| Background Obesity has gradually become a global public health problem.For example,the incidence of obesity in the United States is 17.1% among children and adolescents,and about 32.2% in adults.In China,there are about 21 million overweight children,50% of whom are diagnosed with obesity [1].Obesity are closely associated with hypertension,stroke,type 2 diabetes,coronary heart disease and the occurrence of certain tumors [2].Obesity is closely related to osteoporosis,and estrogen synthase,leptin,adiponectin and various pro-inflammatory cytokines secreted by adipose tissue play an important role in bone reconstruction [3].Osteoporosis is a systemic bone disease,characterized by decreased bone mass,microstructural destruction of bone tissue,increased bone fragility and susceptibility to fracture.Fractures,disability,and chronic pain are ommon clinical complications of osteoporosis [4].Studies have found that most patients with osteoporosis showed increased bone marrow fat content and bone loss [5].As the main type of adult stem cells,MSCs are the common progenitor cells of osteoblasts and adipocytes,so there is a competitive inhibition between osteogenic differentiation and adipogenic differentiation [6].At the same time,fat cells have toxic effects on the function and activity of osteoblasts [7].Micro RNAs(mi RNAs)are a class of endogenous single-stranded non-coding RNAs(about 18-24 nucleotides)discovered in recent years that inhibit post-transcriptional by specifically binding to the 3' non-coding region of the target gene m RNA.Horizontal et al [8] have shown that mi RNAs are important regulators of the body's developmental and physiological processes,involving in energy metabolism,cell differentiation,pancreatic beta cell development,lipid metabolism and so on [9,10].mi RNAs play an important role in the differentiation of mesenchymal stem cells [11,12].Therefore,understanding the effects of mi R-2861 on adipogenesis and adipogenic differentiation of MSCs and possible molecular mechanisms may provide a basis and reference for the prevention and treatment of clinically relevant diseases such as osteoporosis and obesity.Purpose(1)To explore the effect and mechanism of mi R-2861 on adipogenic differentiation of MSCs and 3T3-L1 cells.(2)To explore the effect of mi R-2861 on adipogenic differentiation and osteogenic differentiation of MSCs when co-cultured with OBs or adipocytes.Methods(1)Obese mouse model and normal mice were established by feeding with high-fat diet and normal control chow;Identified obesity through body weight measurement,BMI calculation and adipose tissue weighing,adipose tissue HE staining and RT-q PCR used to detect marker genes of adipogenic differentiation.RT-q PCR was used to detect the differential expression of mi R-2861 in perirenal fat and inguinal adipose tissue between obese mice and normal mice;RT-q PCR was used to examine the expression of mi R-2861 in MSCs and C3H10T1/2 during adipogenic differentiation and in 3T3-L1 during adipogenesis;(2)MSCs were isolated from the compact bone from mouse femur by type II collagenase digestion and cultured in 5% hypoxic environment;cell surface molecular markers were detected by flow cytometry;cell morphology observation,alizarin red staining,Oil red O staining and RT-q PCR used for the expression of key genes for osteogenic and adipogenesis were carried out;Primary osteoblasts were isolated from mouse born with 72 hours by type ? collagenase and trypsin digestion.(3)After over-expression or inhibition of mi R-2861 by cell transfection,adipocytes formation was detected in MSCs,C3H10T1/2 and 3T3-L1 cells by Oil Red O staining,adipocytes counting.RT-q PCR and Western blot were carried out for m RNA and protein expression of adipogenic marker genes.(4)Combining informatics software prediction,dual luciferase reporter gene detection and Western blot experiments were carried out to determine the target gene of mi R-2861.