Monitoring Of P16 Abnormal Methylation By Real-time Fluorescent Quantitative PCR In NSCLC

Objectives:Lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is the most frequent in lung cancer. For different cell types , the survival rate of five years is only 6%～16%. However , to NSCLC , it demonstrates from some dates that the survival rate of five years can increase to 60%-90% if it can be earlier discovered, diagnosed and treated. So in spite of if there is an opportunity of surgical treatment , the best meas of extend the patient's life is early discovery , diagnosis and treatment. The research demonstrated that abnormal CpG methylation of p16 gene has occurred in the stage of hyperblastosis and metaplasia of tumor, which is an important mechanism of p16 gene inactivation and early event of lung cacer, so it has a great value in earlier diagnosing lung cancer . DNA methylation is a process which transfer methylium to 5'terminatio of CpG islands, catalyzed by DNA methylation transferase and donored by SAM. Methylation is negative correlation with genetic transcription , and the mechanism is probably as follows : directly interfere the combination of specific transcripton and recognition site of each promoter ; mediated transcription inhibition by change the construction of chromatin; induced gene silencing by combination of methylation site and transcription inhibition. P16 gene is also called multiple tumor supresser gene , which is the best easily to methylate in the anti-oncogenes. P16 gene locates in 9p21 of mankind-chromatosome , and it's total length is 8.5kb . P16 gene is composed of two intervening sequences and three exons , and encodes p16 protein , which is the inhibiter of CDK4 and CDK6 . They can together prevent phosphorylation deactivation of pRB , and make the cell cycle blocked in G1 stage. P16 gene promoter region has a great of CpG islands , which can be methylated when some adverse factor appears , and so the genetic transcription of p16 can be inhibited , which leads to gene silencing, cell paraplasm, and tumor generate. Belinsky confirmed that abnormalmethylation of p16 gene generally occurs in the early stage of lung cancer, and develops with tumor progress. Our research will make p16 gene of methylation as tumor-marker, and detect 30 NSCLC patients of different status, then discuss clinical significance of the research.Method: 1 . To construct DNA standard preparation of methylation-R and non-methylation-H by means of recombination in vitro . To cultivate Raji cell line (B lymphadenoma) of p16 promoter methylation and Hela cell line (uterine cervix cancer) of p16 promoter non-methylation. When the amount of cell increases to 1×107 , genomic DNA is extracted , in which the nonmethylation C of CpG will shift to U (later to Tfor PCR) after modified by sodium bisulfite. However methylation C will remain original pattern of mC. PCR reaction will start with different specific primer, and the status of CpG methylation will be analyzed. To design and synthesize specific primer of M and U for standard preparation- construction according to gene order . To get destination gene unit of p16 promoter and the first exons which has been methylated by means of ordinary PCR method. The destination gene unit is 262bp long , (include the specific unit for real time PCR), and it is conjuncted to plasmid vector of PMD-18, then transfected in colibacillus competent cell of JM-109. After overnight cultivation at 37℃, we can selective recombinant and extract DNA of plasmid. According to the absorbance of DNA, copy number can be calculated. DNA standardpreparation of plasmid will be diluted proportionally to 107, 106, 105, 104, 103, 102, 101copies/ul , and they are maked as templates for real-time PCR reaction , thus standard curve of methylation p16 and nonmethylation p16 can be obtained. The efficiency of standardpreparation will be detected at the same time. 2 To design and synthesize specific primer of m(methylation system) and u (nonmethylation system )for real time PCR reaction, and quantitation of methylation or nonmethylation can be calculated according to standard curve of two DNA standard preparations. To calculate the percentage of methylation and evaluate the level of methylation .Result: 1 .Satisfactory amplification curve , standard curve and melt curve can be obtained from two DNA standard preparations. Coefficient correlation of standard curve exceeds 0.98, which permit a wide scope for quantitation; efficiency of amplification between 0.8 and 1.2; peak form of melt curve is specific; Tm of methylation p16 is 88.2±1, and nonmethylation p16 is 83.2±1; linearity of standard curve is fine; purity coefficient of plasmidplasmid is perfect by electrophoresis and OD value; the ratio of A260 and A280 is about 2.0, and so standard preparation isconstruct successfully. 2. To analyze methylation level of NSCLC patients by real time PCR, and the conclusion showed there is a significant difference between these patients(P<0.05).Conclusion : The method of fluorescent quantitation PCR is applied in detecting methylation level of NSCLC, and provided the evidence in early diagnosing NSCLC. The method will be available for clinical treatment, and then improve survival rate of NSCLC patients .