To Explore The Effects Of Exercise On Lipid Deposition In The Liver Of Obese Mice And Its Regulatory Mechanism Based On Transcriptomics And Bioinformatics Analysis

Purpose:(1)To compare the effect of different intensity exercise on fat mice liver lipid deposition;(2)Select the best exercise mode to improve liver lipid deposition as the strategy of exercise intervention in the follow-up study,and use transcriptomics to screen the key candidate RNA that exercise affects liver lipid deposition in obese mice;(3)To explore the transcriptional regulatory network of exercise on liver lipid deposition in obese mice by bioinformatics analysis;(4)Based on the RNA interaction network constructed above,observe the role of an RNA pathway(miR-298-5p/G0S2 mRNA)that is important to the network in exercise in improving liver lipid deposition in animals;(5)The mechanism of this RNA pathway in lipid deposition of hepatocytes was verified at the cellular level.Methods:Chapter 1 Research methods:Four-week-old C57BL/6J mice weeks were randomly divided into a high-fat diet group(HFD group,n=48)and a normal diet group(C group,n=12).HFD induced obesity model was established at 12 weeks.HFD group was randomly divided into obesity disease control group(group D,n=12),low-intensity exercise group(group L,n=12),moderate intensity exercise group(group M,n=12),and high-intensity exercise group(H=12).The intervention time was 10 weeks,5 days a week,and each time lasted for 1 hour.After exercise,HE staining was used to detect the pathological characteristics of liver tissue,oil red O staining and liver TG were used to quantitatively observe the liver lipid deposition,liver index was used to observe the proportion of liver and body weight,liver injury index(AST,ALT)was used to observe the liver injury,blood lipid changes were observed through the four items of blood lipid(TC,TG,HDL-C,LDH-C),and serum insulin,glucagon,IPGTT,ITT,liver glycogen content Liver PAS staining was used to observe glucose homeostasis.In conclusion,we compared the effects of different intensity exercise on liver lipid deposition in obese mice.Chapter 2 Research Methods:On the basis of Chapter One,the best exercise mode for improving liver lipid deposition was adopted as the strategy of exercise intervention in the follow-up study.Using high-throughput sequencing technology,mRNA,miRNA,lnc RNA and circ RNA were sequenced in the liver of mice in groups C,D and M(the best exercise mode group for improving lipid deposition),and key candidate RNA was screened to explore the improvement effect of exercise on liver lipid deposition from the transcriptional level.Chapter 3 Research Methods:On the basis of screening class I RNA(mRNA: G0S2,GCK,CYP17A1 and miRNA: miR-298-5p,miR-1a-3p,miR-204-5p)in chapter 2,use databases such as Starbase,Targetscan,Mirdb,Rnanter,etc.to explore class II RNA with potential binding sites to class I RNA(for the convenience of later description,we call the RNA obtained through bioinformatics analysis class II RNA).Next,combine the class II RNA with the binding site of class I RNA to obtain the RNA with the intersection of class I RNA and class II RNA,which is called class III RNA.The RNA RNA interaction network model of exercise improving liver lipid deposition was established based on class III RNA screened in this study.Chapter 4 Research Methods:In the multiple RNA regulatory networks screened in Chapter 3,an important pathway regulating liver lipid deposition was screened,namely miR-298-5p/G0S2 mRNA.The effects of exercise training on miR-298-5p/G0S2 mRNA and downstream pathways were observed in animal experiments.The main indicators included miR-298-5p,target gene(G0S2)of miR-298-5p,liver TG decomposition index(ATGL),liver FAO index(MCAD,ACOX1),and liver DNL index(SREBP1c,FAS).Chapter 5 Research methods:The miR-298-5p/G0S2 mRNA pathway was verified at the cell level.The double luciferase reporter gene was used to confirm that there was a binding site between miR-298-5p and G0S2 mRNA in AML 12 hepatocytes;Palmitic acid was used to stimulate AML-12 to induce hepatocyte lipid deposition