Study On The Function And Mechanism Of MiRNA-542-3p In Heterotopic Ossification

BackgroundHeterotopic ossification(HO)refers to the occurrence of atypical bone formation in muscle or connective tissue.HO form a direct impact on joint mobility,oppression of the peripheral nerve and skin lead to neuralgia and skin ulcers,seriously affecting the rehabilitation of patients,and even lead to disability,bringing a heavy burden to the family and society.At present,the mechanism of HO and treatment strategy for HO are not well understood,previous researches have already proven that endothelial cell to mesenchymal transition(EndMT)plays a major role in the course of HO.Thus,inhibition of EndMT process plays an important role in the treatment of heterotopic ossification.Bone morphogenetic protein 7(BMP7)is an important negative regulator of EndMT process,and regulation of BMP7 protein expression may play an important role in the development of heterotopic ossification.MicroRNAs are a wide range of non-coding micro RNAs,usually negatively regulate gene expression,can be widely involved in a variety of biological behavior.The study of the mechanism of how microRNAs affecting heterotopic ossification by regulating the expression of BMP7 can provide new ideas and methods for the treatment of heterotopic ossification.PurposeThe miRNAs targeting BMP7 were predicted by bioinformatics and verified at the molecular and cellular levels.On this basis,the molecular mechanism of miRNA regulation of EndMT was further explored.Methods1.MiRNA-542-3p,which can inhibit the BMP7 protein,was found by bioinformatics prediction method,and the predicted target gene was verified by luciferase reporter assay.The correlation between miR-542-3p and BMP7 expression was detected in heterotopic ossification clinical samples and heterotopic ossification animal models.2.Construction of lentiviral vector for the stable inhibition of miR-542-3p was established.The expression of miR-542-3p was detected in endothelial cells to confirm the transfection efficiency.3.To stimulate endothelial cells using TGF-β on local EndMT at the cellular level.The expression of endothelial marker E-cadherin and interstitial markers α-SMA,N-cadherin and Vimentin were detected after transfection of Lv-miR-542-3p-inhibitor in the cells.The activation of TGF-β signaling pathway and MAPK signal pathway were also detected.Then the cells were treated with MAPK pathway inhibitors to detect endothelial cell and interstitial markers.The activation of MAPK signaling pathway was also detected in heterotopic ossification clinical samples.4.Rat Achilles tendon injury HO experimental model was established,and X-ray imaging data showed obvious HO after 6 weeks.Immunohistochemical analysis,Western blot and RT-PCR were used to detect the expression of endothelial and interstitial markers.Results1.MiR-542-3p,which can inhibit the BMP7 protein,was found by bioinformatics prediction method.The miR-542-3p was able to bind to the target site of 3’UTR region of BMP7,and inhibit luciferase expression.The expression of miR-542-3p was higher than that of normal tissues and BMP7 was lower than that of normal tissues in heterotopic ossification clinical samples and heterotopic ossification animal models.The expression of miR-542-3p and BMP7 was negatively correlated.2.To construct a lentiviral vector for efficient and stable inhibition of miR-542-3p expression.The expression of miR-542-3p was significantly decreased after transfection of lentiviral vector in endothelial cells.3.After transfection of Lv-miR-542-3p-inhibitor,endothelial cells were stimulated with TGF-β,and the expression of E-cadherin was up-regulated compared with Lv-control group,SMA,N-cadherin and Vimentin were decreased.The phosphorylation of Smad2 and Smad3 in TGF-β signal pathway was decreased and the expression of Smad7 was increased.The phosphorylated ERK and JNK expression of MAPK signal pathway decreased significantly.Cells were transfected with miR-542-3p mimic,then treated with ERK inhibitor(U0126)and JNK inhibitor(SP600125)after TGF-β stimulation.The effects of miR-542-3p mimic are reversed.4.Results indicated the inhibiton of EndMT after the injection of miR-542-3p inhibitor lentivirus package.HO also was controled in rat Achilles tendon injury experimental model after gene therapy.Conclusions1.BMP7 is the target gene of miR-542-3p;the expression of miR-542-3p in heterotopic ossification is increased and the expression of BMP7 is decreased.2.The down-regulation of miR-542-3p expression in endothelial cells can reverse the TGF-β mediated EndMT and is achieved by inhibition of MAPK pathway.