MiR-150 Inhibits Osteogenic Of Human Adipose Stem Cells By Regulating The Notch3/FAK/ERK1/2 Signaling Pathway

objective: In this study,adenovirus or lentivirus was used to infect human adipose stem cells.Through experimental methods such as cell culture,Real-time PCR,immunofluorescence,and Western blotting,it was demonstrated that miR-150 negatively regulates human adipose stem cells by targeting Notch3 / FAK / ERK1 / 2(hADSCs).Provide new ideas for the treatment of bone damage and bone defects.Methods: 1.Human adipose-derived stem cells were cultured in vitro.Three groups of cells: group N,GFP,and miR-150 were cultured in six-well plates.h ADSCs were infected with adenovirus over-expressing miR-150.Realtime-PCR was used to detect the expression of miR-150;Western blotting was used to detect the expression of Notch3.Western blotting was used to detect osteogenic signal pathway related proteins such as FAK,pFAK,ERK1 / 2,pERK1 / 2,and ROHA.The protein expressions of FAK,pFAK,ERK1 / 2,pERK1 / 2,RhoA,and F-actin were detected by immunofluorescence.CCK8 detected the effect of miR-150 on the proliferation of hADSCs.2.Four groups of cells: GFP group,Notch3-siR1 group,Notch3-siR2,Notch3-siR3 group were cultured in a six-well plate.hADSCs were infected with Notch3 knockout lentivirus.Western blotting was used to detect the expression of Notch3.Western blotting was used to detect FAK,pFAK,ERK1 / 2,pERK1 / 2,and RhoA related proteins.3.Three groups of cells: N group,GFP group,miR-150 inhibitor group were cultured in a six-well plate.hADSCs were treated with miR-150 inhibitors.Western blotting was used to detect the expression of Notch3.Western blotting was used to detect FAK,pFAK,ERK1 / 2,pERK1 / 2,and RhoA related proteins.The protein expressions of FAK,pFAK,ERK1 / 2,pERK1 / 2,RhoA,and F-actin were detected by immunofluorescence.4.Culture four groups of cells: group N,group N + OM,group GFP + OM,group miR-150 + OM in six-well plates.Osteogenic induction fluid was used to induce osteogenic differentiation of hADSCs.Western blotting was used to detect the osteogenic marker RUNX-2.Bone formation was identified by alkaline phosphatase(ALP)staining.5.Five groups of cells:N group,GFP group,GFP + OM group,Notch3-siR2 + OM group,Notch3-siR3 + OM group were cultured in six-well plates.Osteogenic induction fluid was used to induce osteogenic differentiation of hADSCs.Western blotting was used to detect the expression level of RUNX-2.Bone formation was identified by alkaline phosphatase(ALP)staining.Bone formation was identified by alkaline phosphatase(ALP)activity.6.Culture four groups of cells: N group,N + OM group,GFP + OM group,and miR-150 inhibitor + OM group in six-well plates.Osteogenic induction fluid was used to induce osteogenic differentiation of hADSCs.Western blotting was used to detect the osteogenic marker RUNX-2.Bone formation was identified by alkaline phosphatase(ALP)staining and activity.Results: 1.Adenovirus packaging miR-150 has successfully infected hADSCs.CCK8 results showed that the absorbance value of each group increased with time(1d,3d,5d,7d),but the cell proliferation trend of mi R-150 group was significantly lower than that of GFP group and N group.Western blotting results showed that the expression levels of Notch3,Pfak,and pERK1 / 2 proteins in mi R-150 group were significantly lower than those in N and GFP groups,while the expression levels of FAK and ERK1 / 2 proteins were not significantly changed.Immunofluorescence results showed that the expression levels of pFAK,pERK1 / 2,ROHA,and Factin in miR-150 group were significantly lower than those in GFP group and N group.2.The results of Western blotting showed that the expression levels of Notch3,pFAK,and pERK1 / 2 in the Notch3-siR2 and 3 groups were significantly lower than those in the GFP group,while the expression levels of FAK and ERK1 / 2 proteins were not significantly changed.3.The results of Western blotting showed that the expression levels of Notch3,pFAK,pERK1 / 2,and RhoA proteins in miR-150 inhibitor group were significantly higher than those in N group and GFP group.The results of immunofluorescence detection showed that the expression levels of pFAK,pERK1 / 2,RhoA,and F-actin proteins in mi R-150 group were significantly higher than those in N group and GFP group.4.After 7 days of osteogenesis induction,Western blotting showed that the expression of RUNX-2 protein in miR-150 + OM group was significantly lower than that in N group and GFP group.And the ALP activity test results showed that the miR-150 group had lower ALP activity than the GFP + OM group.ALP staining results showed that compared with the GFP + OM group,the intensity of ALP staining in the miR-150 group was weakened.5.Seven days after osteogenesis induction,Western blotting showed that the expression of RUNX-2 protein in Notch3-siR2,3 + OM group was significantly lower than that in N + OM group and GFP + OM group.And ALP activity test results showed that the ALP activity of Notch3-siR2,3 + OM group was lower than that of N + OM group and GFP + OM group.ALP staining results showed that compared with the N + OM group and the GFP + OM group,the ALP staining intensity of the Notch3-siR2,3 + OM group was weakened.6.Seven days after osteogenesis induction,Western blotting showed that the expression of RUNX-2 protein in miR-150 inhibitor + OM group was significantly higher than that in N group and GFP group.And ALP activity test results showed that the inhibitor group had higher ALP activity than the N and GFP groups.ALP staining results showed that compared with N group and GFP group,the intensity of ALP staining in inhibitor group was enhanced...