model in vitro,and the model cells were divided into PA group,PA+mimics group(miR-298-5p transfection group),PA+mimics NC group(miR-298-5p no-load transfection group).The mechanism of its role in hepatocyte lipid deposition was verified by comparing the changes of miR-298-5p/G0S2 mRNA and downstream related indicators(the same as those in Chapter 4)in different groups.Results:Chapter 1 Research results:(1)After 12 weeks of high-fat diet(HFD),the weight of mice in HFD group was much higher than that of mice in C group(P<0.01),and the average weight of mice in HFD group was 20% higher than that of mice in C group.The obese mice model was successfully established;(2)Compared with group D,the body weight,liver weight,liver index,ALT and AST of mice in group L,M and H decreased to varying degrees,and the IPGTT and ITT improved.Compared with group L,the improvement in group M and H was more obvious;(3)Compared with group D,oil red O staining showed that the content of TG in the liver of mice in group M and group H was significantly improved;The quantitative results of TG showed that compared with group D,the liver lipid deposition of mice in group L(P>0.05),group M(P<0.05),and group H(P<0.05)decreased to varying degrees.Compared with group L,the liver TG content of mice in group M and group H decreased more significantly;(4)During the exercise,one mouse in Group H died accidentally(was crushed to death by the running platform),and two were injured(one of them was seriously injured and could not be used for the follow-up experiment).The incidence of sports injury was as high as 25%,and the compliance of mice in Group H was low(reacted by the number of electric shocks).Chapter 2 Research results:According to the results of RNA seq sequencing of mouse liver in each group.(1)For differentially expressed mRNAs: nine were screened out because HFD was up-regulated(compared with group C,the mRNA in group D was up-regulated)and down regulated by exercise(compared with group D,the mRNA in group M was down regulated),and five were down regulated by HFD and up regulated by exercise.Among the 14,three were G0S2,GCK,and CYP17A1 with high correlation with liver lipid deposition;(2)For differentially expressed miRNAs: two miRNAs that were up-regulated by HFD and down-regulated by exercise were screened,and one miRNAs that were down-regulated by HFD and up-regulated by exercise,namely miR-298-5p,miR-1a-3p,miR-204-5p;(3)For the differentially expressed lnc RNAs,11 were screened out because HFD was up-regulated and down regulated by exercise,and11 were down regulated and up regulated by exercise due to HFD;(4)For the differentially expressed circ RNA,no relevant differentially expressed circ RNA was identified in group C,group D and group M.Chapter 3 Research results:Based on the results of bioinformatics analysis of differentially expressed RNA in Chapter 2.A total of 15 RNA interaction networks were constructed in databases such as Starbase,Targetscan,Mirdb,and Rnanter,among which miR-298-5p/G0S2 mRNA was highly correlated with liver lipid deposition.Chapter 4 Research results: The effects of exercise on an important RNA pathway(miR-298-5p/G0S2 mRNA)in the above RNA interaction network were observed in animals.The results were as follows:(1)At the level of RNA regulation: compared with group C,miR-298-5p(P<0.01),ATGL(P<0.05),MCAD(P>0.05)and ACOX1(P>0.05)in the liver of group D mice were down regulated to varying degrees;The expression of miR-298-5p target gene G0S2(P>0.05),DNL regulator SREBP1c(P<0.01)and FAS(P<0.05)were significantly up-regulated;Liver lipid deposition was aggravated.After 10 weeks of exercise intervention,compared with group D,liver miR-298-5p(P<0.01),ACOX1(P<0.05),ATGL(P<0.01)in group M were up-regulated in varying degrees;The expressions of G0S2(P<0.05),SREBP1c(P<0.01)and FAS(P<0.05)were significantly down regulated;There was no significant difference between CPT1a(P>0.05)and MCAD(P>0.05);Liver lipid deposition decreased.