(5)Down-regulating protein expression of SIRT1 by cell transfection with small interfering si-RNA-SIRT1 or using SIRT1 activitor CAY10602 to determine the effect of SIRT1 on the adipogenic differentiation of 3T3-L1 by Oil Red O staining,adipocytes counting and by RT-q PCR assay for adipogenic marker gene expression.After transfection of mi R-2861 mimic and si-SIRT1 simultaneously,transfection of mi R-2861 inhibitor and si-SIRT1 simultaneously and transfection of mi R-2861 mimic with CAY10602 administration,Oil Red O staining,adipocytes counting and RT-q PCR and Western blot were carried out to examine whether mi R-2861 modulate adipogenic differentiation by targeting SIRT1.(6)Immunofluorescence and Western blot technique were used to detect the expression of ?-catenin in the nuclear after over-expression and inhibition of mi R-2861 and silencing SIRT1.The expression of ?-catenin in the nuclear was detected by immunofluorescence and Western blot after inhibition of mi R-2861 and silencing SIRT1 simultaneously.(7)Co-culture MSCs with OBs in indirect contact way by transwell chamber.The m RNA expression of a P2,C/EBP ? and PPAR ? and ALP,OCN,RUNX2 were detected by RT-q PCR and Western blot after transfection of MSCs with mi R-2861 mimic or inhibitor under adipogenic induction or osteogenic induction medium;MSCs were co-cultured with adipocytes which were derived from 3T3-L1 cells,and MSCs were transfected with mi R-2861,osteoblastic induction of MSCs were performed simultaneously.Formation of mineralized nodules were observed by Alizarin red staining,and m RNA expression of the osteogenic marker genes(ALP,OCN,RUNX2)was examined by RT-q PCR.Results(1)The weight and BMI of the mice in the high-fat diet group increased by more than 20% compared with the normal group,which was consistent with the obesity standard.The weight of perirenal fat tissue and inguinal fat in the high-fat diet group was significantly higher than that in the normal diet group;HE staining of perirenal adipose tissue and inguinal adipose tissue in the high-fat diet group showed that the volume of single adipocytes was significantly higher than that of the normal conteol chow;RT-q PCR results showed that the adipogenic marker genes adiponectin,a P2,C/EBP? and PPAR? in the high-fat diet group than that of control group.RT-q PCR results showed that the expression of mi R-2861 in the perirenal fat and inguinal adipose tissue of the high-fat diet group was significantly higher than that in the perirenal fat and inguinal adipose tissue of control group;The expression of mi R-2861 was significantly increased during adipogenesis of 3T3-L1 compared with the non-induced group and reached the peak on the fourth day,and then decreased,but still be higher than that of the non-induced group.Mi R-2861 increased significantly during adipogenic differentiation process of MSCs and C3H10T1/2 cells,but decreased significantly during the osteogenic differentiation process.(2)The MSCs isolated from compact bone were spindle-shaped;the results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD44(80%)and CD29(90.5%),and the surface markers of hematopoietic cell were negative for CD45(0.5%).After osteogenic induction of MSCs,Alizarin red staining was positive,and RT-q PCR results showed that the expression of osteogenic marker genes(ALP,OCN,RUNX2)were significantly increased;After adipogenic induction of MSCs,Oil red O staining was positive.At the same time,RT-q PCR results showed that the expression of marker genes of adipogenesis(adiponectin,a P2,C/EBP?,PPAR?)was significantly increased.(3)After over-expression of mi R-2861,Oil red O staining and adipocytes counting were performed for adipocytes formation,RT-q PCR and Western blot were performed to detect the adipogenic marker gene and protein expression(adiponectin,a P2,C/EBP?,PPAR?),and the results showed that over-expression of mi R-2861 can promote adipogenic differentiation of MSCs and C3H10T1/2;Conversely,inhibition of mi R-2861 expression significantly inhibited adipogenic differentiation of MSCs and C3H10T1/2 compared with negative control group.After over-expression of mi R-2861,adipocytes formation was detected by Oil Red O staining and adipocytes counting.RT-q PCR and Western blot were performed for detecting the adipogenic marker gene and protein expression(adiponectin,a P2,C/EBP?,PPAR?),and the results showed that over-expression of mi R-2861 can promote adipogenesis of 3T3-L1 cells;Conversely,inhibition of mi R-2861 expression significantly inhibited adipogenesis of 3T3-L1 cells compared with negative control group.