| Object:After obesity,the content of miR-548ag increases,which can up regulate Dipeptidyl peptidase-4（DPP4）and Fatty Acid Synthase（FASN）,resulting in the disorder of liver glucose and lipid metabolism.The specific molecular mechanism is not very clear.After the prediction of bioinformatics software in the early stage of the research group,it was found that there was miR-548ag binding site in the 3’untranslated region（3’UTR）of DNA methyltransferase 3B（DNMT3B）,up regulation of miR-548ag in human hepatoma cell line HepG2can inhibit the expression of DNMT3B,and Cp G sites were found in the promoter regions of DPP4 and FASN genes.Based on the cultivation of human hepatoma cell line HepG2 and human hepatoma cell line L02 and the construction of high-fat diet induced obesity mouse model,this study determined whether miR-548ag promoted the expression of downstream glucose and lipid metabolism related genes DPP4 and FASN through targeted inhibition of DNMT3B,which eventually led to the disorder of glucose and lipid metabolism.It provides a new theoretical basis for finding molecular targets for preventing and treating obesity and Type2 Diabetes Mellitus（T2DM）.Method:1.In vitro cell experiments:（1）The bioinformatics software targetscan was used to predict whether there was a binding site between miR-548ag and DNMT3B;Human hepatoma cell HepG2 was cultured in vitro,miR-548ag overexpression model was constructed,and 3’UTR luciferase reporter gene plasmid of DNMT3B was constructed in vitro.Double luciferase reporter gene experiment was used to verify whether miR-548ag can bind to DNMT3B.（2）Human hepatoma cells HepG2 and human hepatocyte L02 were cultured in vitro,after up regulating and inhibiting miR-548ag,the expression of downstream target gene DNMT3B,the expression of DPP4 and FASN and the ability of glucose and lipid metabolism were detected.（3）Human hepatoma cell HepG2 and human hepatocyte L02 were cultured in vitro to up/down regulate DNMT3B,and up regulate DNMT3B while up regulating miR-548ag.The effects on the expression of DPP4and FASN and the ability of glucose and lipid metabolism were detected.（4）After up regulation of miR-548ag and up/down-regulation of DNMT3B,Bisulfite sequencing PCR（BSP）method was used to detect the effects on the methylation modification of DPP4 and FASN promoter region in human hepatoma cell HepG2.2.In vivo animal experiments:（1）16 4-week-old male C57BL/6 mice were fed with normal diet（n=8）and high-fat diet（n=8）respectively after 1 week of adaptive feeding.The obesity related signs such as weight,body length and Lee’s index were dynamically monitored and continued to be fed until the 12th week.（2）16 4-week-old male C57BL/6 mice were fed with normal diet until the 16th week.They were divided into an intraperitoneal injection empty adenovirus group（n=8）and an intraperitoneal injection of miR-548ag overexpression adenovirus vector group(n=8,1×1011VP/mice,1 time/week),continuously injected for 6weeks to 22 weeks,and dynamically monitored the weight,body length,Lee’s index and other obesity related signs of mice.（3）16 4-week-old male C57BL/6 mice were fed with high-fat diet until the 16th week.They were divided into an intraperitoneal injection empty adenovirus group（n=8）and an intraperitoneal injection of miR-548ag inhibitor adenovirus vector group(n=8,1×1011VP/mice,1 time/week),continuously injected for6 weeks to 22 weeks,and dynamically monitored the weight,body length,Lee’s index and other obesity related signs of mice.（4）12 4-week-old male C57BL/6 db/db mice were fed with normal diet,and after 1 week of adaptive feeding.They were divided into an intraperitoneal injection empty adenovirus group（n=8）and an intraperitoneal injection of miR-548ag inhibitor adenovirus vector group(n=6,1×1011VP/mice,1 time/week),continuously injected for 6 weeks,and dynamically monitored the weight,body length,Lee’s index and other obesity related signs of mice.（5）Intraperitoneal Glucose Tolerance Trial（IPGTT）and Insulin Tolerance Test（ITT）were used to evaluate the glucose tolerance and insulin sensitivity of mice.The serum of mice was collected by orbital blood sampling method.The contents of blood glucose,FFA,TG,TC,HDL and LDL were measured.After the mice were killed,the liver tissues of mice were collected,and the expression levels of miR-548ag,DNMT3B,DPP4 and FASN in serum and liver tissues were detected by q RT-PCR and Western blot.（6）The effects of miR-548ag overexpression and inhibitor on the degree of methylation modification of DPP4 and FASN promoter region in mouse liver tissue were detected by BSP method.3.Statistical methods:SPSS 17.0 statistical analysis software was used for data analysis.If the data conform to the normal distribution,the t-test is used for analysis,and if the data do not conform to the normal distribution,the nonparametric rank-sum test is used for analysis.P<0.05,the divergence was regarded as statistically significant.Results:1.After obesity,the expression of miR-548ag in serum and liver tissue increased significantly,the expression of DNMT3B decreased significantly,and the expression of FASN and DPP4 increased significantly.