Screening Of MC4R Downstream Gene Expression Profiles By High-throughput Sequencing And Validation

BackgroundObesity is a multi-pathogenic chronic disease characterized by excessive body fat content and/or abnormal distribution,always accompanied by an increase in body mass.In recent years,with the improvement of living standards,obese people have grown year by year,and obesity has become a worldwide epidemic.The latest obesity epidemiological studies show that the global male and female obese population has increased to 266 million and 375 million respectively,while the number of obese people in China has reached nearly 90 million,ranking first in the world.At the same time,obesity can lead to hyperinsulinemia,which are mutually causal and form a vicious circle.Although the scientific community in the 19 th century has proposed brain tissue to play a leading role in glucose homeostasis,with the discovery of insulin,the entire 20 th century domestic and foreign research mainly focused on the role of islets in maintaining blood glucose homeostasis.covering the brain as a key point to regulate the status of blood glucose homeostasis.Foreign studies have shown that low-dose leptin,which has no effect on peripheral tissues,is directly injected into the ventricles of insulin-deficient animals,and it has been found that central leptin can still normalize blood glucose in diabetic animal models.This striking conclusion is inconsistent with the islet-centered glucose homeostasis pattern.It is suggested that the maintenance of peripheral energy metabolism and glucose homeostasis is not only directly affected by peripheral metabolic organs,but also the role of central nervous system and central energy regulation factors.The hypothalamic melanocortin receptor 4(MC4R)belongs to the seven-transmembrane G protein-coupled receptor and is an important component of the central melanocortin pathway.MC4 R can regulate appetite and energy metabolism by interacting with its endogenous agonist ?-melanocyte stimulating hormone(?-MSH)and endogenous antagonist squirrel-associated protein(AgRP).It has been pointed out that MC4 R is a key regulator of mammalian energy homeostasis and body weight.It can sense and integrate external stimuli and regulate the activity of appetite,autonomic and neuroendocrine systems to control energy metabolism.The MC4 R heterozygous mutation is the most common cause of human monogenic obesity.And the previous study of our group showed that the activation of MC4 R in the brain can improve the functional damage of skeletal muscle mitochondria in the animal model of diabetes and correct mitochondrial oxidative stress.Other studies have indicated that MC4 R knockout rats showed a significant increase in serum lipid levels,increased visceral white adipose tissue weight,increased fat cell numbers,and impaired glucose tolerance.In summary,brain MC4 R plays a crucial role in peripheral energy metabolism.However,MC4 R is only a transmembrane receptor on the surface of neurons in the central nervous system and cannot directly act on the periphery.Therefore,we speculate that MC4 R may act on certain intermediate mediators,so as to play a regulatory role on peripheral metabolic organs.High-throughput sequencing technology,also known as second-generation sequencing technology,is characterized by the ability to sequence hundreds of thousands to millions of DNA molecules in parallel and generally read short lengths.Among them,transcriptome sequencing can detect the sum of all mRNAs that a particular cell can transcribe under a certain functional state.Gene function and gene structure can be studied at an overall level,and genes differentially expressed in cells,tissues or individuals under different physiological or pathological conditions can be found.Therefore,we intend to screen differentially expressed mRNAs in MC4 R overexpressing cells by high-throughput sequencing,and we carry out bioinformatics analysis and preliminary mechanism exploration,to lay the foundation for further study on the specific mechanism of energy expenditure and glucose metabolism in central MC4 R and peripheral metabolic organs.ObjectiveIn this study,MC4 R overexpressing adenoviral vector was constructed and transfected into mouse hypothalamic GnRH neuron cells(GT1-7 cells).High-throughput sequencing technology was used to screen differentially expressed genes downstream of MC4 R.Real-time quantitative PCR and Western Blot were used to verify some differentially expressed mRNAs.A new target for MC4 R affecting peripheral energy metabolism and insulin sensitivity was explored in order to explore a new brain-centered metabolic pathway associated with obesity,laying a solid theoretical foundation for targeting obesity and insulin resistance-related diseases.MethodsMouse hypothalamic GnRH neuronal cells(GT1-7 cells)were cultured.The MC4 R overexpressing adenoviral vector was constructed and transfected into mouse hypothalamic GT1-7 cells.High-throughput sequencing technology was used to detect mRNAs differentially expressed in MC4 R overexpression group(n=3)and control group(n=3)GT1-7 cells,and differential expression profiles were constructed.GO analysis and KEGG Pathway analysis of differential mRNAs were performed to screen for metabolic pathways such as energy metabolism,endocrine and metabolic diseases,and related differentially expressed mRNAs.In addition,MC4 R overexpressing and interfering expression of adenoviral vectors were constructed and transfected into mouse hypothalamic GT1-7 cells.Real-time quantitative PCR and Western Blot were used to verify the expression of MC4 R and some differentially expressed genes in MC4 R overexpression group,MC4 R interference group and control group,and to determine the mechanism by which MC4 R regulates peripheral energy metabolism through downstream pathway.Results1.Compared with the control group GT1-7 cells,the MC4 R overexpression group GT1-7 cells showed nearly one thousand mRNA changes.There were 629 mRNAs with up-regulated expression and 260 mRNAs with down-regulated expression,and the fold change was more than 2 times(Q<0.001).2.GO analysis of differentially expressed mRNAs showed that biological processes mainly focused on biological regulation,cellular processes,cellular tissue organization or biogenesis,developmental processes,metabolic processes,stimulation responses,signaling pathways,etc.;molecular functions are mainly concentrated in molecules.Binding,catalytic activity,transcriptional regulator activity,transporter activity,signal transduction protein activity,molecular function regulators,and the like.KEGG Pathway analysis shows that gene regulation mainly focuses on endocrine and metabolic diseases,infectious diseases,environmental adaptive regulation,nonalcoholic fatty liver,Cushing's syndrome,energy metabolism,lipid metabolism,glycan synthesis and metabolism,cAMP pathway,calcium signaling pathway,cGMP-PKG pathway,cell transport and decomposition and other metabolic pathways.3.Real-time quantitative PCR and Western Blot confirmed some differentially expressed mRNAs.The results showed that MC4 R,ADCY3,AGT mRNA and protein were up-regulated in MC4 R over-expression group,while MC4 R interference group was reversed.Contrary to the expression of ADCY3 and AGT,the expression of HCRT mRNA and protein in MC4 R overexpression group was down-regulated,and then the expression of HCRT mRNA and protein in MC4 R interference group was increased.Conclusion1.The mRNA expression profiles related to endocrine and metabolic diseases pathway,environmental adaptation pathway,energy metabolism pathway,and signal transduction pathway changed significantly in MC4 R overexpressing GT1-7 cells.2.Activation of MC4 R in the brain may activate downstream ADCY3 and AGT molecules,while inhibiting downstream Hypocretin factors,and indirectly exert energy regulation.