Knockdown Of Ckip-1 Promotes Proliferation And Osteogenic Differentiation Of Mesenchymal Stem Cells By Regulating Osteogenesis-related Signaling Pathway

Backgrounds: Therapies of a series of bone defect-related diseases caused by craniofacial bone tumors,fractures and bone abnormalities were hot topics in the field of oral medicine.More and more studies have shown that mesenchyme stem cells(MSCs)are the main cellular source in bone formation,and their proliferation and osteogenic differentiation are the keys to repairing bone defects.Important signaling pathways and genes can significantly influence the proliferation and osteogenic differentiation of MSCs.It is reported that the five osteogenesis-related signaling pathways(WNT pathway,BMP pathway,FGF pathway,HH pathway,NOTCH pathway)are closely related to the proliferation and osteogenic differentiation of MSCs.According to recent reports,Ckip-1(Casein Kinase-2 Interaction Protein-1)is the negative regulate molecule associated with bone formation.The mice knocked out Ckip-1 showed increased bone mass,and the osteogenic ability of osteoblast is enhanced.However,can the knockdown of Ckip-1 affect the proliferation and osteogenic differentiation of MSCs? And whether it can regulate osteogenesis-related signaling pathways during osteogenic differentiation of MSCs? All these questions need to be investigated.Aims: 1.To explore the regulation of Ckip-1 on the proliferation and osteogenic differentiation of MSCs;2.To investigate the effect of the knockdown of Ckip-1 on the osteogenesis-related signaling pathway in the process of osteogenic differentiation of MSCs;3.To investigate whether the knockdown of Ckip-1can regulate proliferation and osteogenic differentiation of MSCs through the key molecule Lrp5 in WNT signaling pathway.Methods: 1.RNA interference and lentivirus infection were used to construct the C3H10T1/2 cell line with stable over expression of Ckip-1 and the C3H10T1/2 cell line with knockdown of Ckip-1;2.QPCR and Western Blot were used to detect the transcription level and the expression level of Ckip-1 gene in C3H10T1/2 cells;3.Immunofluorescence technique was used to locate the position of Ckip-1 protein in C3H10T1 /2 cells;4.MTT assay and Annexin V staining were used to detect the effect of Ckip-1 on proliferation and apoptosis of C3H10T1/2 cells;5.QPCR technique was used to detect the effect of Ckip-1 on the transcriptional level of osteogenic genes in C3H10T1/2 cells;6.Osteogenic induction and ALP staining were used to detect the effect of Ckip-1 on the osteogenic ability of C3H10T1/2 cells;7.QPCR technique was used to detect the effect of the knockdown of Ckip-1 on transcription level of key molecule in osteogenesis-related signaling pathway in C3H10T1/2 cells;and detect the effect of the knockout of Ckip-1 on transcription level of key molecule in osteogenesis-related signaling pathway in BMMSCs;8.QPCR technique was used to detect the effect of the knockdown of Ckip-1 on transcription level of key molecules in osteogenesis-related signaling pathway in C3H10T1/2 during osteogenic differentiation;and detect the effect of the knockout of Ckip-1 on transcription level of key molecules in osteogenesis-related signaling pathway in bone;9.Western Blot was used to detect the effect of the knockdown of Ckip-1 on the expression of Lrp5 and ?-catenin in the WNT signaling pathway during osteogenic differentiation of C3H10T1/2 cells;10.MTT assay,Annexin V staining and QPCR technique were used to analyze the effects of the knockdown of Lrp5 and the knockdown of Lrp5 and Ckip-1 on the proliferation,apoptosis and osteogenic activity of C3H10T1/2 cells.Results: 1.Fluorescence microscopy showed that the transfection efficiency of each gene was more than 99%;the cell growth was fine,and the morphology was not different;2.QPCR and Western Blot showed that the transcription level and protein level of Ckip-1 in sh Ckip-1 group were lower than those in sh Ctrl group.Compared with EV group,the transcription level and protein level of Ckip-1 in Ckip-1 group were increased;3.Immunofluorescence staining showed that the protein expression level of Ckip-1 in sh Ckip-1 group was decreased compared with sh Ctrl group,and it was aggregated in cytoplasm and nucleus.Compared with EV group,Ckip-1 