Functional Effects And Mechanism Of MiR-3940-5p On Gingival Stem Cell Proliferation And Osteogenic Differentiation Under Nifedipine

Objectives:Drug-induced gingival hyperplasia is an increase in gingival hyperplasia and volume caused by long-term use of ceratin drugs.However,the mechanism of DIGO mediated by nifedipine（NIF）is still unclear.Studies have shown that in NIF mediated DIGO,cell proliferation and cell cycle imbalance may be related to the occurrence of diseases.With the discovery of miRNA,more and more studies have proved that miRNA can regulate cell proliferation,apoptosis and cell cycle.This study preliminarily investigated the function and mechanism of miR-3940-5p in gingival mesenchymal stem cells（GMSCs）stimulated by NIF.Material and Methods:1.Clinically,healthy adult gums were collected and GMSCs were isolated and cultured in vitro by enzyme digestion culture method.2.Establishment of stable transfected cell lines:GMSCs was transfected with lentivirus transfection method,and the overexpressed miR-3940-5p（miR-3940-5p mimics）and the empty vector control group（Consh）were obtained in the experimental group.The expression of miR-3940-5p at the transcription level was detected by real-time RT-PCR,and the transfection efficiency was determined to determine the effect of overexpression.3.Flow cytometry-CFSE method was used to detect the proliferation of the control group（Consh）and the experimental group（miR-3940-5p mimics）after48h of NIF stimulation at the concentrations of 0ug/ml,1ug/ml,2ug/ml and 3ug/ml,so as to determine the optimal stimulation concentration of NIF.4.Flow cytometry-PI assay was used to detect the changes of cell cycle level in the control group（Consh）and the experimental group（miR-3940-5p mimics）after 48h of stimulation at the optimal concentration of NIF,and real-time RT-PCR and Western Bolt techniques were used to detect the expression of cell cycle related genes.To detect the effect of miR-3940-5p on the directional differentiation function of GMSCs.Use osteogenesis/tooth differentiation inducing culture,in vitro under the condition of stable transfection of miR-3940-5p mimics and Consh GMSCs for osteogenesis/into a tooth to induction,observe osteogenesis/into tooth differentiation index,including the determination of alkaline phosphatase activity,alizarin red staining and determination of Ca2+ion concentration,and applied to real-time RT-PCR test in the process of osteogenesis key transcription factors DLX5（Distal-Less Homeobox 5）on osteogenesis/dispersion of the tooth.And detection of osteogenesis/into tooth differentiation markers dentine salivary phosphoprotein（Dentin sialophosphoprotein,DSPP）,Dentin matrix protein 1（Dentin matrix acidic phosphoprotein 1,DMP1）,to observe effects on osteogenesis/differentiation into teeth.Results:1.Flow cytometry-CFSE assay showed that the proliferation of GMSCs was promoted when the concentration of NIF was 1ug/ml,2ug/ml and 3ug/ml.Under the stimulation of different concentrations of NIF（0ug/ml,1ug/ml,2ug/ml,3ug/ml）,overexpression of miR-3940-5p can inhibit the proliferation of GMSCs.2.The detection cycle results of flow cytometry-PI method showed that the GMSCs cell cycle was blocked at G0/G1 phase when the NIF concentration of miR-3940-5p was 0ug/ml and 1ug/ml.3.Real-time RT-PCR results showed that the expressions of p15^INK4b,p18^INK4cNK4c and p19^INK4dNK4d in the overexpression group of miR-3940-5p were up-regulated when the concentration of NIF was 0ug/ml and 1ug/ml,while the expression of p21^Cipip was only up-regulated when the concentration of NIF was 1ug/ml.Western Bolt results showed that when the concentration of NIF was 0ug/ml and 1ug/ml,the expression of CyclinA was up-regulated and the expression of CyclinE was down-regulated in the overexpression of mir-3940-5p group,while the expression of CDK2 and CKD4 was not different.CyclinD expression is down-regulated when NIF concentration is0ug/ml and up-regulated when NIF concentration is 1ug/ml.4.The detection results of osteogenic/odontogenic differentiation showed that overexpression of miR-3940-5p promoted the osteogenic/odontogenic differentiation of GMSCs in vitro.The results showed that the activity of alkaline phosphatase,an early indicator of osteogenic/odontogenic differentiation,increased in the experimental group compared with the control group,and the activity of alialiin red staining and calcium ion quantitative analysis,an indicator of late mineralization,significantly increased in the experimental group compared with the control group.Real-time rt-pcr results showed that overexpression of miR-3940-5p promoted the expression of DSPP,DMP1,and DLX5,the key transcription factor for osteogenic/odontogenic differentiation of GMSCs in vitro,at the mRNA level.Conclusions:1.Different concentrations of NIF can promote cell proliferation of GMSCs2.Overexpression of miR-3940-5p inhibited the proliferation of GMSCs cells under the stimulation of different concentrations of NIF3.Overexpression of miR-3940-5p inhibits the cell cycle of GMSCs in GO/G1 phase by up-regulating the expression of p15^INK4b,p18^INK4c,p19^INK4dNK4d and CyclinA,and down-regulating the expression of CyclinD and CyclinE.Overexpression of miR-3940-5p under the stimulation of NIF（1ug/ml）blocked the GMSCs cell cycle in the GO/G1 phase by up-regulating the expression of p15^INK4b,p18^INK4c,p19^INK4d,p21^Cip1,CyclinA and CyclinD,and down-regulating the expression of CyclinE4.Overexpression of miR-3940-5p promoted the osteogenic/odontogenic differentiation potential of GMSCs in vitro...