| Introduction: The steady state of lipid metabolism plays a significant role in maintaining the body’s nutritional balance.Unhealthy lifestyles and long-term intake of high-fat diets lead to lipid metabolism disorders,which lead to a series of lipid metabolism correlation diseases such as obesity,non-alcoholic fatty liver(NAFLD),type2 diabetes,cardiovascular disease,mental illness disease and the likes.At the same time,lipid metabolism also plays a critical role in the occurrence and development of tumors.Therefore,studying the molecular mechanisms related to lipid metabolism has significant clinical value for preventing lipid metabolism-related diseases.MYCT1(Myc target 1)is a new gene cloned by our team from laryngeal squamous cell carcinoma(LSCC),which encodes 235 amino acid residues.MYCT1 is lowly expressed in gastric cancer,liver cancer,laryngeal cancer and other tumor tissues.However,In acute myeloid leukemia,MYCT1 is activated and highly expressed to play the role of oncogene as the downstream of oncogene RUNX1-ETO and MYC.Our previous research results showed that MYCT1 was lowly expressed in SMMC7721 cells and significantly inhibited cell proliferation and migration,which was linked to age and HBV infection.The over expression of MYCT1-TV,a new transcript of MYCT1 in Bel7402 cells of liver cancer,can inhibit growth and invasion,and promote cell apoptosis,but its specific mechanism is still unclear.In order to further study the function of MYCT1,our team used Cas-9 technology to construct Myct1 knockout mice.In the analysis of the model mice,it was found that the body weight,body fat and liver weight of the litter Myct1 knockout mice were significantly higher than those of the wild type.Therefore,it speculats that MYCT1 is related to liver lipid metabolism.There are two ways for liver tissue to synthesize lipids.One is to obtain it from food,and the other is to synthesize it by itself,which is called de novo lipogenesis(DNL).DNL is the primary way.Fatty acid synthase(FASN)is the key rate-limiting enzyme in the DNL process,and it is also a specific protein expressed in liver tissue.Its main role is to catalyze the synthesis of hexadecimal acid from malonyl-Co A and acetyl-Co A with nicotinamide adenine dinucleotide phosphate(NADPH).FASN protein is a homogeneous dimer with a molecular weight of 273 k Da,each containing seven domains that are active only in the form of a polymer.Normally,FASN converts excess carbohydrates into fatty acids and then esters them into triglycerides for storage,which,if necessary,provides energy through β-oxidation.In normal human tissues,FASN is almost not expressed,except for the liver,fat,lactating breast tissue and hyperplastic uterine tissue,but it is highly expressed in tumor cells.Studies have shown that FASN is expressed in various human epithelial cancers and precancerous lesions.Tissues such as breast cancer,colon cancer,prostate cancer,lung cancer,bladder cancer,ovarian cancer,stomach cancer,endometrial cancer,kidney cancer,skin cancer,pancreatic cancer,head and neck cancer and tongue cancer have extremely high levels of FASN.FASN has the potential to become a cancer treatment target.Therefore,studying the regulatory relationship of FASN is of great importance to the treatment of cancer and lipid metabolism-related diseases.N-acetyltransferase 10(N-acetyltransferase 10,NAT10)is a histone acetyltransferase that participates in many cellular processes,such as DNA damage repair,tubulin acetylation,autophagy,m RNA acetylation,etc.It is the only protein identified currently with both an acetylase domain and an RNA binding domain.It has RNA acetylase and lysine acetyltransferase activities,which can promote the stability of downstream target gene m RNA and promote its translation efficiency.Studies have shown that NAT10 is highly expressed in liver cancer,leukemia,melanoma and other malignant tumors,suggesting that NAT10 is associated with the occurrence and development of cancer and is a potential target for the treatment of malignant tumors.Studies in this group have shown that MYCT1 inhibits the migration of laryngeal cancer cells by down-regulating the expression of NAT10 in Hep-2 cells of laryngeal cancer,but its specific mechanism and the regulatory relationship between MYCT1 and NAT10 in liver tissue are not