| With the development of China's economy and society,chronic and non-communicable diseases represented by diabetes and malignant tumors have seriously threatened people's health.Type 2 diabetes mellitus(T2DM)is a complex polygenic genetic disease.Epidemiological investigations show that T2DM can increase the prevalence of certain cancers,the most common being digestive system tumors,of which colon cancer first place.In the past,genes associated with T2DM risk were identified primarily by genome-wide association studies(GWAS),including the ADP-ribosylation factor-like 15,ARL15 gene.ARL15 is a member of the ADP-ribosylation factor(ARF)family.The function of the ARF family is involved in vesicle trafficking and actin recombination.There are 40%to 60%sequences between ARL and ARF members.Homology.ARL15 regulates membrane transport,lipid composition and insulin signal transduction pathway,and participates in inflammatory response.The interaction mechanism between T2DM and tumor includes high insulin and insulin-like growth factors 1(IGF-1).),high blood sugar,obesity,and chronic inflammation.Therefore,ARL15 is likely to be involved in the pathogenesis of T2DM and colon cancer.This study used population genetics to explore the relationship between ARL15 gene polymorphism and ARL15 levels and adiponectin,insulin,physiological and biochemical indicators and the risk of T2DM,but polygenic genetic diseases have the characteristics of micro-effect gene accumulation,so it will be another A T2DM susceptibility gene PGC-1? interacts with ARL15 to observe the correlation of the above indicators.It is envisaged to use cytology,immunohistochemistry and molecular biology techniques to study whether ARL 15 participates in colon cancer by glucose metabolism or fat metabolism in tumor cells.To explore the biological functions of ARL15,this study was conducted in two parts.Part oneAssociation analysis between ARL15 and PGC-1?gene polymorphisms of T2DMObjective:To investigate the association between ARL15 and PGC-la gene single nucleotide polymorphisms(SNPs)rs255758,rs7656250 additive effect and T2DM,and ARL15 gene SNP Correlation with ARL15 levels and indicators of adiponectin(PA),insulin(INS),blood lipids,high body mass index(BMI)and T2DM.Methods:In this study,a case-control enrolls 102 patients with normal glucose tolerance(NGT)and 256 patients with T2DM was conducted.SNP typing and sequencing were performed using LDR and SnaPshot(?)Multiplex;ARL15,insulin(INS)and adiponectin(PA)levels were measured by ELISA;body mass index(BMI)and waist-to-hip ratio(WHR)were calculated by collecting physiological and biochemical indexes.Use the Hardy-Weinberg equilibrium law to determine the population sample of the study sample.The frequency distribution of alleles and genotypes was performed by using SHEsis online software.The data were statistically analyzed by SPSS 25.0.The results of each group of clinical indicators were statistically analyzed by two independent sample rank sum tests.M(P25,P75)indicates.Spearman correlation method was used to analyze the correlation between ARL15 level and other factors.Binary regression analysis was used to analyze risk factors for T2DM.Results:1.The allele frequencies and genotype frequencies of rs255758 in the NGT group,T2DM group and high BMI group had reached the genetic balance(p>0.05),which was representative for the population.2.There was no statistical difference in the frequency of alleles and genotype frequencies of rs255758 in the NGT group,T2DM group and high BMI group,p>0.05.The frequency of rs7656250 allele C was significantly higher in the high BMI group than in the NGT group,the difference was statistically significant,p<0.05.The frequency of TC genotype in the high BMI group was significantly higher than that in the NGT group,the difference was statistically significant,p<0.05,the results also showed that the frequency of T2DM group carrying TC genotype was significantly higher than that of NGT group,the difference was statistically significant,p<0.05.3.When AA-TT genotypes were combined,there was a significant difference between the high BMI group and the NGT group,p<0.05,OR=0.526,95%CI=0.306-0.902;when the AA-TC genotype was combined,the high BMI group There was also a significant difference from the NGT group,p<0.05,OR = 1.865,95%CI=1.018-3.216.4.There were significant differences in WHR between the rs255758 loci in the NGT group,p<0.05,which was statistically significant,and the WHR in the CC genotype group was lower than the WHR of the AC genotype.There was a significant difference in the PA index in the T2DM group,p<0.05,which was statistically significant,and the PA in the CC genotype group was smaller than the PA genotype in the AC genotype,suggesting that the PA level was lower when the CC genotype was present.There was no significant difference in the correlation analysis between rs7656250 loci and NGF,T2DM and high BMI in all NGF,T2DM and high BMI groups,p>0.05,no statistical significance.5.FPG,ARL15 and T2DM are positively correlated and are risk factors for T2DM.6.The level of ARL15 in the high BMI group was correlated with TC,PA and INS.The ARL15 level was positively correlated with TC and INS,and the r values were 0.154 and 0.190.ARL15 levels were negatively correlated with PA with an r-value of-0.426.Conclusion:1.There was no correlation between the allele frequency and genotype frequency distribution of rs255758 locus in ARL15 gene and T2DM group and high BMI group.Although the allele frequency and genotype frequency of PGC-1? gene rs7656250 were distributed in T2DM group There is a correlation with the high