(2)At the level of protein regulation:we found that the expression trend of the above factors was consistent with the corresponding mRNA expression level,but only the difference between G0S2 and ACOX1 was statistically significant(P<0.05).Chapter 5 Research results: The results were as follows:(1)Compared with wt+mimics NC group,the relative luciferase activity of wt+mimics NC group was significantly decreased(P<0.01);Compared with the mut+mimics NC group,the relative luciferase activity in the mut+mimics group decreased significantly(P<0.01);The results showed that miR-298-5p could specifically bind to the 3 ’-UTR target site of G0S2 mRNA.(2)After PA stimulation,the lipid deposition in AML-12 hepatocytes increased,which indicated that the establishment of AML-12 hepatocyte lipid deposition model was successful;In addition,PA stimulation decreased the expression of miR-298-5p in AML 12 hepatocytes and increased the expression of G0S2 mRNA and protein(P<0.01);After transfection of miR-298-5p(overexpression),the expression of miR-298-5p in PA+mimics group was significantly up-regulated(P<0.01),the expression of G0S2 and FAS was significantly decreased(P<0.01),the expression of ATGL and MCAD was significantly up-regulated(P<0.05),the expression of SREBP1 c,ACOX1 was not significantly changed(P>0.05),and the lipid deposition of liver cells was reduced.At the level of protein regulation: we found that the expression trend of the above factors was consistent with their corresponding mRNA expression levels,but only G0S2,ATGL and MCAD were statistically significant(P<0.05).Conclusions:Chapter 1 Research Conclusion:C57BL/6J strain mice will have abnormal blood glucose and lipid,impaired systemic insulin sensitivity,increased expression of liver injury markers and obesity accompanied by liver lipid deposition when fed with HFD.Different intensity exercise can ameliorate these metabolic abnormalities to some extent,and moderate intensity exercise and high-intensity exercise have more obvious improvement effects.When considering factors such as exercise injury and exercise compliance,Moderate intensity exercise is the best choice to improve liver lipid deposition in obese mice.Chapter 2 Research Conclusion: The abnormal expression of mRNA,nc RNA(miRNA,lnc RNA,circ RNA)and other transcriptome in the liver of HFD induced obese mice leads to the imbalance of liver lipid metabolism and excessive lipid deposition;Liver lipid homeostasis is related to abnormal expression of lipid metabolism genes,and the molecular regulation mechanism of exercise beneficial liver is related to changes in liver transcriptome caused by exercise.Chapter 3 Research conclusion: Exercise affects the expression of genes related to liver lipid deposition by affecting multiple nc RNA/mRNA regulatory networks.Although nc RNA cannot encode proteins,it can indirectly regulate the generation of proteins.In many RNA regulatory networks,miR-298-5p/G0S2 is not only closely related to liver lipid deposition caused by obesity,but also can be reversed by exercise,thereby improving liver lipid deposition.Chapter 4 Research Conclusion:In animal(in vivo)experiments,the expression of miR-298-5p in the liver of obese mice was down regulated,and the expression of G0S2 mRNA and protein with binding sites to miR-298-5p was up regulated,and the liver lipid deposition was aggravated.Exercise can not only up regulate the expression of miR-298-5p in the liver of obese mice,but also down regulate the expression of G0S2 mRNA and protein,thus affecting the liver lipolysis and FAO,and then reducing the liver lipid deposition of obese mice.Chapter 5 Research conclusion: In the cell(in vitro)experiment,miR-298-5p has a binding site with G0S2 mRNA,and miR-298-5p negatively regulates the expression of its target gene G0S2,thereby affecting the lipolysis of AML 12 hepatocytes,FAO and DNL,and reducing the lipid deposition of hepatocytes.In conclusion,on the basis of screening the best exercise intensity,this study confirmed that exercise can induce abnormal expression of liver transcriptome in obese mice,and revealed the potential role of miR-298-5p/G0S2 mRNA pathway in exercise regulation of liver lipid deposition in obese mice in vivo and in vitro.