Mechanism Of Ring Finger Protein 11 Regulating Akt Signaling Pathway To Promote Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

ObjectiveBone marrow derived mesenchymal stem cells(BMSCs)have multidirectional differentiation potential.Osteogenic differentiation is an important direction of BMSCs differentiation,which can be used to treat osteoporosis,osteoarthritis,bone defect and other diseases.A variety of signal pathways can regulate the osteogenic differentiation of BMSC,including BMP,Wnt,notch,NELL signal pathway and so on.However,the molecular mechanism regulating the osteogenic differentiation of BMSCs has not been fully clarified,which hinders the further development of bone defect cell therapy based on BMSCs.Serine / threonine protein kinase Akt signaling pathway plays an important role in cell proliferation,differentiation and apoptosis,and also participates in BMSC osteogenic differentiation.Ring finger protein 11(RNF11)is one of the important members of the ring finger protein family.It participates in a variety of physiological and pathological processes through E3 ubiquitin ligase properties.It is highly expressed in bone and has Akt binding sites.We speculate that it may be involved in regulating the osteogenic differentiation of BMSCs,and there is no relevant report at present.Therefore,this study intends to explore whether RNF11 is involved in the regulation of osteogenic differentiation of BMSCs,and further clarify the molecular mechanism of RNF11 regulating osteogenic differentiation.Methods(1)BMSCs were isolated,cultured and subcultured from fresh bone marrow of healthy human body.The fourth generation cells were identified by flow cytometry,and then induced,cultured and identified into osteogenesis,chondrogenesis and lipogenesis.(2)BMSCs were cultured for 0 ～ 14 days.The degree of osteogenic differentiation was detected by alizarin red staining and ALP staining,and the expression of RNF11 protein was detected by Western blot.(3)The fourth generation BMSCs were divided into blank control group(group A),empty lentivirus(LV NC)group(group B)and knockdown RNF11(LV sh RNF11)group(Group C).Osteogenic differentiation was induced and cultured for 0 ～ 14 days.The expression of RNF11 protein was detected by Western blot,and the degree of osteogenic differentiation was detected by alizarin red staining and ALP staining,At the 14 th day,real-time fluorescence quantitative PCR was performed to detect the osteogenic markers of BMSCs: the relative expression of Runx2,osteocalcin(OCN)and osteopontin(OPN)mRNA;Akt,Smad1/5/8 and β-The relative expression of catenin signaling pathway protein was expressed by the ratio before and after phosphorylation.(4)In order to study the effect mechanism of RNF11 on Akt signaling pathway,the fourth generation BMSCs were divided into LV NC transfection group(group A1),LV sh RNF11 transfection group(Group B1)and LV sh RNF11 transfection group(Group C1)supplemented with Akt signaling pathway activator sc79.At 14 days,the relative expression of RNF11 and Akt signaling pathway protein was detected by Western blot,and the related indexes of osteogenesis were detected by alizarin red staining,ALP staining and QRT PCR.Results(1)Flow cytometry and osteogenic,chondrogenic and adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs.The relative expression of RNF11 protein increased gradually with the extension of osteogenic differentiation time(P<0.05);After down-regulation of RNF11,the number of positive calcium nodules and ALP activity in BMSCs were significantly lower.QRT PCR showed that the relative expression of Runx2,OCN and OPN mRNA decreased(P<0.05),suggesting that down-regulation of RNF11 expression in BMSCs will inhibit its osteogenic differentiation ability.(2)The relative expression of RNF11 and Akt signaling pathway protein increased with the extension of osteogenic differentiation time(P<0.05).After down regulating RNF11,the relative expression of Akt signaling pathway protein decreased significantly(P<0.05),while Smad1 / 5 / 8 and β-The relative expression of catenin signaling pathway protein had no significant effect(P>0.05).Further,on the basis of lv-sh RNF11,Akt signal pathway activator sc79 was used.It was found that it could activate Akt signal pathway,but the expression of RNF11 protein was not affected.The number of positive calcium nodules and ALP activity were higher than those in the control lv-sh RNF11 group,suggesting that Akt activator can reverse the effect of RNF11 and promote the osteogenic differentiation of MSC.At the same time,the number of positive calcium nodules and ALP activity were higher than those in the lvsh RNF11 group,and the relative expressions of Runx2,OCN and OPN mRNA were also higher.The above results suggest that RNF11 acts through Akt,which affects the osteogenic differentiation of BMSCs.ConclusionRNF11 promotes the differentiation of BMSCs into osteoblasts by activating Akt signaling pathway.RNF11 is expected to become a new target to improve the osteogenic differentiation efficiency of BMSC and guide clinical treatment in the future.