（1）After feeding male C57BL/6 mice with a high-fat diet for 12 weeks,the body weight and Lee’s index of the mice in the high-fat diet group were significantly higher than those in the normal diet group;and the liver,mesenteric adipose tissue（MAT）,perirenal adipose tissue（PAT）,epididymal adipose tissue（Epi）,subcutaneous adipose tissue（Sub）weight of the mice in the high-fat diet group,and serum blood glucose,TG,TC,HDL and LDL levels were significantly higher than the normal diet group.According to the above results,the mouse obesity model has been successfully constructed.P<0.05,the divergence was regarded as statistically significant.（2）After 12 weeks of high-fat diet feeding,compared with the normal diet-fed mice,the expression of miR-548ag in the serum and liver tissue of the obese mice was significantly increased;the expression level of DNMT3B protein in the liver tissue of the mice was significantly decreased,The protein expression levels of FASN and DPP4 were significantly increased.P<0.05,the divergence was regarded as statistically significant.2.Overexpression of miR-548ag can inhibit the expression of DNMT3B,up-regulate the expression of DPP4and FASN,reduce the glucose consumption and insulin sensitivity of HepG2 and L02 cells,and promote cell lipid synthesis.（1）Using the bioinformatics software Target Scan,it was found that there is a miR-548ag binding site in the 3’UTR region of DNMT3B;the 3’UTR luciferase reporter gene plasmid of wild-type DNMT3B was co-transfected with miR-548ag mimic After reaching HepG2 cells,compared with the mutant plasmid group,the 3’UTR luciferase activity of DNMT3B in cells was significantly decreased,and the difference was statistically significant,P<0.05.（2）After 50n M miR-548ag mimic was transfected into HepG2 and L02 cells for 24h,the protein expression level of DNMT3B in cells was significantly decreased,and the protein expression levels of glucose metabolism-related gene DPP4 and lipid metabolism-related gene FASN were significantly increased,and The glucose consumption capacity and insulin sensitivity of the cells were significantly decreased,and the lipid synthesis capacity was significantly increased.P<0.05,the divergence was regarded as statistically significant.3.miR-548ag inhibitor can increase the expression of DNMT3B,down-regulate the expression of DPP4 and FASN,increase the consumption of glucose and insulinum susceptibility of HepG2 and L02 cells,and inhibit the cell lipid synthesis.（1）Refer to reagent instructions,after 100n M miR-548ag inhibitor was transfected into HepG2 and L02cells for 24 hours,the protein expression level of DNMT3B in the cells was significantly increased,and the protein expression levels of the glucose metabolism-related gene DPP4 and lipid metabolism-related gene FASN were significantly decreased.In addition,the glucose consumption capacity and insulin sensitivity of cells were significantly increased,and the lipid synthesis capacity was significantly decreased.P<0.05,the divergence was regarded as statistically significant.4.miR-548ag can down-regulate DNMT3B,promote the expression of DPP4 and FASN,reduce the glucose consumption capacity and insulin sensitivity of hepatocytes,and increase the lipid synthesis capacity.（1）After transfecting 4μg/ml and 1μg/ml DNMT3B overexpression plasmids into HepG2 and L02cells for 24 hours,the results showed that DNMT3B was successfully up-regulated,and after up-regulation of DNMT3B,the protein expression levels of DPP4 and FASN in HepG2 and L02 cells were significantly reduced.P<0.05,the divergence was regarded as statistically significant.（2）After transfecting 80 n M and 40 n M DNMT3B interference fragments into HepG2 and L02 cells for24 hours,the results showed that the intracellular DNMT3B was successfully down regulated,and after down-regulation of DNMT3B,the protein expression levels of DPP4 and FASN in HepG2 and L02 cells were significantly increased.P<0.05,the divergence was regarded as statistically significant.（3）Compared with the NC group,overexpression of miR-548ag can significantly inhibit the expression of DNMT3B,promote the protein expression levels of DPP4 and FASN,and significantly reduce the consumption of glucose and insulinum susceptibility of HepG2 and L02 cells,and reduce lipid synthesis.Compared with the simple overexpression group of miR-548ag,the overexpression of miR-548ag and the up-regulation of DNMT3B can significantly inhibit the protein expression levels of DPP4 and FASN in HepG2 and L02 cells,and make the consumption of glucose and insulinum susceptibility was significantly increased,and lipid synthesis capacity was significantly decreased.P<0.05,the divergence was regarded as statistically significant.5.miR-548ag can significantly inhibit the expression of DNMT3B in the liver,increase the expression of DPP4 and FASN,reduce the tolerance of glucose and insulinum susceptibility in mice,and induce lipid metabolism disorders in mice.