Study On The Role And Mechanism Of Adipose Tissue-derived MicroRNA-450a-5p Inducing T2DM By Down-regulating DUSP10

Objective:Based on the collection of general data and serum samples of normal-weight,obesity,and Type 2 Diabetes Mellitus（T2DM）subjects,and the establishment of diet-induced obesity mouse models,this study analyzed the correlation between the expression levels of microRNA-450a-5p and FPG,blood lipids and general data;on the basis of culturing HepG2cells,L02 cells and 3T3-L1 cells in vitro,observe whether microRNA-450a-5p targeted down-regulates dual specificity phosphatase 10（Dual Specificity Phosphatase 10,DUSP10）lead to the disorders of glucose and lipid metabolism,induce insulin resistance（IR）and T2DM,and explore possible molecular mechanisms to provide a new theoretical basis for clinical treatment to find molecular targets.Methods:（1）September 2016-October 2019,the normal weight（n=72）,simple obesity（n=120）and T2DM（n=58）individuals serum samples of Uyghur in the 51 regiment of the third Division of Xinjiang Production and Construction Corps,Kazakh in Qingshuihe Township of Manas County and and Han nationalities in the first affiliated Hospital of Shihezi University Medical College were collected.The blood glucose and lipids levels and microRNA-450a-5p levels in the serum of subjects were detected.（2）Selected four-week-old male C57BL/6 mice to construct an obesity model induced by high-fat diet（n=8）,the C57BL/6 mice in normal diet（n=8）were used as controls,and dynamically monitor the weight and Lee’s index of mice.To detect blood glucose and lipids contents in mouse serum,using qRT-PCR to detect the expression levels of microRNA-450a-5p in mouse serum,liver,adipose and skeletal muscle tissues in the 12th week.（3）Using mi RDB,Target Scan and mi R.MICT to predict downstream target genes that may be targeted by microRNA-450a-5p and participate in the body’s glucose and lipids metabolism.（4）Culture HepG2 cells in vitro,construct microRNA-450a-5p over-expression model and DUSP10 3’Untranslated Region（3’Untranslated Region,3’UTR）luciferase reporter gene plasmids,and detect whether microRNA-450a-5p has a targeted regulatory effect on DUSP10 by using luciferase reporter gene experiment.（5）HepG2,L02and 3T3-L1 cells were cultured in vitro,and mimic or inhibitor transfected to up-regulate/inhibit microRNA-450a-5p,and up-regulate the expression of microRNA-450a-5p while over-expressing DUSP10,qRT-PCR and Western-Blot technology to detect the expression levels of DUSP10/JNK/IRS/PI3K/AKT/GLUT4 in cells;glucose consumption test,insulin sensitivity test and lipid synthesis test to detect the effects of microRNA-450a-5p on HepG2,L02 and 3T3-L1 cells glucose and lipid metabolism.（6）Twenty-two in aged 4 weeks male C57BL/6 mice were selected and fed in normal diet（n=10）and high-fat diet（n=12）.After 16 weeks,the normal diet was used as the control group（n=5）and intraperitoneal injection of over-expressing microRNA-450a-5p adenovirus vector group(n=5,1×10¹¹VP/mice,once a week),the high-fat diet is the control group（n=6）and intraperitoneal injection of microRNA-450a-5p inhibitor adenovirus vector group(n=6,1×10¹¹VP/mice,once a week),dynamically monitor the weight and Lee’s index of mice,using IPGTT（Intraperitoneal Glucose Tolerance Trial）and ITT（Insulin Tolerance Test）evaluate the glucose tolerance and insulin sensitivity of mice after continuous injection for 6weeks,collects mouse serum,liver,white adipose tissue and brown adipose tissue,and detects serum FPG,TG,TC,HDL and LDL content at sacrifice;qRT-PCR technology detects the mRNA expression level of microRNA-450a-5p/DUSP10/GLUT4 in mouse serum,liver and epididymis white adipose tissue,Western-Blot technology detects DUSP10/JNK/IRS/in mouse liver and epididymis white adipose tissue PI3K/AKT/GLUT4 protein expression and phosphorylation level.（7）Eighteen 4-week-old male C57BL/6 mice were selected and fed with normal diet（n=6）and high-fat diet（n=12）for 16 weeks.The high-fat diet was used as the control group（n=6）and the gallnut（GA）gavage treatment group（n=6,200 mg/kg body weight,3 times a week）,dynamically monitor the weight and Lee’s index of mice,and use IPGTT and ITT evaluate the glucose tolerance and insulin sensitivity of mice after continuous gavage for 6 weeks.The serum,liver and adipose tissue of mice were collected to detect microRNA-450a-5p expression levels.The FPG、TG、TC、HDL and LDL contents in serum were detected,and the effects of GA on glucose and lipid metabolism of obese body were determined.