Molecular Mechanism Of CACUL1 Regulating Adipogenic And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

With the aging of the population,osteoporosis has become an important public health problem in China.At present,most of the commonly used drugs to treat osteoporosis are antiresorptive drugs,and there are few drugs to promote bone formation.Therefore,further in-depth research on the mechanism of osteoporosis in order to find more effective osteogenesis drugs,has a very important clinical value.CACUL1(CDK2-associated cullin1)is a gene closely related to cell cycle regulation,and recent studies have found that CACUL1 inhibits 3T3-L1 cells differentiation into adipocytes by inhibiting the adipogenic marker PPAR?.BMSCs(bone marrow derived mesenchymal stem cells),like 3T3-L1 cells,are precursors of adipocytes and have the ability to differentiate into lipids.If CACUL1 has the same ability to inhibit adipogenic differentiation in BMSCs,CACUL1 can promote differentiation of BMSCs into osteoblasts and play a role in osteoporosis.This study includes the following three parts:(1)The difference of expression of CACUL1 gene in BMSCs in osteoporosis patients and normal people was analyzed using gene chip data from GEO database.Decreased expression of CACUL1 in patients with osteoporosis was verified by examining the bone marrow tissue of patients with osteoporotic fracture and normal fracture.(2)The expressions of CACUL1 in the process of adipogenic and osteogenic differentiation were analyzed in rat BMSCs.The effects of CACUL1 on adipogenic and osteogenic differentiation of BMSCs were analyzed using adenovirus overexpression and knockdown CACUL1 vector.(3)Analysis of the role and molecular mechanism of CACUL1 in BMSCs osteogenic differentiation.Part ? Expression of CACUL1 in BMSCs of osteoporosispatients Objective:The expressions of CACUL1 in BMSCs in osteoporosis patients and normal people were analyzed by bioinformatics methods and CACUL1 was demonstrated to be reduced in BMSCs in osteoporosis patients.The decrease of expression of CACUL1 gene in BMSCs in patients with osteoporosis was verified by clinical collection of bone marrow tissue of patients with osteoporotic fracture and normal fracture.Methods:Search and download the raw data of microarray GSE35958 from the GEO database.Data preprocessing and analysis using Bioconductor software based on R.The differential expression of CACUL1 in BMSCs between osteoporosis patients and normal people were identified.Recruitment of 6 patients with osteoporotic fractures and 6 patients without osteoporotic fractures according to inclusion and exclusion criteria.Collection of residual bone marrow tissue which used for isolation and culture of BMSCs during surgical reduction of fracture.The expression of CACUL1 protein was detected by Western blotting test.Results:After analyzing the data of GSE35958,a total of 805 differentially expressed genes were screened,and CACUL1 expression was 3.5 times lower in BMSCs in patients with osteoporosis than in the control group.Western blotting results showed that the expression of CACUL1 in BMSCs in osteoporosis patients was lower than that in normal control group.Conclusion:The expression of CACUL1 in osteoporosis patients is lower than that in normal people,and CACUL1 may play an important role in the occurrence and development of osteoporosis.Part ? Effect of CACUL1 on adipogenic and osteogenic differentiation of BMSCs Objective:This research aims to study the expression trend of CACUL1 gene in early stage of adipogenic and osteogenic differentiation of BMSCs in rats.Then,the effects of CACUL1 on adipogenic and osteogenic differentiation of BMSCs were studied using adenovirus knockdown or overexpression of CACUL1.Methods:Classic adipogenic and osteogenic induction protocols were applied to induce BMSCs in SD rats.The expression of CACUL1 was detected by qRT-PCR and Western blotting at 1 d,2 d,3 d,4 d and 5 d after induction.The markers of adipogenesis,PPAR?2,LPL and FABP4,and markers of osteogenesis,RUNX2,OCN and OPN,were detected after adenovirus vector knockdown and overexpression of CACUL1 gene.The roles of CACUL1 on adipogenesis and osteogenesis of BMSCs were explored.Results:CACUL1 mRNA and protein expression decreased 5 days after adipogenic differentiation,but increased 5 days after osteogenic differentiation.CACUL1 knockdown augmented the expressions of adipogenic markers,PPAR?2,LPL and FABP4,downregulated the expressions of osteoblast markers,RUNX2,OCN and OPN.However,CACUL1 overexpression had an opposite effect.Conclusion:CACUL1 expression decreased in early stage of adipogenic differentiation,but increased in early stage of osteogenic differentiation of BMSCs.CACUL1 inhibited adipogenesis and promoted osteogenesis.CACUL1 may be a key factor in the directional differentiation of BMSCs,and its osteogenesis may be an important potential target for the treatment of osteoporosis.Part ? Molecular mechanism of CACUL1 in BMSCs osteogenic differentiation Objective:Analysis of molecular mechanism of CACUL1 in BMSCs osteogenic differentiation.Methods:The expressions of p-ERK1/2 and ERK1/2 were detected by Western blotting in CACUL1 overexpression BMSCs.The expression of RUNX2,a marker of osteogenic differentiation,was detected by Western blotting after using ERK1/2 pathway-specific inhibitor U0126.Results:After overexpression of CACUL1 by adenovirus,the protein expressions of RUNX2 and p-ERK1/2 increased,however,the expression of EKR1/2 remained unchanged.The expressions of p-ERK1/2 and osteogenic marker gene RUNX2 decreased significantly after using the inhibitor U0126.Conclusion:CACUL1 promoted differentiation of BMSCs into osteoblasts and bone formation by activating the ERK1/2 signaling pathway.Owing to the osteogenic effect of CACUL1,it may be a potential drug target for the treatment of osteoporosis.