The Experimental Study On Transcription Factor KLF9 Promoting Osteogenic Differentiation And Participating In Bone Metabolism

Objective:With the gradual aging of our society,the incidence rate of age-related diseases,especially osteoporosis,has increased significantly in recent years.Therefore,it is particularly important to attach importance to the treatment of osteoporosis and deepen the exploration of the mechanism of bone metabolism.Krüppel like factors(KLFs)are a family of evolutionarily conserved and widely expressed transcription factors,which play an important role in regulating cell proliferation,differentiation,apoptosis,and normal development of the body.In recent years,the role of KLFs family members(KLF2,KLF4,KLF5,KLF10,KLF15)in bone metabolism is gradually revealed.KLF9 is a member of the KLFs family.Current studies have shown that KLF9 regulates the physiological and pathological processes of nervous,cardiovascular,digestive,respiratory,hematopoietic,immune,and other systems,but there is no research on its role in bone metabolism.This study explored the function of KLF9 in mouse preosteoblast(MC3T3-E1),the bone phenotype of Klf9-/-mice,and the role of KLF9 in thyroid hormone induced osteogenic differentiation,to provide new targets for the prevention and treatment of osteoporosis and metabolic bone diseases.Methods:Part Ⅰ:KLF9 knockdown and overexpression MC3T3-E1 stable cell line was constructed by lentivirus transfection.The transfection efficiency was verified by real-time quantitative PCR(qRT-PCR)and Western blotting(WB).KLF9 knockdown and its control group were expressed by Lv-shKlf9 and Lv-shCtrl,and overexpression and its control group were expressed by Lv-Klf9 and Lv-GFP.The effect of KLF9 on cell proliferation was detected by cell counting kit-8(CCK8).Osteoblast differentiation medium was used to induce cells to differentiate into osteoblasts.Alkaline phosphatase(ALP)staining,and alizarin red staining were performed on the day 7 and day 21 respectively to detect the effects of KLF9 on osteoblast differentiation and mineralization.The mRNA and protein expression levels of osteogenic differentiation related parameters were detected by qRT-PCR and WB.The binding of KLF9 to Alpl and Osx gene promoters was detected by luciferase reporter gene experiment.Part Ⅱ:The animals used were 4-week-old Klf9-/-mice with C57BL/6J background and littermate wild-type(WT)mice.Alizarin red and alcian blue staining was used to evaluate the bone and cartilage development of newborn male mice.Micro-CT was used to evaluate the bone microstructure of trabecular bone and cortical bone in the distal femur of 4-weekold male and female mice.H&E staining,toluidine blue staining,and von Kossa staining were used to evaluate the bone histomorphology of the distal tibia or proximal femur of mice.The rate of bone formation was detected by calcein double label test.Enzyme linked immunosorbent assay(ELISA)was used to detect the serums concentrations of Ctelopeptide of type Ⅰ collagen(CTX),procollagen type Ⅰ amino terminal peptide(P1NP)and bone alkaline phosphatase(BALP).The maximum load of femur was measured by small animal bone strength tester.Part Ⅲ:MC3T3-E1 cells were cultured in osteogenic medium for 7 days,then added T3 and continued to culture for 48 hours.ALP staining was performed,and Alpl mRNA expression was detected by qRT-PCR to prove the role of T3 in promoting bone differentiation.MC3T3-E1 cells were treated with different concentrations of T3(0,1,10,100,1000 nm)to detect the expression of Klf9 mRNA.Lv-shCtrl cells and Lv-shKlf9 cells were induced to differentiate in osteogenic differentiation medium for 7 days,and then cultured in medium with or without T3 for 48 hours.The level of osteogenic differentiation was detected by ALP staining.The mRNA expression levels of Alpl,THRA and THRB were detected by qRT-PCR,and the protein expression levels of ALP,THRA and THRB were detected by WB.Lv-Klf9 and control group cells were induced into osteogenic differentiation for 9 days,and the mRNA and protein expression levels of THRA and THRB were detected.Results:Part Ⅰ:(1)CCK8 experiment found that compared with the control group,overexpression of Klf9 significantly inhibited the proliferation of MC3T3-E1 cells from the 24h,knockdown of Klf9 had the trend of promoting cell proliferation from the 48h,and significantly promoted cell proliferation at the 72h.