（8）Use SPSS 20.0 software for data analysis.Normally distributed data analyzed by t-test,used rank-sum test to analyze non-normally distributed data,and multi-groups data mutual comparison used single-factor analysis of variance.P<0.05 considered the difference to be statistically significant.Results:1.Compared with normal-weight individuals,serum microRNA-450a-5p is significantly higher in obese and T2DM individuals,and is significantly positively correlated with the blood glucose and lipids levels of the subjects.（1）In the serum of Uyghur,Kazakh and Han subjects,the expression level of microRNA-450a-5p showed a gradual increase trend in normal（n=24;n=24;n=24）and obese（n=48;n=48;n=24）and T2DM（n=24;n=10;n=24）individuals;non-ethnic analysis showed that the expression level of microRNA-450a-5p in the serum of obese individuals was significantly higher than normal weight individuals（P<0.05）,compared with normal weight individuals,serum microRNA-450a-5p expression in T2DM patients was far higher（P<0.01）.（2）Serum microRNA-450a-5p contents showed a significant positive correlation in FPG（r=0.175,P<0.01）,TG（r=0.192,P<0.01）,TC（r=0.185,P<0.01）and LDL（r=0.199,P<0.01）of subjects.2.In obesity state,microRNA-450a-5p is far high expressed in liver and white adipose tissue of mice.（1）Compared with normal diet group,after feeding male C57BL/6 mice with 60%high-fat diet for 12 weeks,the body weight and Lee’s index of the mice showed a significantly increase（P<0.05）;the serum levels of FPG,TG,TC,HDL and LDL level were far higher than normal diet group（P<0.001）.（2）After 12 weeks of high-fat feeding,the weight of liver,visceral adipose tissue（VAT）,subcutaneous adipose tissue（Sub）,epididymis adipose tissue（Epi）,perirenal adipose tissue（KAT）,and brown adipose tissue（BAT）were all significantly higher than the normal diet group（P<0.05）.（3）After 12 weeks of high-fat feeding,compared with normal diet group,microRNA-450a-5p was highly expressed in liver,Sub,Epi,VAT,and KAT,and low expression in BAT.3.microRNA-450a-5p can target down-regulation of DUSP10 expression in HepG2 cells.（1）The results of the bioinformatics database（mi RDB、Target Scan、mi R.MICT）suggested that DUSP10 may be the downstream target gene of microRNA-450a-5p related to glucose and lipid metabolism.（2）Construct 3’UTR wild type（Wild type,WT）and mutant（Mutation type,MUT）luciferase reporter gene plasmids of DUSP10,and transfect WT plasmids while over-expressing microRNA-450a-5p in HepG2 cells,compared with the control group,the DUSP10 3’UTR luciferase activity value was significantly reduced in HepG2（P<0.001）.（3）The MUT plasmid was transfected while over-expressing microRNA-450a-5p in HepG2cells,compared with the group of over-expressing microRNA-450a-5p and transfected with WT plasmid,the intracellular DUSP10 3’UTR luciferase activity value significantly increased（P<0.05）.4.Over-expression of microRNA-450a-5p can inhibit the expression of DUSP10,reduce glucose consumption and insulin sensitivity of HepG2,L02 and 3T3-L1 cells,and promote lipid synthesis.（1）microRNA-450a-5p was up-regulated in HepG2,L02 and 3T3-L1 cells,the mRNA and protein expression of DUSP10 and GLUT4 which is a key gene of glucose metabolism in the cells were significantly inhibited（P<0.05）,and the protein phosphorylation level of IRS-tyr895/p-PI3K/AKT-ser473/AKT-thr308 decreased,the protein expression level of p-JNK and IRS-ser302/307 increased.（2）After up-regulating microRNA-450a-5p,the glucose consumption（P<0.05）and insulin sensitivity（P<0.05）were markably inhibited in HepG2,L02 and 3T3-L1 cells,compared to the control group;The lipid droplet content and TG content in the HepG2,L02 and 3T3-L1cells were markably higher than the control group（P<0.05）.5.The microRNA-450a-5p inhibitor can promote the expression of DUSP10,increase the glucose consumption and insulin sensitivity of HepG2,L02 and 3T3-L1 cells,and inhibit lipid synthesis.