the shRNA-lncRNA 4732465J04Rik virus and fed with lithogenic diet;Group 5(G5)seven mice were fed with standard diet only without being injected anything;Group 6(G6)seven mice were fed with lithogenic diet only3.Concentrations of cholesterol(TC),phospholipid(PL),bile acid(TBA)and Bile bilirubin(BL)in the gallbladder bile between model groups and the control groups were detected,and the total bile lipid content was calculated based on the content of TC,PL and TBA.Then CSI was calculated in the table of Carey et al based on the total lipid concentration(TLC)and the ratio of PL/(PL+TBA)4.The effect of lncRNA 4732465J04Rik on lithogenic gene expression was detected at the in vitro level.Hepal-6 cells were transfected with siNC or si4732465J04Rik,and then treated with cholesterol for 48 hours.Then RT-qPCR assays were conducted to detect the expression of cyp7al,Cckar,Fxr2,Srebf2,Hmgcr,Scarb1 and Abcb11 after different cell treatments5.Western blot assays were used to detect the protein expression of cyp7al,Cckar,Scarb1 and Abcb11 in Hepal-6 cells transfected with siNC or si4732465J04Rik6.To explore the downstream targets of 4732465J04Rik,miRDB,Starbase and Targetscan were used to predict its targeted miRNAs.Luciferase reporter assays were conducted to verify the interaction between lncRNA and miRNA.Then,Hepal-6 cells were transfected with siNC,si4732465J04Rik,miR-761 inhibitor or si4732465J04Rik+miR-761 inhibitor.And the expressions of 4732465J04Rik,miR-761 and SCARB1 were detected by RT-PCR7.The effect of 4732465J04Rik on the formation of cholesterol gallstones was validated by the mice cholesterol gallstones model.The expression of lncRNA4732465J04Rik in mice was knocked down by shRNA lentivirus injection through tail vein,and the mice were divided into 6 groups:standard diet group,lithogenic diet group,standard diet group with shNC,lithogenic diet group with shNC,standard diet with shRNA-4732465J04Rik and lithogenic diet with shRNA-4732465J04Rik.TC,TBA and TBL were detected by ELISA,and the expressions of Cckar,Cyp7a1,Fxr2,Srebf2,Hmgcr,Scarb1 and Abcb11 were detected by RT-PCR.Results:1.The mice model of cholesterol gallstones was successfully established.Compared with the control group,the total cholesterol,total cholesterol/total bile acid,total cholesterol/phospholipid and CSI of cholesterol gallstone mice were increased,while the total bile acid of cholesterol gallstone mice was decreased.The heat map of differentially expressed lncRNA was constructed by bioinformatic analysis.Based on the differential expressions of the lncRNAs and gallstone-associated mRNAs,we used miRanda software to predict potential targeted miRNAs,and hypothetically built up the ceRNA network map2.RT-qPCR further verified the expressions of C030037D09Rik,4732465J04Rik and 9030622O22Rik in cholesterol gallstone mice.And the expression of 4732465J04Rik was significantly increased in the hepatic tissue of cholesterol gallstone mice,while the others showed no differences.we found that the expressions of Srebf2,Hmgcr,Scarb1 and Abcb11 increased significantly in hepatic tissues.However,the expression of Cyp7a1 decreased and Cckar and Fxr2 showed no differences3.Hepa1-6 cells were first transfected with siNC or si4732465J04Rilk and then treated with cholesterol for 48 h.and the expressions of Cckar,Cyp7a1,Fxr2,Srebf2,Hmgcr,Scarb1 and Abcb11 were detected by RT-qPCR.Results showed that the cholesterol treatment can significantly increase the expressions of Cckar,cyp7al,Scarb1,Fxr2,Screbr2 and Abcb11,while si4732465J04Rik transfection effectively inhibit the expressions of raised Cckar,Cyp7a1,Scarb1,Fxr2 and Hmgcr by cholesterol treatment4.Western blot assays were used to detect the protein expressions of Cyp7a1,Cckar,Scarb1,Abcb11 in Hepal-6 cells transfected with siNC or si4732465J04Rik.The results showed that knocking down 4732465J04Rik could significantly inhibit the expressions of Cyp7a1,Cckar,Scarb1,Abcb11.5.The binding sites of miR-761 and 4732465J04RIK were predicted by software,and and the direct binding of miR-761 and 4732465J04Rik were validated by the luciferase reporter assays.And Scarb1 was predicted to be the target of miR-761,and their binding sites were also verified by the luciferase reporter assays.And then Hepal-6 cells were then transfected with siNC,si4732465J04Rik,miR-761 inhibitor or si4732465J04Rik+miR-761 inhibitor.RT-qPCR shows that knocking down 4732465J04Rik can up-regulate miR-761 and down-regulate Scarb1 expression,while miR-761 inhibitor treatment can reverse the effect of si4732465J04Rik on Scarb1 expression6.The effect of 4732465J04Rik on the formation of cholesterol gallstones was demonstrated by the mouse model of cholesterol gallstones.The experiment result shows that the contents of TCH,TBA and TBL were significantly increased in the cholesterol gallstone group fed with lithogenic diet,while knocking down the expression of 4732465J04Rik by lentivirus can effectively reduce the levels of TCH,TBA,TBL in the cholesterol gallstone group.RT-PCR assays were performed to detect the expressions of Cckar,Cyp7a1,Fxr2,Srebf2,Hmgcr,Scarb1,Abcb11.Compared with the standard diet groups,the expressions of Cckar,Cyp7a1,Scarb1 increased significantly in lithogenic diet group(p<0.05),while knocking down the expression of 4732465J04Rik effectively suppressed the expressions of Cyp7a1,Cckar,Scarb1,Abcb11 fed with lithogenic dietConclusion:Feeding of lithogenic diet can up-regulate the expression of lncRNA 4732465J04Rik in the mice liver,which is involved in the formation of cholesterol gallstones in the gallbladder of mice.In terms of mechanism,4732465J04Rik can remove the inhibitory effect of miR-761 on the important lithogenic gene Scarb1 by sponging miR-761,and up-regulate the expression of Scarb1,thus affecting the occurrence and development of cholesterol gallstone disease.