was mainly expressed in the cell membrane and the nucleus without aggregation in Ckip-1 group;4.MTT assay and Annexin V staining showed that the cell proliferation activity of sh Ckip-1 group was decreased,and the apoptotic ability was decreased compared with those in sh Ctrl group.Compared with EV group,the cell proliferation activity was decreased,and the apoptotic ability was enhanced in Ckip-1 group;5.QPCR showed that the transcriptional level of Runx2,Osx,Col1,Ocn,Bsp in sh Ckip-1 group was higher than that in sh Ctrl group.Compared with EV group,the transcriptional level of Runx2,Osx,Col1,Ocn was decreased and the activity of Alp was decreased in Ckip-1 group;6.The Alp staining showed that the Alp activity of sh Ckip-1 was enhanced compared with sh Ctrl group.Compared with EV group,the activity of Alp in Ckip-1 group was decreased;7.QPCR showed that the transcriptional levels of Lrp5,Tcf1 and Lef1 in the WNT pathway in sh Ckip-1 group were higher than those in sh Ctrl group,and the transcription level of Bmpr1 a and Smad4 in BMP pathway were increased,and the transcription level of Fgfr1 and Fgfr2 in FGF pathway were decreased,and the transcription level of Gli2 in HH pathway was increased,while the transcription level of Ptch1 decreased,and the transcription level of Hes1 in NOTCH pathway was decreased,but the transcription level of Notch2 was not significant;Compared with BMMSCs in WT group,the changes of WNT pathway and BMP pathway in Ckip-1-/-group were consistent with the above results.However,the transcription levels of Fgfr1 and Fgfr2 in FGF pathway were increased,and the transcription level of Gli2 and Ptch1 in HH pathway was increased,and the transcription levels of Hes1 in NOTCH pathway was decreased,but the transcription level of Notch2 was not significant;8.QPCR showed that the transcriptional levels of Lrp5,Tcf1 and Lef1 in the WNT pathway in sh Ckip-1 group were higher than those in sh Ctrl group during osteogenic differentiation,and the transcription level of Bmpr1 a and Smad4 in BMP pathway were increased,and the transcription level of Fgfr1 and Fgfr2 in FGF pathway were increased,and the transcription level of Gli2 in HH pathway was increased,while the transcription level of Ptch1 was decreased,and the transcription level of Notch2 in NOTCH pathway was not significant,but the Hes1 decreased;Compared with bone in WT group,the transcriptional levels of Lrp5 and Tcf1 in the WNT pathway in Ckip-1-/-group were increased,but the transcription level of Lef1 was decreased.The transcription level of Bmpr1 a in BMP pathway was not significant,but the transcription level of Smad4 was increased,and the transcription level of Fgfr1 and Fgfr2 in FGF pathway were increased,and the transcription level of Gli2 in HH pathway was increased,while the transcription level of Ptch1 was decreased,and the transcription level of Notch2 and Hes1 in NOTCH pathway were decreased;9.Western Blot showed that the expression of Lrp5 in sh Ckip-1 group was significantly higher than that in sh Ctrl group after osteogenic induction of 7 days,but the expression level of ?-catenin did not change;The expression level of Lrp5 and ?-catenin in sh Ckip-1 group were significantly higher than those in sh Ctrl group after 14 days of induction;10.MTT assay,Annexin V staining and QPCR showed that the cell proliferation activity and apoptotic ability in sh Lrp5 group were significantly decreased than that in sh Ctrl group.Compared with sh Ctrl group,the cell proliferation activity was significantly high and the apoptotic ability was decreased in sh Lrp5+sh Ckip-1 group.Compared with sh Lrp5 group,the cell proliferation activity was enhanced,and the apoptotic ability was significantly decreased and the transcription level of osteogenesis-related gene was significantly increased in sh Lrp5+sh Ckip-1 group.Conclusions: 1.Knockdown of Ckip-1 can promote the proliferation of MSCs and inhibit its apoptosis,while enhancing osteogenic differentiation;2.Knockdown of Ckip-1 can enhance the osteogenesis-related WNT,BMP,FGF,HH signaling pathway activity of MSCs during osteogenic differentiation,and inhibit the NOTCH signaling pathway;3.Knockdown of Ckip-1 can enhance proliferation and osteogenic differentiation of MSCs through Lrp5 in WNT signaling pathway.