Effect Of LncRNA H19 On The Mesenchymal Characteristics Of Buffalo Bone Marrow Mesenchymal Stem Cells Under Hypoxic Culture

Bone marrow mesenchymal stem cells(BMSCs)have the advantages of self-renewal,pluripotency and immunosuppression.After long-term cultivation in vitro,BMSCs will gradually lose their mesenchymal characteristics due to mesenchymal epithelial transformation(MET),which requires BMSCs to undergo epithelial mesenchymal transformation(EMT)to maintain their mesenchymal characteristics.Studies have shown that hypoxia is one of the important reasons for activating EMT.In addition,it has been reported that hypoxic conditions are more conducive to the regulation of multiple lncRNA participating in EMT of tumor cells.Among them,lncRNA H19 is the key gene for hypoxic EMT promotion.Therefore,in this study,the effect of hypoxia culture conditions on the mesenchymal characteristics of Buffalo fetal BMSCs and the expression level of lncRNA H19 were examined,and then the effect of lncRNA H19 on maintaining the mesenchymal characteristics of Buffalo fetal BMSCs under hypoxia culture conditions and its mechanism also were explored,so as to further understand the molecular mechanism of maintaining the mesenchymal characteristics of Buffalo fetal BMSCs.First,Buffalo fetal BMSCs were obtained by whole bone marrow adherence method,and proliferation,apoptosis and migration of Buffalo fetal BMSCs in hypoxic culture were detected by CCK8,Annexin V-FITC/PI and Transwell respectively.The results showed that the proliferation rate,survival time and migration ability of Buffalo fetal BMSCs were increased under hypoxia culture.qPCR and Western blot results showed that the expression level of Vimentin gene m RNA and protein was up regulated,the expression level of E-Cadherin gene m RNA and protein was down regulated.lncRNA H19 expression was up regulated(P<0.01),and EMT was promoted.Then,the interference vectors of lncRNA H19 were constructed,and p Sico R-sh RNA-NC and p Sico R-sh RNA1324/1614/2096 were transfected respectively.The interference efficiency was analyzed by qPCR.The results showed that the interference efficiency of p Sico R-sh RNA1324 was the highest(P < 0.01).the proliferation rate,survival time and migration ability of lncRNA H19 of Buffalo fetal BMSCs were significantly reduced after transfection with p Sico R-sh RNA1324 in hypoxic culture,the expression level of Vimentin gene m RNA and protein was down regulated,the expression level of E-Cadherin gene m RNA and protein was up regulated,and EMT was inhibited.Finally,the mechanism of lncRNA H19 affecting the mesenchymal characteristics of Buffalo fetal BMSCs was explored.The results showed that the genes and proteins(PI3K,AKT1 and BCl2)involved in PI3K/Akt signaling pathway were inhibited by lncRNA H19,which interfered with Buffalo fetal BMSCs.IGF-1(10 ng/m L),an activator of PI3K/Akt signaling pathway,was added to eliminate the inhibition of PI3K/Akt signaling pathway caused by lncRNA H19 interference,and the effects of lncRNA H19 interference on cell proliferation,apoptosis,migration ability,EMT related genes and proteins were also partially eliminated.The above results indicated that the expression of BMSCs lncRNA H19 in buffalo fetus was promoted under hypoxia culture.In addition,lncRNA H19 plays an important role in maintaining the mesenchymal characteristics of Buffalo fetus BMSCs,which is mainly due to the fact that lncRNA H19 regulates the EMT of Buffalo fetal BMSCs through the PI3K/AKT pathway,which is conducive to the maintenance of mesenchymal features.