| Psoriasis is a common, chronic, inflammatory skin disorder. Histopathologically the disease is characterized by hyper-proliferation of the epidermis, accumulation of inflammatory cells and hyperplasia, elongation, dilatation and increased tortuosity of dermal papillary blood vessels. Owing to the unclear pathogenesis, psoriasis is the keystone in the study on the skin diseases. With the development of modern molecular biology, study on cell cycle progress rapidly. It has been shown that the CDK4-cydinD kinase complex promotes progression entering the S of the cell cycle from G1 phase. In normal cells, the retinoblastoma tumor suppressor protein regulates cell proliferation by binding and sequestering transcription factors. Phosphorylated Rb, thereby allowing cells to enter S phase, releases these transcription factors at late Gl phase. The main function of the CDK4-cyclinD kinase complex may be to phosphorylate Rb at late Gl phase. P16 protein coded by tumor suppressor gene p16 has been characterized by specifically binding to and inhibiting CDK4/CDK6, and thus P16 protein may regulate phosphorylation of Rb, thereby p16 gene may play an important role in the negative regulation of cell proliferation. Studies suggest that promoter methylation usually exist near the tumor suppressor gene loci. Therefore, someone thought that CpG island hypermethylation occurs in relation to tumorigenesis or aging. In the past studies, someone usually focused on therelationship between the status of the exon methylation and the tumorigenesis. However, the links between the promoter CpG island methylation status of p16 gene and clinicopathological parameters has been very limited in psoriasis vulgris. Our former study shows that the frequencies of LOH at the microsatelite in p16 gene are associated with the development of psoriasis. It also suggested that the high reach in p16 gene has close relationship with psoriasis, may have other forms of changes.Recently, development of new techniques-Methylation-specific PCR (MSP) has enabled more hypermethylation locus be studied. Compared with Southern Blotting Method and Digestion with methylation-sensitive enzymes followed by PCR, it can not only compensate their shortages but also possess many striking advantages: avoiding the use of restriction enzymes and resultantly problems associated with incomplete enzymatic digestion, high sensitivity and specialty and being suitable for small, heterogeneous samples. In this study, MSP was used to explore the status of the promoter CpG island methylation of the p16 gene in psriasis vulgris tissues, non-lesion skin of psoriasis and normal skin samples. The frequency of CpG island hypermethylation of p16 gene promoter and the relationship between hypermethylation and origination as well as the development of psriasis vulgris were discussed.Materials and MethodsTwenty-six psoriasis vulgris patients, 15 male and 11 female, age from 14 to 62(31.77± 10.34), 8 with familial history and 18 without familial history, 10 early onset and 16 late onset, 12 in progressive phase and 14 in stationary phase. They are all diagnosed as psoriasis vulgris according to the Third Edition of Clinical Dermatology written by Zhao Bian and pathology. Keratinocyte were cultured primarily from epidermis of the lesions and non-lesion epidermis respectively by the apparatus of epidermis transplanting for treating vitiligo, and normal epidermis were surgically removed and collected from Orthopaedics, First Affiliated Hospital of Zhengzhou University, China. Genome DNA was obtained by phenol extraction and ethanol precipitation following proteinase K digestion. All samples were immediatelyused to be separated keratinocyte and cell cultured MSP, non-denaturing PAGE and the detection to DNA sequencing of MSP products were used to detect the promoter methylation in p16 gene in a series of 26 psoriasis vulgris, 26 non-lesion epidermis tissues and 26 normal epidermis samples and the data was studied associating with clinic factors.The data was analysized by SPSS10.0 and a=0.05 was taken as significant criteria of test.Results1. Methylation of p16 promoter in psoriatic lesion region and non-lesion region was found in 23.08% (6/26) and 19.23% (5/26), none in normal skin. The frequency of methylation of p16 promoter was higher in psoriatic lesion region than that of normal kerotinocyte (P<0.05). The frequency of methylation of p16 promoter was higher in non-lesion region than that of normal kerotinocyte (P<0.05). There is no significant difference about the frequency of methylation of p16 promoter between lesion region and non-lesion region(P>0.05).2. Methylation of p16 promoter was found 41.67% (5/12) in psoriatic lesion in progress phase, while in stationary phase 7.14% (1/14). The frequency of methylation of p16 promoter in psoriatic lesion was higher in progress phase than that in stationary phase and normal kerotinocyte (P<0.05). Methylation of p16 promoter was found 7.14% (1/14) in psoriatic lesion in stationary phase. It was higher in psoriatic lesion in stationary phase than that of non-lesion in stationary phase and normal skin. But there are no significant diference between them (P>0.05 ) .3. Methylation of p16 promoter were found 41.67% (5/12) in non-lesion in progress phase, while in stationary phase (0/14) and 0% (0/14). The frequency of methylation of p16 promoter in non-lesion was higher in progress than that in non-lesion in stationary phase and normal kerotinocyte (P<0.05). Methylation of p16 promoter was found 0% (0/14) in non-lesion in stationary phase, no higher than that of normal skin. And there is no significant difference between non-lesion in stationary pahse and normal kerotinocyte (P>0.05) .4. Methylation of p16 promoter was found 41,67% (5/12) in psoriatic lesion and non-lesion in progress phase; there is no significant difference between them. Methylation of p16 promoter were found 7.14%(1/14) and 0%(0/14) in psoriatic lesion and non-lesion respectively in stationary phase; there is no significant difference between them (p>0.05 ) .5. Methylation of p16 promoter were found 25.00% in male patients and 21.43% in female patients; there is no significant difference between them (P>0.05) . Methylation of p16 promoter were found 20.00% in early onset patients and 25.00% in late onset patients; there is no significant difference between them(P>0.05) . Methylation of p16 promoter were found 27.27% in patients with family history and 20.00% in patients without family history; there is no significant difference between them (P>0.05) .Conclusions1. There are methylations of CpG islands in p16 promoter. Methylation of CpG islands in p16 promoter is related with the genisis and progress of psoriasis.2. The frequency of methylation in CpG islands of p16 promoter in lesion region was higher in progress phase than that in stationary phase. The frequency of methylation in CpG islands of p16 promoter in non-lesion region was higher in progress phase than that in stationary phase. It indicates that there may be significant correlation between methylation of the regulating sequence of p16 gene and severity as well as development of psoriasis.3. The frequency of methylation in CpG islands of p16 promoter is associated with clinical phase and has no association with origins of issue. This shows that methylation in CpG islands of p16 promoter may be an important molecular mark in the development of psoriasis.4. Methylation in CpG island of p16 promoter in psoriasis patients has nothing to do with age, first onset age, sex and family history.
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