| Objective: The bidirectional connection between diabetes mellitus and periodontitis has been widely accepted.Micro RNAs(mi Rs)play a regulatory role in both diseases,but the pathogenesis of periodontitis concomitant with diabetes is still unknown.This study was to detect the high glucose-induced expression of mi R-126 and relevant genes in human gingival fibroblasts(HGFs);to analyze the influence of mi R-126 on inflammatory factors by transfecting mi R-126 mimic;to find out the candidate target gene of mi R-126 by bioinformatics;to explore its regulatory pathway in HGFs stimulated by high glucose;in order to clarify the function and mechanism of mi R-126 in the influence of diabetes on periodontitis,and provide a reference for a novel treatment strategy of periodontitis concomitant with diabetes.Methods:1.After informed consent,healthy gingival tissues from systemic healthy volunteers were collected while extracting the third molar,washed in phosphate buffer saline(PBS)solution three times,cut into small pieces(about 1 mm3).After HGFs migrated from tissue explants in vitro,their morphology and growth were observed by inverted phase contrast microscope.2.HGFs were cultured with low(5.5 mmol/l),medium(15 mmol/l)and high(25mmol/l)glucose in vitro,respectively.After cultured for 12 h,24 h and 72 h,the levels of mi R-126,interleukin-6(IL-6),tumor necrosis factor ?(TNF?),chemical chemokine ligand 2(CCL2)and IL-10 were detected by real time polymerase chain reaction(real time PCR).3.After transfected with miR-126 mimic,the transfection efficiency was detected.The cells were stimulated with low,medium and high glucose,then total RNA was collected,and above-mentioned genes were verified by real time PCR.Also,total protein was collected for Western Blot.4.After transfected with miR-126 mimic,the cells were stimulated with low,medium and high glucose,and the supernatants of culture medium were utilized for enzyme linked immunosorbent assay(ELISA)to detect the production of cytokines of HGFs.5.We screened and analyzed the downstream genes of mi R-126 through bioinformatics and selected a gene with a highly predicted score.6.The binding sites of the downstream gene were verified by dual luciferase reporter assay to check whether it was the direct target of mi R-126.Results:1.The primary cells were migrated from the gingival tissue,tended to be long fusiform and swirled,and grew vigorously.2.The mRNA levels of IL-6,TNF?,CCL2 increased while IL-10 decreased as concentration of glucose increasing.3.At 12 h,24 h and 72 h,increasing glucose significantly suppressed miR-126 expression in HGFs(P<0.05).According to bioinformatics,we chose TNF receptor associated factor 6(TRAF6)for further signaling pathway exploration.High glucose increased TRAF6 level(P<0.05).Mi R-126 and TRAF6 showed an opposite trend.4.After transfection of mi R-126 mimic in HGFs,the level of mi R-126 significantly increased(P<0.05);both m RNA and protein levels of TRAF6 significantly reduced(P<0.05);TRAF6 was the direct target of mi R-126 through binding to the site 348-354 in 3'-untranslated region(3'-UTR)of TRAF6.5.After transfection of mi R-126 mimic in HGFs,the m RNA levels of IL-6,TNF? and CCL2 significantly decreased while IL-10 significantly increased(P<0.05).The secretion of IL-6,TNF?,CCL2 and IL-10 from HGFs showed the similar trend with the results of real time PCR.Conclusion:1.High glucose induced low expression of miR-126 in HGFs.2.MiR-126 inhibited the secretion of proinflammatory cytokines from HGFs via targeting TRAF6.3.As a protective factor,miR-126 might be a potentially effective therapeutic target for periodontitis accompanied with diabetes. |
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