obevious.Therefore,we assume that MYCT1 affects liver lipid metabolism by regulating the expression of FASN through NAT10,which providing new ideas and theoretical basis for the treatment of lipid metabolism-related diseases.Here,to investigated the effect of MYCT1 on lipid metabolism we detected physiological indicators such as body weight,body fat and the liver weight,biochemical indicators like plasma ALT,AST,liver tissue triglycerides and total cholesterol and pathological indicators including HE staining of liver tissue,Oil red O staining of liver tissue in Myct1 knockout mice and wide-type mice.Next,to clarify the regulatory relationship of MYCT1 with NAT10 and FASN,we detected the changes in the expression of Nat10 and Fasn in the liver tissues of Myct1 gene knockout mice and wide-type mice.Finally,to verify the results of animal experiments,we repeated these experiments in HepG2 cells,including tested the regulatory relationship of MYCT1 on NAT10,FASN and NAT10 on FASN.Lastly,in order to further explore the mechanism of MYCT1/NAT10/FASN in liver lipid metabolism,we conducted Oil red O staining,triglyceride to detect the role of MYCT1/NAT10/FASN axis in liver tissue metabolism.Materials and methods:1.Experimental materials: Myct1 knockout mouse liver tissue,human liver cancer cell HepG2,human normal liver cell LO2.2.Test methods: HE staining,Oil red O staining,enzymatic method detecting triglyceride,enzymatic method detecting total cholesterol,cell culture,gene transient transfection,Real-time PCR,Western Blot.Results:1.The test results of physiological indicators of mice showed that compared with wild-type mice,the body weight,body fat and liver weight of Myct1 gene knockout mice increased significantly(P<0.05).2.The blood biochemical and liver biochemical test results of mice showed that compared with wild-type mice,the plasma AST and ALT of Myct1 knockout mice were significantly increased(P<0.05),and the content of liver triglycerides and total cholesterol were significantly increased(P<0.05).3.The results of HE staining and Oil Red O staining of mouse liver tissue sections showed that compared with wild-type mice,Myct1 knockout mice had liver damage and significantly increased lipid droplet content(P<0.05).4.The results of Real-time PCR and Western blot showed that compared with wild-type mice,the expression of Fasn,a key fat synthesis enzyme in the liver tissue of Myct1 knockout mice,was significantly increased(P<0.05).5.The results of Real-time PCR and Western blot showed that compared with wild-type mice,the expression of Nat10 in the liver tissue of Myct1 knockout mice was significantly increased(P<0.05).6.The results of Real-time PCR and Western blot showed that compared with the control group,when MYCT1 was overexpressed,the expression of NAT10 and FASN were significantly reduced(P<0.05),and when MYCT1 was knocked down,the expression of NAT10 and FASN was significantly reduced(P<0.05),in liver cancer cells HepG2 and normal liver cells.7.Correlation analysis results showed that NAT10 and FASN are positively correlated in m RNA expression level(P<0.05).8.The results of Real-time PCR and Western blot showed that compared with the control group,when NAT10 was over-expressed or knocked-down in HepG2 cells and normal liver cells,the expression of FASN was increased or decreased significantly(P<0.05).9.The results of Oil red O staining experiment showed that compared with the control group,the lipid content of cells was significantly reduced after overexpression of MYCT1(P<0.05);after NAT10 was overexpressed,the lipid content of cells was significantly increased(P<0.05);and overexpression of NAT10 can significantly restore the inhibitory effect of MYCT1 on lipid content(P<0.05).After knocking down MYCT1,the cell lipid content was significantly increased(P<0.05);after knocking down NAT10,the cell lipid content was significantly reduced(P<0.05);and knocking down NAT10 can significantly reduce the promotion of MYCT1 on lipid content(P<0.05).Conclusions:1.MYCT1 affects liver lipid metabolism in mice.2.MYCT1 inhibits the expression of NAT10 and FASN.3.NAT10 promotes the expression of FASN.4.NAT10 is positively correlated with FASN.5.MYCT1 regulates the expression of FASN via NAT10 and affects liver lipid metabolism. |
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