（1）Transfected HepG2,L02 and 3T3-L1 cells with microRNA-450a-5p inhibitors,the intracellular mRNA and protein expression of DUSP10 and GLUT4,a key gene of glucose metabolism were significantly up-regulated（P<0.05）,IRS-tyr895/p-PI3K/AKT-ser473/AKT-thr308 increased levels of protein phosphorylation,the protein expression level of p-JNK and IRS-ser302/307 decreased.（2）Transfected HepG2、L02 and 3T3-L1 cells with microRNA-450a-5p inhibitors,the glucose consumption（P<0.05）and insulin sensitivity（P<0.05）of HepG2,L02 and 3T3-L1cells were far higher than the control group;The lipid droplet content and TG content were far lower than the control group in the HepG2,L02 and 3T3-L1 cells（P<0.05）.6.microRNA-450a-5p down-regulates DUSP10 by targeting,inhibits cellular glucose metabolism signaling pathway,glucose consumption and insulin sensitivity,and promotes lipid synthesis.（1）Over-expression of microRNA-450a-5p while up-regulating DUSP10 in HepG2,L02,and3T3-L1 cells,compared with the simply over-expressed microRNA-450a-5p group,the mRNA and protein expression of DUSP10 and GLUT4,a key gene of glucose metabolism were markably up-regulated（P<0.05）,the protein phosphorylation level of IRS-tyr895/p-PI3K/AKT-ser473/AKT-thr308 increased,and the protein expression levels of p-JNK and IRS-ser302/307 decreased.（2）Over-expression of microRNA-450a-5p while up-regulating DUSP10,compared with the simply over-expressed microRNA-450a-5p group,the glucose consumption（P<0.05）and insulin sensitivity（P<0.05）of HepG2,L02 and 3T3-L1 cells were significant increased;the lipid droplet content and TG content in the cells were markably decreased（P<0.05）.7.microRNA-450a-5p can inhibit the expression of DUSP10/IRS/PI3K/AKT/GLUT4 in liver and adipose tissues,and reduce the glucose tolerance and insulin sensitivity of mice.（1）Compared with the control group（NC）,intraperitoneal injection of microRNA-450a-5p over-expression adenovirus vector to mice fed with normal diet after 6 weeks,,the expression level of microRNA-450a-5p in mouse serum,liver and adipose tissue significantly increased,and the expression levels of DUSP10 and GLUT4 in liver and adipose tissues were significantly decreased（P<0.05）,and the protein expression levels of IRS-tyr895/p-PI3K/AKT-ser473 and AKT-thr308 decreased,p-JNK and IRS-ser302/307 protein expression levels increased.（2）Intraperitoneal injection of microRNA-450a-5p over-expression adenovirus vector to mice fed with normal diet after 6 weeks,compared with NC,the weight of mice,liver weight and serum FPG,TG levels increased significantly（P<0.05）,glucose tolerance was significantly reduced（P<0.05）.（3）Intraperitoneal injection of microRNA-450a-5p inhibitor adenovirus vector to the diet-induced obese mice after 6 weeks,compared with the control group（NC）,the expression levels of DUSP10 and GLUT4 were significantly increased in the liver and adipose tissues of the mice（P<0.05）,the protein expression levels of IRS-tyr895/p-PI3K/AKT-ser473 and AKT-thr308 increased,and the protein expression levels of p-JNK and IRS-ser302/307reduced.（4）Compared with NC,intraperitoneal injection of the microRNA-450a-5p inhibitor adenovirus vector to the of diet-induced obese mice after 6 weeks,the liver weight,adipose tissue weight of mice and serum FPG and TG levels were significantly decreased（P<0.05）,glucose tolerance and insulin sensitivity were significantly enhanced（P<0.001）.8.The effects of Gallnut（GA）on microRNA-450a-5p expression and glucose metabolism in diet-induced obese mice.（1）After gavaged with GA in diet-induced obese mice for 6 weeks,compared with the control group,the levels of microRNA-450a-5p in serum,liver and adipose tissue of mice were significantly reduced（P<0.05）.（2）After gavaged with GA in diet-induced obese mice for 6 weeks,compared with the control group,the body weight,white adipose tissue weight and FPG levels were significantly reduced（P<0.05）,and glucose tolerance and insulin sensitivity were significantly improved（P<0.05）.Conclusion:（1）Under the obesity state,the high expression of microRNA-450a-5p in serum is closely related to the occurrence T2DM.（2）Under the obesity state,the increase of microRNA-450a-5p levels can inhibit the expression of DUSP10 in liver and adipose tissue leading to inactivation of p-JNK/p-IRS/p-AKT/GLUT4 signal transduction pathway,and induce IR and T2DM.