（1）After continuous intraperitoneal injection of miR-548ag-overexpressing adenovirus vector for 6weeks in normal diet-fed mice,compared with the control group,the body weight,liver and adipose tissue weight of the mice were significantly increased,and serum and liver tissue were significantly increased.The expression of miR-548ag was significantly increased,the protein expression level of DNMT3B in liver tissue was significantly decreased,and the DPP4 and FASN were significantly increased.P<0.05,the divergence was regarded as statistically significant.（2）After continuous intraperitoneal injection of miR-548ag-overexpressing adenovirus vector for 6weeks in normal diet-fed mice,compared with the control group,the blood glucose level of the mice was significantly increased,and the tolerance of glucose and insulinum susceptibility were significantly decreased.Blood lipid levels such as TG,FFA and LDL were significantly increased.P<0.05,the divergence was regarded as statistically significant.（3）After continuous intraperitoneal injection of miR-548ag inhibitor adenovirus vector in high-fat diet-fed mice for 6 weeks,compared with the control group,the body weight,liver and adipose tissue weight of the mice were significantly reduced,and serum and liver tissue were significantly reduced.The protein expression level of DNMT3B in liver tissue was significantly increased,and the protein expression levels of DPP4 and FASN were significantly decreased.P<0.05,the divergence was regarded as statistically significant.（4）After continuous intraperitoneal injection of miR-548ag inhibitor adenovirus vector in high-fat diet-fed mice for 6 weeks,compared with the control group,the blood glucose level of the mice was significantly decreased,and the tolerance of glucose and insulinum susceptibility were significantly increased.Blood lipid levels such as TC,TG and LDL-C were significantly reduced.P<0.05,the divergence was regarded as statistically significant.6.miR-548ag inhibitor can up-regulate the expression of DNMT3B in the liver tissue of db/db mice,down-regulate the expression of DPP4 and FASN,enhance the tolerance of glucose and insulinum susceptibility of the mice,and improve the lipid metabolism disorder in the mice.（1）After continuous intraperitoneal injection of miR-548ag inhibitor adenovirus vector to db/db mice for 6 weeks,compared with the control group,the body weight,Lee’s index,liver and adipose tissue weight of the mice were significantly reduced;The protein expression level of DNMT3B was significantly increased in liver tissue,and the protein expression levels of DPP4 and FASN were significantly decreased.P<0.05,the divergence was regarded as statistically significant.（2）After intraperitoneal injection of miR-548ag inhibitor adenovirus vector into d B/db mice for 6weeks,the blood glucose of the experimental group was significantly lower than that of the control group,the glucose tolerance and insulin sensitivity increased significantly,and the blood lipid levels such as TC and TG decreased significantly.P<0.05,the divergence was regarded as statistically significant.7.miR-548ag and DNMT3B had no significant effect on the methylation status of Cp G sites in DPP4 and FASN genes promoter site.miR-548ag and DNMT3B had no significant effect on the methylation status of Cp G sites in DPP4 and FASN genesin promoter region（1）Compared with the control group,overexpression of miR-548ag had no significant effect on the methylation status of Cp G sites in DPP4（0 vs 1.2%）and FASN（1.7%vs 0）genes promoter site of HepG2cells（P>0.05）.（2）Compared with the control group,up-regulation of DNMT3B had no significant effect on the methylation status of Cp G sites in DPP4（1.2%vs 0）and FASN（0.5%vs 0.5%）genes promoter site of HepG2cells（P>0.05）;Compared with the control group,down-regulation of DNMT3B had no significant effect on the methylation status of Cp G sites in DPP4（0 vs 1.7%）and FASN（0.5%vs 1.0%）genes promoter site of HepG2 cells（P>0.05）.（3）Compared with the control group,intraperitoneal injection of miR-548ag-overexpressing adenoviral vector has no significant effect on the methylation of Cp G sites in DPP4（0.5%vs 0.5%）and FASN（0.5%vs0.1%）genes promoter site of mouse livers（P>0.05）;Compared with the control group,intraperitoneal injection of miR-548ag inhibitor adenovirus vector had no significant effect on the methylation of Cp G sites in DPP4（0.5%vs 0.3%）and FASN（1.1%vs 1.3%）genes promoter site of mouse livers（P>0.05）.Conclusion:1.The increase of serum miR-548ag content after obesity can specifically inhibit DNMT3B,increase the expression of DPP4 and FASN,and finally lead to the disorder of liver glucose and lipid metabolism.2.The promoting effect of miR-548ag and DNMT3B on the expression of DPP4 and FASN is not achieved by directly changing the methylation state of the Cp G site in the above gene promoter site. |
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