The Role Of Hsa＿circ＿0000711 In The HiBEpic Primary Biliary Cholangitis Model

Objective:To screen circular RNA(circRNA)of susceptibility genes associated with primary cholangitis(PBC).To investigate the expression of circRNA in serum of patients with PBC.The role of circRNA in human intrahepatic bile duct epithelial cell(HiBEpic)PBC model is further investigated.Method:1.Disease-related related databases(DisGeNET,circBank and High-throughput sequencing database of serum miRNA in patients with PBC)were used to screen susceptible genes of PBC.Then,combined with the existing serum micro RNA(miRNA)high-throughput sequencing of PBC patients,the circRNA related to PBC was obtained.2.The differential expression of hsa＿circ＿0000711 between 6 PBC patients and 3healthy subjects was detected by qPCR.Subsequently,the differential expression was verified by qPCR of serum samples,among the PBC group(40 PBC patients),the healthy group(40 healthy subjects),and the other liver disease group(30 chronic hepatitis B patients,10 primary hepatocellular carcinoma patients).Receiver operating characteristic(ROC)curves were drawn to explore the diagnosis value of hsa＿circ＿0000711 in PBC.3.A PBC HiBEpic cell model was established by inducing HiBEpic cells with 1 mM GCDCA for 24 hours.qPCR,Western blot,and CCK-8 assays were used to verify whether the PBC HiBEpic cell model was successfully constructed.qPCR was also used to reflect the expression of hsa＿circ＿0000711 in the PBC HiBEpic cell model.4.The possible interaction sites of hsa＿circ＿0000711 with miR-185-5p and miR-361-3p were predicted,and the dual luciferase reporter gene vector was constructed to verify the interactions between them.5.The overexpression vector and siRNA of hsa＿circ＿0000711 were constructed and transfected into HiBEpic cells.The expression of hsa＿circ＿0000711 in each group was detected by fluorescence microscopy and qPCR to verify the overexpression or knockdown efficiency.The normal HiBEpic cell group,hsa＿circ＿0000711 transient transfected HiBEpic cell group,and hsa＿circ＿0000711 transient transfected PBC HiBEpic model cell group were set up.qPCR and Western blot were used to detect the mRNA and protein expression levels of PDC-E2 and NFATc3 in the three groups.CCK-8 assays were used to observe the survival rate of cells in each group.6.The data in accordance with normal distribution were compared and analyzed by using two independent samples T-test and one-way ANOVA.Wilcoxon rank sum test and Kruskal-Wallis rank sum test were used to compare and analyze the data in accordance with non-normal distribution.For the classified variables,chi-square test is used to analyze.The above analysis showed P < 0.05,which means the difference was statistically significant.Results:1.Through the disease-related database combined with miRNA high-throughput sequencing data,hsa＿circ＿0000711 with NFATc3 as the target gene was obtained,and the miR-185-5p and miR-361-3p might bind to it.2.The pre-experiment showed that the expression level of serum hsa＿circ＿0000711in PBC patients had an upward trend compared with that in healthy subjects.The validation group experiment showed that the expression level of serum hsa＿circ＿0000711in PBC patients was significantly higher than in other groups.ROC curves showed that hsa＿circ＿0000711 had a certain clinical value in distinguishing PBC patients from healthy subjects and patients with other liver diseases.3.After the intervention of GCDCA on HiBEpic cells,qPCR and Western blot showed that the expression of PDC-E2 in PBC HiBEpic model cells was higher than that in healthy HiBEpic cells.CCK-8 assays showed that the cell survival rate of PBC HiBEpic model cells was lower than that of normal HiBEpic cells.qPCR results showed that the expression of hsa＿circ＿0000711 in PBC HiBEpic model cells was upregulated compared with that in healthy HiBEpic cells.4.Dual-luciferase reporter gene assays showed that both miR-185-5p mimics and miR-361-3p mimics could significantly reduce the luciferase activity of cells transfected with the hsa＿circ＿0000711 wild-type vector.However,it could not reduce the luciferase activity of cells transfected with the hsa＿circ＿0000711 mutant vector.5.After the hsa＿circ＿0000711 overexpression plasmid and siRNA were constructed and transfected into HiBEpic cells,fluorescence microscopy qPCR showed that the transfection efficiency of related vectors was high.6.qPCR,Western blot,and CCK-8 assays showed that the expression of PDC-E2 mRNA and protein increased in both HiBEpic cells and PBC model HiBEpic cells after overexpression of hsa＿circ＿0000711,and the cell survival rate was decreased.Knockout of hsa＿circ＿0000711 had the opposite effect.qPCR and Western blot experiments also showed that the expression of NFATc3 mRNA and protein in HiBEpic cells and PBC model HiBEpic cells was also increased after overexpression of hsa＿circ＿0000711,while knockout of hsa＿circ＿0000711 had the opposite effect.Conclusion:1.In this study,PBC-related susceptibility gene NFATc3 was selected as the target gene of hsa＿circ＿0000711,which may be a potential biomarker of PBC.2.Hsa＿circ＿0000711 promotes the occurrence and development of primary cholangitis by regulating the expression of NFATc3 and possibly targeting miR-185-5p and miR-361-3p.