BMI group,but the allele frequency and genotype frequency of the NGT group do not reach the genetic balance,and there is no representativeness of the group.In the future,it is necessary to expand the sample and reduce the age gap to determine the correlation.2.People with AA-TC genotype will increase the risk of high BMI by about 1.9 times.When carrying AA-TT genotype,it will reduce the risk of high BMI,suggesting that when ARL15 gene rs255758 and PGC-la gene rs7656250 AA-TC combined genotype can increase the risk of developing T2DM and BMI.3.ARL15 is a risk factor for T2DM.It is associated with TC,PA and INS.High INS and obesity are risk factors for tumorigenesis.Part twoStudy on the mechanism of ARL15 in glucose metabolism and fatmetabolism in colon cancer cellObjective:To explore the mechanism of ARL15 through the development of glucose metabolism or fat metabolism and colon cancer.Methods:The mitotic blocker Nocodazole was used to induce HUVEC cell arrest in mitosis and interphase.Immunohistochemistry and cellular immunofluorescence confirmed the expression of ARL15 in tissues and cells.ARL15 plasmid and siRNA were constructed respectively.After transfection,the expression of glucose and lipid metabolism related proteins in colon cancer cells was detected by immunoblotting.The expression of ARL15 in colon cancer cells was detected by qRT-PCR.mRNA transcription level of lipid metabolism-related genes;JIB-04 was applied to colon cancer cells,and the expression of glucose and lipid metabolism-related proteins in ARL15 and colon cancer cells was detected by immunoblotting,and ARL15 and colon were detected by qRT-PCR.mRNA transcription levels of genes involved in sugar and lipid metabolism in cancer cells.Results:1.The expression of ARL15 in HUVEC cells at mitotic phase was higher than that at interphase(p<0.05).2.Immunohistochemistry showed high expression of ARL15 in colon cancer tissues,which was dark brown,while normal colon tissue ARL15 was also expressed.The basal layer was light brown and expressed in the cytoplasm.3.ARL15 was expressed in FHC cells,HCT116 and SW620 cells.ARL15 is expressed in the same position in cells,mainly in the cytoplasm4.The plasmid pCMV-3Tag-2-ARL15 was transfected into HCT116.After 36 hours of culture,the proteins of the control group and the experimental group were extracted and detected by immunoblotting.After ARL15 overexpression,FSAN,AKT,P-AKT,The expression of P-GSK.SREBP-1(p125)and AMPK protein was higher than that of the normal control group,and the difference was statistically significant.The qRT-PCR technique showed that after overexpression of ARL15,the mRNA levels of FASN,GSK,AMPKal and SREBP-1 were up-regulated,and the difference was statistically significant compared with control(p<0.05).5.siRNA was transfected into HCT116.After 36 hours of culture,the proteins of the control group and the experimental group were extracted.The results of immunoblotting showed that after low expression of ARL15,PKM2,PFK,FSAN,AKT,P-AKT,P-GSK The expression level of AMPK protein decreased,and the difference was statistically significant The qRT-PCR technique showed that after low expression of ARL15,the mRNA levels of FASN,GSK and AMPKal mRNA were down-regulated,and the difference was statistically significant compared with control.6.The expression of ARL15 protein in three different colon cancer cells decreased after adding 2?M JIB-04.The trend of ARL15 expression in different cell colon cancer cell lines was not the same with time.7.After adding 2?M JIB-04,the expression levels of AKT,P-GSK,FASN,AMPK and SREBP-1 in HCT116,HT29 and SW620 cells decreased significantly after 24 hours of culture,and the difference was statistically significant8.qRT-PCR detection,using PGK1 as an internal reference,using 2^-??Ct method to analyze the data,found that the mRNA levels of AKT,P-GSK,FASN,AMPK,SREBP-1 gene in the drug-added group The expression level was significantly lower than that of the normal control group,and the difference was statistically significant.Conclusion:1.The expression of ARL15 protein in mitosis is higher than that in interphase,and ARL15 may be involved in cell proliferation.2.ARL15 is positively correlated with glucose and lipid metabolism related proteins during overexpression and low expression.ARL15 may be involved in the sugar and lipid metabolism pathways of colon cancer cells.3.The expression of ARL15 protein in colon cancer tissues is higher than that in normal colon tissues.ARL15 may promote the occurrence and development of colon cancer by increasing the synthesis of glycogen and fat in the downstream metabolic pathways of AKT and AMPK.Targeting effect on ARL15 may be Treatment of colon cancer will have a certain effect.4.After JIK-04 acts on colon cancer cells,the expression of ARL15 is significantly decreased,and the expression of glucose and lipid metabolism-related proteins in downstream signaling pathways of AKT and AMPK is affected.Summary:1.The AA-TC combined genotype increased the risk of T2DM and high BMI by about 1.9 times after the interaction of the rs255758 locus of the ARL15 gene with the rs7656250 locus of the PGC-1? gene.2.ARL15 is a risk factor for T2DM and has a correlation with TC,PA and INS.ARL15 promotes the development of colon cancer by increasing the synthesis of glycogen and fat in the downstream metabolic pathways of AKT and AMPK.JIB-04 acts on colon cancer cells.After that,the expression level of ARL15 protein was decreased,and the expression of sugar and lipid metabolism-related proteins in the downstream signaling pathways of AKT and AMPK was affected. |
PDF Full Text Request