(4)The results of dual luciferase reporter experiment and Western blot showed that SIRT1 is a direct target gene of mi R-2861.(5)After MSCs and 3T3-L1 were transfected with si-SIRT1,the protein expression of SIRT1 decreased.After down-regulation of SIRT1,Oil red O staining and adipocytes were significantly increased.RT-q PCR results showed that si-SIRT1 caused increased m RNA expression of a P2,C/EBP? and PPAR?;CAY10602 as an activator of SIRT1 reduced m RNA expression of a P2,C/EBP? and PPAR?,indicating that SIRT1 inhibits adipogenic differentiation.Compared with the co-transfection group of mi R-2861 mimic negative control and si-SIRT1,the number of adipocytes and adipogenic marker genes expression increased significantly in the co-transfection group of mi R-2861 mimic and si-SIRT1 under adipogenic induction;after transfection of mi R-2861 mimic and use of CAY10602 under adipogenic induction,adipogenic marker genes expression were significantly decreased compared with mi R-2861 mimic transfection.;compared with co-transfection of mi R-2861 inhibitor negative control and si-SIRT1,mi R-2861 inhibitor and si-SIRT1 co-transfection showed decreased Oil Red O staining and adipogenic marker genes expression.(6)Immunofluorescence and Western blot results showed that the expression of ?-catenin in the nuclear was decreased when over-expression of mi R-2861 mimic compared with the transfection of mimic negative control group.Conversely,the expression of ?-catenin in the nucleus was higher when transfection with mi R-2861 inhibitor than transfection of inhibitor negative control.The nucleus expression of ?-catenin in cells transfected with mi R-2861 inhibitor and si-SIRT1 was lower than that in the nuclear of cells transfected with mi R-2861 inhibitor and the si RNA negative control group.(7)Adipogenic differentiation of MSCs was decreased when MSCs were co-cultured with OBs for 4 days.Adipogenic marker genes(adiponectin?a P2?PPAR??C/EBP?)expression was increased after co-cultured with OBs and transfected with mi R-2861 mimic for 4 days,PPAR? expression of MSCs was decreased after transfected with mi R-2861 inhibitor.Adipogenic differentiation of MSCs was increased when MSCs were co-cultured with OBs for 8 days.Adipogenic marker genes(adiponectin?a P2?PPAR??C/EBP?)expression was increased after co-cultured with OBs and transfected with mi R-2861 mimic for 8 days.Conversely,expression of a P2?PPAR? and C/EBP? were decreased in MSCs after transfected with mi R-2861 inhibitor.Western blot results showed that mi R-2861 mimic promoted the protein expression of a P2 and C/EBP? in MSCs,whereas mi R-2861 inhibitor inhibited the expression of a P2 and C/EBP protein.Mi R-2861 mimic significantly decreased the m RNA expression of OCN and Osterix in MSCs when MSCs were co-cultured with OBs for 8 days,whereas mi R-2861 inhibitor promoted the expression of ALP?OCN?Runx2 and Osterix.mi R-2861 mimic significantly decreased the osteogenic marker genes(ALP?OCN?Runx2 and Osterix)expression of MSCs when MSCs were co-cultured with adipocytes for 8 days,whereas mi R-2861 inhibitor promoted the m RNA expression of ALP and Runx2.Alizarin red staining showed that mineralized nodules in mi R-2861 mimic group were significantly less than those in NC-mimic group.More mineralized nodules were observed in mi R-2861 inhibitor group than in NC-inhibitor group.Conclusion(1)mi R-2861 promotes adipogenic differentiation by targeting SIRT1 through Wnt/?-catenin signal pathway.(2)mi R-2861 promoted adipogenic differentiation and inhibited osteogenic differentiation of MSCs when co-cultured with OBs;mi R-2861 inhibited osteogenic differentiation of MSCs when co-cultured with adipocytes. |
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