The Roles Of Histone Demethylase Kdm2a Regulation On Adipocyte Proliferation And Differentiation In Mice

With the development of society and the continuous improvement of living standards,diet-induced obesity and related metabolic diseases are increasing,which seriously affect the health of humans or animals.Therefore,obesity and related metabolic diseases have become the focus of attention in the world.Current studies have shown that adipose tissue is treated as an important "energy metabolism balancer" and it’s excessive deposition is the main cause of obesity.In addition,studies have shown that epigenetic regulation plays an important role in adipose tissue deposition.Therefore,further studies on epigenetic regulation factors and their mechanisms of adipose tissue deposition will provide new ideas for the treatment of obesity prevention and related metabolic diseases.Histone demethylase 2A(KDM2A),as one of the important epigenetic modifications,plays an important role in the growth and development of oocytes and embryos,as well as in the occurrence and development of diseases.However,the role of Kdm2a in adipose tissue deposition remains unclear.Here,the expression pattern and subcellular localization of Kdm2a in different types of mouse adipocytes were firstly detected,and then the effects of Kdm2a on proliferation and differentiation of preadipocytes were studied using3T3-L1 preadipocytes as a model.Finally,Adipoq-Cre mediated adipose tissue specific knockout of Kdm2a was used to study the effect of Kdm2a on the differentiation of primary adipocytes in mice.The following results are obtained:1.Expression pattern and subcellular localization of Kdm2a in different types of adipose tissue of mice1)Kdm2a was expressed in inguinal white adipose tissue(iWAT),epididymal white adipose tissue(eWAT)and brown adipose tissue(BAT)of mice at different ages(E18.5 d,4 d,45 d,60 d and 150 d).The mRNA expression level of Kdm2a reached the peak in epididymal white adipose tissue(eWAT)of mice at 45 days(p<0.0001).In addition,with the increase of age,the expression of Kdm2a showed a downward trend.During the differentiation of mouse primary adipocytes and 3T3-L1 preadipocytes,the level of the mRNA for PPARγ was up-regulated,while the expression of Kdm2a showed a downward trend.2)The expression and localization of KDM2A protein in preadipocytes and adipocytes differentiated at 0 d,6 d and 13 d were detected by immunofluorescence technique,and it was found that KDM2A protein was mainly localized in the nucleus.2.The effect of Kdm2a on the proliferation and differentiation of 3T3-L1 adipocytes1)Using siRNA technique to interfere with the expression of Kdm2a,it was found that the interference efficiency of siRNA2 was about 60%,and the protein expression of KDM2A was significantly decreased by immunofluorescence technique(p<0.05).2)CCK-8 detected the proliferation of 3T3-L1 preadipocytes after siRNA2 interference at 0 h,12 h,24 h,48 h and 72 h,and it was found that siRNA2 interference at 48 h and 72 h significantly inhibited the proliferation of 3T3-L1adipocytes(p<0.01),and Ki67 and Ed U staining results were consistent with the result of CCK-8.At the same time,the expression levels of cell cycle related genes were detected after siRNA2 interfered with Kdm2a,and the mRNA expressions of CCND1,CCND2 and CCNB1 were down-regulated,but the differences were not significant.In conclusion,interference with Kdm2a inhibited the proliferation of3T3-L1 preadipocytes.3)The relative expression of C/EBPα in adipocytes differentiation was significantly up-regulated by siRNA2 interference with Kdm2a(p<0.01),and the mRNA levels of triglyceride synthesis and decomposition related genes,such as GPAM,DGAT1,ATGL,were also significantly up-regulated(p<0.0001).In addition,the fluorescence signal of PPARγ was enhanced after interference with Kdm2a.However,when the relative expression of brown fat specific marker genes such as Ucp1,Prdm16,Cidea and Pdk4 were measured,it was found that the expression levels of brown fat specific marker genes increased after the interference of Kdm2a,but the differences were not significant.3.Effect of Kdm2a knockout on differentiation of primary adipocytes in mice1)Compared with control mice(WT),the relative expression of Kdm2a was significantly down-regulated in inguinal white adipose tissue(iWAT),epididymal white adipose tissue(eWAT),brown adipose tissue(BAT)and scapular white adipose tissue(AsWAT)of knockout mice(p<0.01),but had no change in other tissues.2)After the specific deletion of Kdm2a in adipose tissue,primary adipocytes were isolated and cultured for inducing differentiation for 10 days.After oil red O and Bodipy staining,it was found that Kdm2a knockout significantly promoted the differentiation of the preadipocytes.The results showed that Kdm2a was expressed in different types of adipose tissue of mice,but the mRNA expression of Kdm2a showed a decreasing trend with the increase of age.During the differentiation of mouse primary adipocytes and 3T3-L1 preadipocytes,the expression level of PPARγ was up-regulated,while the expression of Kdm2a was decreased.KDM2A protein was localized in the nucleus of preadipocytes and mature adipocytes at different stages of differentiation.After interference with Kdm2a by siRNA,proliferation of 3T3-L1 preadipocytes was inhibited and differentiation of 3T3-L1 preadipocytes was promoted.We found that knocking out of Kdm2a promoted the differentiation of primary adipocytes in mice,which laid a foundation for further elucidating the specific mechanism of Kdm2a in the process of fat deposition in mice.