The Effect Of MiR-34a On Inflammatory Response Of Human Periodontal Ligament Cells

ObjectiveTo detect the gene expression of miR-34a in the inflamed periodontal tissues.To investigate the effect of miR-34a on LPS-induced inflammation and osteogenic differentiation of human periodontal ligament cells(hPDLCs).The research had been divided into four parts:Part ? The expression changes of miR-34a in inflamed periodontal tissues26 cases of inflamed periodontal tissues were obtained from the Hospital of Stomatology of Wuhan University.The total RNA were extracted and the expressions of miR-34a,Notchl and SIRT1 were detected by quantitative real-time PCR(qRT-PCR).qRT-PCR assay showed that miR-34a was expressed in normal periodontal tissues.The level of miR-34a expression showed an significant increase in periodontitis tissues(1.3418±0.1663)compared with normal tissues(0.9988±0.011)(p<0.05).Notchl and SIRT1 expression both showed an obvious decrease in periodontitis tissues(p<0.001).Part ? The effects of miR-34a on inflammation of hPDLCsHPDLCs were obtained through in vitro culture combined with enzyme digestion method and tissue pieces culture method.First,the cells were stimulated by 1?g/ml E.coli LPS for different time points(3,6 and 12 hours)or by different doses(0.01,0.1,1 and 10?g/ml)for 3 hours.The expression of miR-34a was detected by qRT-PCR.Second,hPDLCs were transfected with lentiviral vector containing pre-miR-34a or control to build the miR-34a overexpression model or control model(hPDLCs/34a,hPDLCs/pCDH)and were stimulated by 1?g/ml E.coli LPS for different time points(3,6,12 and 24 hours)or by different doses(0.01,0.1,1 and 10?g/ml)for 3 hours.Quantification of IL-6,IL-1? and TNF-? were used to assess the influence of miR-34a on the inflammation of hPDLCs.qRT-PCR assay showed that miR-34a expression slightly increased at different time points with 1?g/ml E.coli LPS or with different doses of E.coli LPS for 3 hours and the differences were of statistical significance(p<0.05).The expressions of IL-6,IL-1? and TNF-? significantly increased in both time-dependent and dose-dependent manner(p<0.05).Part? The effects of miR-34a on proliferation of hPDLCsMiR-34a overexpression model or control model(hPDLCs/34a,hPDLCs/pCDH)were constructed and the cell proliferation were assessed by Cell Counting Kit-8(CCK-8).CCK-8 assay showed that hPDLCs/34a exhibited much slower growth than hPDLCs/pCDH and the proliferation of hPDLCs were repressed by miR-34a.PartIV The effects of miR-34a on osteogenic differentiation of hPDLCsFirst,the cells were continuously induced by mineralization induction medium for 3,7,14 days.The expression of miR-34a was detected by qRT-PCR.Second,miR-34a overexpression model or control model(hPDLCs/34a,hPDLCs/pCDH)were constructed and induced by mineralization induction medium for 3,7,14,21 days.Quantification of osteogenic genes(ALP,Runx2,OCN and BSP)expression,ALP activity detection,ALP staining and Alizarin red staining were used to assess the influence of miR-34a on the osteogenic differentiation of human periodontal ligament cells.The expression of miR-34a significantly increased in a time-dependent manner(p<0.001)during the osteogenic differentiation.The mineralization nodule formation and ALP activity were all attenuated by miR-34a.The early osteogenic genes(ALP and Runx2)expression slightly increased within 7 days in hPDLCs/34a(p<0.05).In the late stage of osteogenic differentiation,the level of BSP gene expression exhibited much lower at 14 days(p<0.001)and much higher at 21 days(p<0.01)in hPDLCs/34a.The level of OCN gene expression was not observed significant differences between hPDLCs/34a and hPDLCs/pCDH at 14 days and showed an obvious decrease in hPDLCs/34a at 21days(p<0.01).ConclusionsMiR-34a was detected in normal periodontal tissues,and the expression was up-regulated in inflamed periodontal tissues as well as in LPS-stimulated hPDLCs.As target genes of miR-34a,Notch1 and SIRT1 expression both showed an obvious decrease in inflamed periodontal tissues and Notch1 expression was also down-regulated in LPS-stimulated hPDLCs.MiR-34a overexpression could enhance the inflammation response characterized by increased expressions of pro-inflammatory cytokines including IL-6,IL-? and TNF-?.Furthermore,miR-34a may have a negative effect on the proliferation and osteogenic differentiation of hPDLCs.Our study showed that miR-34a may have a pro-inflammatory role and inhibit the proliferation and osteogenic differentiation of hPDLCs through targeting Notch1.During the development of periodontitis,miR-34a may exert a negative effect on periodontal tissue regeneration,restoration and remodeling.Taken together,we supposed miR-34a as a potential therapeutic target of periodontitis.