Study Of The Role Of MicroRNA On NK Cells In Primary Biliary Cholangitis

Aims: Patients with primary biliary cholangitis(PBC)have significantly increased numbers of Natural Killer(NK)cells in the liver and peripheral blood,which may be involved in their pathogenesis.miRNA regulation of NK cells plays a role in other autoimmune diseases,but not in the pathogenesis of PBC.The role of miRNA regulation of NK cells in other autoimmune diseases and in the pathogenesis of PBC has not been reported.The aim of this study was to investigate whether miRNAs in PBC have a role in the activation and protein secretion of NK cells and whether they are involved in their pathogenesis.Methods: By using gene prediction software to predict NK regulation-related miRNAs,we screened 10 miRNAs with strong association with nuclear factor of activated T cell cytoplasmic 1(NFATC1),which can bind to CD16 on the surface of NK cells.Peripheral blood specimens were collected from 168 PBC patients and 74 healthy controls(HC).After sorting out NK cells by magnetic bead,RNA was extracted by lysis and qPCR was applied to quantify the ten miRNAs in NK cells.Dual luciferase gene reporter was applied to verify whether the miRNAs could bind to NFATC1.The mimics and inhibitors of the differential miRNAs were constructed to overexpress or inhibit the expression of the three differential miRNAs.The constructed system was transferred into NK cells,and after completion,the cells were lysed to obtain the supernatant,and ELISA was used to determine the perforin and granzyme secreted by NK.Flow cytometry was used to determine the sorting purity of NK cells and to verify the effect of miRNA on CD molecules on the surface of NK cells.SPSS and Graphpad were used for statistical analysis and graphing.Results: The Ct values of hsa-miR-137-3p,hsa-miR-124-3p and hsa-miR-506-3p in peripheral blood NK cells of the PBC group were lower than those of HC(11.33±4.40 vs11.85±4.40,P<0.01;8.61±2.22 vs 8.94±2.99,P<0.01 and 7.18±1.89 vs 8.29±1.46,P<0.05),indicating that NK cells from PBC patients expressed these three miRNAs higher than HC.The dual luciferase reporter gene assay verified that the fluorescence intensity of miR-137-3p bound to NFATC1 Wild type(WT)plasmid was weaker compared to miR-137-3p Negative control(NC)(P < 0.05),while hsa-miR-124-3p and hsa-miR-506-3p had no significant effect,showing that miR-137-3p was able to bind NFATC1.The granzyme B expressed by NK cells after miR-137-3p mimics transfection was decreased compared to that of miR-137-3p inhibitor((348.59 ± 7.47 pg/ml)vs(373.92 ± 15.50 pg/ml),P < 0.05).NK cells were transfected by miR-137-3p mimic/inhibitor and FCM was applied to measure CD16 and CD56 on the surface of NK cells.The results showed an increase in CD3-CD16+ NK cells after overexpression of miR-137-3p compared to the miR-137-3p NC group(73.2% vs 46.8%,P < 0.05),possibly indicating that the binding of miR-137-3p to NFATC1 increased CD16+ on the surface of NK cells has while CD56 molecules decreased.Conclusions: There were differences in hsa-miR-137-3p,hsa-miR-124-3p,and hsa-miR-506-3p in NK cells from PBC patients and HC,and the differences were statistically significant.Hsa-miR-137-3p was able to bind to the NFATC1 gene and may promote the expression of CD16 for NK cells.The overexpression of hsa-miR-137-3p promoted the secretion of granzyme B in NK cells from PBC patients.miR-124-3p,miR-506-3p had no effect on either CD16 molecules or perforin/granzyme B.In conclusion,miRNA-137-3p may play an important role in NK cell activation in PBC,possibly by binding to the NFATC1 gene on the expression of CD molecules on the surface of NK cells and the secretion of granzyme B granzyme B by binding to the NFATC1 gene.