MiRNA-335-5p Regulates Pyk2-mediated Autophagy Induced By Porphyromonas Gingivalis

Objectives: Porphyromonas gingivalis is recognized as the main pathogen of chronic periodontitis.Previous studies showed that during the infection of epi-4 cells by P.gingivalis,autophagy was enhanced and the activation of protein-rich tyrosine kinase2(Pyk2).Moreover,the autophagy level in epi-4 cells was significantly decreased after the treatment with the specific Pyk2 inhibitor TAE226,suggesting that Pyk2 mediated the autophagy of epi-4 cells induced by P.gingivalis infection.Some scholars have found that miRNA-335-5p can reduce the degree of inflammation of endothelial cells in the mouse inflammatory model by enhancing autophagy.In this study,we built overexpression and silencing of miRNA-335-5p gingival epithelial cells.Through the analysis on the expression of LC3 and Pyk2 in gingival epithelial cells infected by P.gingivalis,the pathogenic mechanism of the bacteria was revealed to provide new targets and ideas for the prevention and treatment of chronic periodontitis.Methods: 1.Epi-4 cells were infected by P.gingivalis(MOI 100:1)for 0,1,2,4,6,and 12 h.The non-infected cells were used as the control group to detect the expression of miRNA-335-5p by tail-PCR.2.Liposome transient transfection technology was employed to transfect miRNA-335-5p mimic,inhibitor and their counterpart into epi-4 cells.The transfection efficiency was detected by flow cytometry and tail-PCR.3.q RT-PCR was used to detect the expression of LC3 and Pyk2.Western blot was used to analysis LC3Ⅱ/LC3 I and Pyk2 protein levels.4.miRWalk software was used to forecast the target gene of miRNA-335-5p.GTRD software was used to predict transcription factors that bind simultaneously to LC3 and Pyk2 promoter regions.After the intersection of the two results,transcription factors with negative regulatory effects were selected by combining literature review.Results: 1.After P.gingivalis infection,the expression of miRNA-335-5p increased,reaching a peak at 1 h,6.8 times of the control group(P<0.001).2.The transfection efficiency of miRNA-335-5p mimic detected by flow cytometry was 73.1% and that of inhibitor was 71%,respectively.tail-PCR results showed that miRNA-335-5p expression increased in mimic group,456 times higher than that in NC group,and decreased in inhibitor group,0.42 times lower than that in NC-in group(P<0.05).3.After mimic pretreatment,the m RNA expressions of LC3 and Pyk2 increased,meanwhile,the protein expressions of LC3Ⅱ/LC3 I and Pyk2 was up-regulated.After inhibitor pretreatment,the m RNA expressions of LC3 and Pyk2 decreased.At the same time,the protein expressions of LC3Ⅱ/LC3 I and Pyk2 was down-regulated.(P<0.05).4.Four transcription factors,FOXA2,WT1,SP1 and MAX,were obtained after the intersection of the predicted results of miRWalk software and GTRD software,in which SP1 had a negative regulatory effect.Conclusion: P.gingivalis infection can up-regulate the expression of miRNA-335-5p in epi-4 cells,and the expression of miRNA-335-5p can promote Pyk2-mediated autophagy in epi-4 cells.