(2)Compared with the control group,the ALP staining and alizarin red staining of Lv-shKlf9 cells were weakened;Overexpression of Klf9 significantly enhanced ALP staining,and alizarin red staining suggested the increase of mineralized nodules.(3)After knockdown of Klf9,the expression levels of Osx and Alpl mRNA and protein decreased significantly compared with the control group.On the contrary,overexpression of Klf9 significantly increased the expression levels of Osx and Alp mRNA and protein.In addition,the expression level of bone sialoprotein(BSP)mRNA in Lv-shKlf9 group was significantly lower than that in the Lv-shCtrl group.Overexpression of Klf9 significantly upregulated the ratio of RANKL/OPG mRNA expression.(4)The results of double luciferase reporter gene experiment showed that the fluorescence value of co-transfected KLF9+Alpl promoter group was significantly higher than that of Alpl promoter group;The fluorescence value of co-transfected KLF9+Osx promoter group was significantly higher than that of Osx promoter group.Part Ⅱ:(1)There was no significant abnormality in the development of bone and cartilage in male KO mice at embryonic stage.(2)Compared with littermate WT mice,the bone volume fraction(BV/TV)and the number of trabeculae(Tb.N)of male KO mice were significantly reduced,the structure model index(SMI)was significantly increased,and the trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp)were not significantly changed.There was no significant change in trabecular bone parameters between female WT and KO mice.The area and thickness of cortical bone showed a downward trend in both female and male KO mice.However,there were no statistical difference.(3)H&E staining and toluidine blue staining showed no significant changes in the histomorphology of the proximal tibia.(4)The Von Kossa staining showed that the degree of bone mineralization decreased in female and male KO mice.(5)There was no significant change in bone formation rate in calcein double label test.(6)The results of ELISA showed that the level of P1NP decreased in female KO mice and increased in male KO mice,and there was no significant change in serum CTX and BALP.(7)There was no significant change in the maximum bending stress of femur in male and female KO mice.Part Ⅲ:(1)T3 can promote osteogenic differentiation and promote Alpl gene expression in a dose-dependent manner.(2)T3 significantly promoted the expression of Klf9 mRNA from 2 hours of treatment;Klf9 mRNA expression was promoted in a dose-dependent manner.(3)After adding T3,the ALP staining of Lv-shKlf9 group was still weaker than that of the control group,and the expression of Alpl was also significantly lower than that of the control group.(4)KLF9 had no effect on the expression levels of THRA mRNA and protein.The levels of THRB mRNA and protein increased with the decrease and overexpression of KLF9.Conclusion:(1)Overexpression of KLF9 inhibited the proliferation of MC3T3-E1 cells,and knockdown of KLF9 promoted the proliferation of MC3T3-E1 cells.Overexpression of KLF9 promotes osteogenic differentiation of MC3T3-E1 cells,and knockdown of KLF9 inhibits osteogenic differentiation.KLF9 regulates Alpl and Osx expression by binding with their promoters,thus affecting osteogenic differentiation.(2)The number and volume fraction of trabecular bone in 4-week-old male Klf9-/-mice decreased;PlNP levels increased.PlNP levels of the female Klf9-/-mice decreased.The mineralization of bone tissue in Klf9-/-female and male mice decreased.(3)T3 promotes the osteogenic differentiation of MC3T3-E1 cells.T3 promoted KLF9 expression in MC3T3-E1 cells in a dose-dependent manner,and the expression level of KLF9 was the highest after 2 hours treatment of T3.KLF9 partially mediated the osteogenic effect of T3 on MC3T3-E1 cells.KLF9 partially mediates the promoting effect of T3 on THRB gene expression.