Expression Of MiR-20a-5p In Peripheral Blood Mononuclear Cells Of Primary Biliary Cirrhosis And Study On Its Function

Primary biliary cirrhosis is a chronic liver disease characterised by intrahepaticbile-duct destruction, cholestasis, and, in some cases, cirrhosis. The highly specificantimitochondrial antibodies (AMAs) can be found in the serum of patients. PBC mainlyaffects middle-age women with a female to male ratio of up to10:1. The incidence ofprimary biliary cirrhosis has increased over recent decades, the pathogenesis is still unclearyet. The routine use of ursodeoxycholic acid is currently the only accepted treatment ofprimary biliary cirrhosis, and patients have poor response on immunosuppressants.microRNAs (miRNAs) are small noncoding RNAs that widely participate inposttranscriptional gene regulation. There have been a lot of researchers confirmed thatmiRNAs played important roles in the development and the process of treatment of manyautoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus,multiple sclerosis and ulcerative colitis. The role of miRNAs in primary biliary cirrhosiswas also gradually got attention inrecent years. Our group found that17microRNAs weredifferentially expressed in peripheral blood mononuclear cells of primary biliary cirrhosispatients using a microRNA array. Among these microRNAs, miR-20a-5p, miR-106b-5pand miR-93-5p, which belong to the family of miR-17, were located in the center of themiRNA-gene-network.Interleukin-8(IL-8) is an important mediator of inflammation. Zimmermann HWfound that serum IL-8levels were significantly increased in CLD patients, especially incholestatic disease and alcoholic cirrhosis, and IL-8correlated with non-invasive fibrosismarkers.For this study, miR-20a-5p and IL-8expression levels were detected in patients withPBC, and we tried to explore the possible regulatory relationship between them.Part I.Detection of miR-20a-5p and IL-8expression level in PBC patientsWe collected PBMCs of PBC, RA, SLE, VHC hospitalized patients and normalcontrols,each20cases in Changzheng Hospital, then detected the miR-20a-5p and IL-8expression level of each disease using RT-PCR, and the plasma levels of IL-8expressionwere detected by ELISA. Results showed that the expression levels of miR-20a-5p andIL-8were significantly higher than the normal control group and other groups (P<0.05).The relative expression of miR-20a-5p was6.33times of the normal control group, therelative expression of IL-8was7.96times of the normal controls, the average plasma levels of IL-8in patients with PBC was89.5±36.6ng/L.Part II.Changes of IL-8expression in THP-1cells after miR-20a-5p transfectionSix hours before the transfection of miR-20a-5p mimic and inhibitor, THP-1cellswere pretreated with100ng/ml of LPS, the changes of IL-8expression levels were detectedusing RT-PCR. Results showed that the relative expression of IL-8was reduced in THP-1cells after transfection of miR-20a-5p mimic, approximately0.25times of the controlgroup, and the relative expression of IL-8was increased in THP-1cells after transfectionof miR-20a-5p inhibitor, almost4.02times of the control group, differences werestatistically significant (P <0.05).Part III.Possible regulatory mechanism between miR-20a-5p and IL-8To further explore the possible regulatory relationship between miR-20a-5p and IL-8,we predicted the target genes of miR-20a-5p using miRDB, TargetScan andMicroRNA.org database and validated the targeting relationship between miR-20a-5p andIFN regulatory factor9using dual luciferase reporter assay. At last, we designed smallinterfering RNAs to interfere the expression of IRF9, then detected the changes of IL-8levels using RT-PCR. MicroRNA.org database showed there were nine consecutivecomplementary base pairs between miR-20a-5p and IRF9, and the relative fluorescencevalues lowered by30.3%after co-transfection of miR-20a-5p mimic and the reporter genevectors (P <0.05), both implicated a close targeting relationship between miR-20a-5p andIRF9. IL-8relative expression lowered after transfection of IRF9siRNA, this trend wasconsistent with the RT-PCR results after transfection of miR-20a-5p mimic. Both theprotein expression after transfection of miR-20a-5p mimic and IRF9siRNA were detectedusing Westernblot experiment.CONCLUSIONS: miR-20a-5p and IL-8expression level were elevated in PBMCsfrom PBC patients. IL-8expression was inhibited by miR-20a-5p in LPS-pretreated THP-1cells, and this inhibition may be achieved through the targeting effect on IRF9.miR-20a-5p may play a protective role in PBC patients by suppression the continuousrising of IL-8，and by what means IRF9could affect the expression of IL-8needs furtherstudy.