The Study Of Expression Of Mmp-2-related Mirna Regulating By Arachidonyl Ethanolamide In Human Odontoblast-like Cells

Objective The cannabinoid(CB)system,as a major neurochemical system in the human body,has a wide range of biological effects,including analgesia,neuroprotection,immunity,and endocrine.Endogenous cannabinoid anandamide(AEA)is one of the main endogenous CB-like substances.AEA has important biological functions,mainly through its combination with specific receptor,which not only induces noxious stimulation to tissues but also promotes inflammation and tissue repair.Matrix metalloproteinase(MMPs)are a family of zinc-dependent endogenous enzymes that play a leading role in extracellular matrix degradation.Matrix metalloproteinase-2(MMP-2),which can activate a variety of inflammatory cytokines,such as interleukin-1,tumor necrosis factor-?,and transforming growth factor-? is one of the most important proteolytic enzymes.Therefore,MMP-2 may be an important virulence factor involved in dental pulp and periapical lesions.Our previous study presented that human odontoblasts(HODs)functionally express the CB specific receptor cannabinoid type1 receptor(CB1)have been reported in odontoblasts(ODs)in several literatures.In this study,human odontoblast(HOD)like cells were established in vitro,and AEA-induced MMP-2 production in HOD-like cells was observed by Western blot(WB).Prediction and validation of micro ribonuclenic acid(mi RNA)regulating MMP-2 gene expression demonstrates that mi RNA are involved in the regulation of MMP-2 expression in HODs-like cells.The aim of this study was to investigate the molecular mechanism of AEA regulating MMP-2 expression in HODs-like cells,and to lay a foundation for clinical treatment of pulp disease.Methods In this study,HOD-like cells were cultured as described by About et al.An OD phenotype was confirmed by expression of specific markers(e.g.,DSPP,nestin)by immunofluorescence(IF)and real-time quantitative polymerase chain reaction(q PCR).Confluent HOD-like cells were treated with 10 ?mol/L AEA for 0?12?24?48?72h.Tumor necrosis factor(TNF)-?(10 ng/m L)was used as positive control of MMP-2 induction in HOD-like cells.MMP-2 production was evaluated by Western blot assay(WB).To identify the effect of AEA on MMP-2 protein expression in HOD-like cells,we treated confluent cells at various concentrations of AEA for72 h.MMP-2 production and expression were assessed by WB.Subsequently,Target Scan and mi RDB were used to predict mi RNAs regulating MMP-2 gene expression,and mi RNA was screened for detection based on gene function.First,the enhanced green fluorescent protein(EGFP)fluorescence reporter vector was used to verify that mi RNA can directly regulate the gene expression of MMP-2.The effect of overexpression of mi R-136 on MMP-2 m RNA and protein levels was further determined by q PCR and WB.Finally,q PCR was used to detect the effect of AEA on the expression of mi RNA at different times.The effects of overexpression of mi RNA on the m RNA and protein levels of MMP-2 in HODs-like cells were examined by q PCR and WB.Results IF and q PCR results showed that DSPP and nestin were expressed positively on cultured HODs-like cells.WB analysis showed that the MMP-2 protein levels began to increase after 24 h of exposure and reached a level comparable with that of the positive control after 72 h.In addition,MMP-2 m RNA and protein levels were significantly reduced in response to AEA in a dose-dependent manner.The 10 mmol/L AEA treatment group first reached the level comparable to the positive control(TNF-?).The 12mmol/L AEA treatment group reached the highest peak of protein level,which confirmed that the MMP-2 protein level was up-regulated with the increase of AEA concentration.According to Target Scan and mi RDB,MMP-2 is a predicted target gene of mi R-136.To verify that mi R-136 can directly target MMP-2,the EGFP reporter plasmids containing MMP-2 wild-type or mutant 3?-UTR were constructed.In HOD-like cells,the relative EGFP activity was significantly reduced in both the pri-mi R-136 and wild-type 3?-UTR of MMP-2 cotransfected group and cotransfection with ASO-mi R-136,and wild-type 3?-UTR of MMP-2 increased the relative EGFP activity.However,mi R-136 overexpression or knockdown did not play an important role in the EGFP activity of the 3?-UTR mutation of MMP-2.In addition,significant downregulation of MMP-2 was observed at both m RNA and protein levels in HOD-like cells expressing pri-mi R-136 and the upregulation of MMP-2 at both m RNA and protein levels in the cells expressing ASO-mi R-136.Rescue experiments were performed to further confirm the effects of AEA on MMP-2 production mediated by mi R-136 in HOD-like cells.Mi R-136 levels decreased after 24 h treated with 10 ?mol/L AEA.Upregulation of MMP-2 was observed at both m RNA and protein levels in AEA-confluent HOD-like cells.Downregulation of MMP-2 was observed at both m RNA and protein levels in HOD-like cells expressing pri-mi R-136,whereas AEA treatment partially rescued the phenotypes caused by mi R-136 in HOD cells.These results demonstrated that AEA affects mi R-136 on MMP-2 production in HOD-like cells.Conclusions This study showed that AEA significantly up-regulated MMP-2 expression levels in HODs-like cells.Mi R-136 may be involved in the process of AEA-induced MMP-2 expression.