| Background: Acne vulgaris is an inflammatory disease of the sebaceous glands of the hair follicle,and its pathogenesis involves androgen metabolism,sebum secretion,lipophilic microbial infection,and immune-inflammatory response.It has been shown that androgen receptor(AR)and insulin-like growth factor 1 receptor(IGF1R)are abnormally expressed in acne vulgaris and are involved in the pathogenesis of acne vulgaris,but the mechanism of their abnormal expression is not clear.DNA methylation regulates gene expression,regulates lipid metabolism and inflammatory response.In the early stage of this research group,gene methylation chip technology was used for screening.It was found that the AR gene and IGF1 R gene had differential methylation in patients with acne vulgaris,but the methylation sites and methylation levels need further study.Objective: To identify the methylation sites and methylation levels of AR gene and IGF1 R gene in patients with acne vulgaris,and to preliminarily clarify the association between AR gene and IGF1 R gene methylation and acne vulgaris.Method:1.Specimen collection: with the consent of the ethics committee of shenzhen hospital of southern medical university.The inclusion criteria were established to select patients with moderate to severe acne vulgaris who were in the outpatient department of shenzhen hospital of southern medical university and had a family history of acne.The blood of patients with moderate to severe acne vulgaris was routinely extracted(acne blood group,CCXY),and all whitehead and blackhead tissues were collected with acne needles(acne organization group,CCZZ).In the normal control group,peripheral blood(normal blood group,ZZXY)and skin lesions(normal tissue group,ZCZZ)were all from the patients who underwent resection of pigmented nevus on the back of the dermatology department of shenzhen hospital of southern medical university(age and gender matched with the patient group,excluding systemic diseases and other skin diseases).All patients participating in this study signed written informed consent.2.DNA extraction and quality testing: DNA extraction kits were used to extract the whole genomic DNA from peripheral blood samples and skin tissues.The concentration and purity of DNA were measured by UV spectrophotometer,and the integrity of DNA was detected by 1.5% agar gel electrophoresis.The ratio of OD260/OD280 was between 1.7 and 2.1,and the bands were clear and intact,which were qualified DNA.Store in refrigerator at-80?.3.CpG sites selection: the CpG sites of AR?IGF1R were selected by Gene CpG v1.0 software.4.Bisulfite treatment:After processing the DNA sample with the EZ DNA Methylation-Gold ? Ki Bisulfite Conversion Kit,unmethylated cytosines are converted to uracil.The minimum amount of human genomic DNA required for bisulfite treatment and subsequent PCR amplification is 500 pg.5.Primer design and optimization: The geneCpG software was used to analyze the genomic regions of AR and IGF1 R,and converted them into bistable DNA sequences.Primer3 software(http://primer3.ut.ee/)from bisulfite-convert DNA was used to design PCR primers.a special method based on capillary electrophoresis was used to optimize the primer composition and concentration in the multiplex PCR panel.6.Multiplex PCR reaction of sample target fragments: Multiplex PCR amplification was performed using the optimized multiplex PCR primer panel with the transformed sample genome as a template.7.Specific tag sequences were added to the samples: primers with Index sequences were used to introduce specific tag sequences compatible with illumina platform to the end of the library by PCR amplification.8.Quantitative library and on-line sequencing: The final MethylTarget sequencing library was obtained after sample extraction and gel recovery.The Agilent2100 Bioanalyzer verified the fragment length distribution of the library.After precise library molar concentration,high-throughput sequencing was performed on the Illumina Hiseq platform to obtain FastQ data.Results:1.A total of 20 patients with acne vulgaris of han nationality in shenzhen were collected,including 7 males(35%)and 13 females(65%),with an average age of25.50±1.70 years.There were 20 patients in the normal control group,including 4males(20%)and 16 females(80%),with an average age of 23.90±2.94 years.2.Qualified DNA: 13 cases of CCZZ,18 cases of CCXY,9 cases of ZCZZ,19 cases of ZZXY.The AR gene contains 3 CpG islands and 40 CpG sites.the IGF1 R gene contains 6 CpG islands and 135 CpG sites.3.Comparing the acne group with the normal control group,it was found that the AR gene had an average methylation level of 37 CpG sites,and all of them were present in the acne tissue group.the IGF1 R gene had an average methylation level of51 CpG sites.The average methylation level of 3 CpG sites decreased.4.After comparison between the acne group and the normal control group,methylation differences were found in a total of 7 gene regions,all of which were found in the acne tissue group.The average methylation level in 3 CpG island regions of AR gene increased,and that in 4 CpG island regions of IGF1 R gene decreased.5.The difference in methylation of AR gene was found between the acne group and the normal control group,and the average methylation level of AR gene was increased.Differences in methylation of IGF1 R gene were found between the acne group and the normal blood group,and the average methylation level of IGF1 R gene was reduced.Conclusion:1.Abnormal methylation of AR gene and IGF1 R gene was found in patients with acne vulgaris.2.The AR gene was hypermethylated and the IGF1 R gene was hypomethylated in acne vulgaris3.There was no difference in the average methylation level of AR gene and IGF1 R gene in peripheral blood of acne patients compared with normal control group.4.The hypermethylation status of the AR gene and / or the hypomethylation status of the IGF1 R gene may be involved in the pathogenesis of acne vulgaris by regulating the expression of corresponding genes,but the exact mechanism needs further research and clarification. |
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