| Objective1. To explore the mutation of PUMA promoter binding site with p53, site BS1 and BS2, in lung cancer tissue.2. To explore the relationship between PUMA promoter methylation and lung cancer or clinical pathological type.Methods1. By exploring the reaction conditions of polymerase chain reaction, we optimized and identified amplification systems of PUMA BS1, BS2 site squence which bound with p53 to make the sequence that GC content 76.3% be amplificationed successfully. The system was used to amplify 43 cases of lung cancer tissue, and the purified products were sequenced to analyse the mutation.2. DNA methylation detection assay in this study was METHYLSCREEN technology. Methylation-sensitive restrictive enzyme (MRSE) and methylation-dependent restrictive enzyme (MRDE) were used. Productions of enyme digestion were analysed by fluorescence quantitative RT-PCR.35 cases of lung cancer tissue and 10 cases of lung benign lesion were measured the relative ratios of methyaltion (the percentage of hypermethylation, unmethyaltion, intermediamethylation) in PUMA promoter CpG island.Results1. The amplification systems and reaction conditions of PUMA BS1, BS2 site squence which bound with p53 were:dNTP 250μmol/L, MgCl2 1.5mmol/L, forward primer and reverse forward primer each 250nmol/L, TaqDNA polymerase (High GC 2U/μL) 0.04U/μl, templates 2000ng,5×Buffer 10μL, the total volum was 50μl. The cycling conditions were as follows:After 5 min pre-denaturation at 95℃, reaction were cycled through 1 min denaturation at 95℃,1 min annealing at 65℃,72℃1 min extension at 72℃for 33; followed 72℃for 5 minutes. The sequences of 43 lung cancer tissue cases were amplified successfully. The amplified products were depurated and sequenced to detect mutation. The sequencing results contrast to standard DNA sequence by using Blast2. None of mutations were found.2. In the study of PUMA DNA promoter methyaltion, the enzyme digestion system of PUMA were:DNA 250ng joined in restrictive enzyme digestion buffer solution 26ul, added nuclease free water to total volum 120ul. By adding MSRE or MDRE 4 restrictice enzyme digestion Mo, Ms, Md and Msd were established. No enzyme was added in Mo. MSRE lul was added in Ms. MDRE lul was added in Md. MSRE and MDRE each lul were added in Msd. Prepared DNA templates 28ul, nuclease free water was added to total volum 30ul. The real-time quantitative PCR amplification systems and reaction conditions of PUMA methylation were:PCR primer Mix 1.1ul, Enzyme digested product was 5ul, PCR Master Mix 13.75ul, nuclease free water 7.15ul, the total volum was 27ul. The cycling conditions were as follows:After 10 min active the Hotstart DNA polymerase at 95℃, reaction were cycled through 1 min denaturation at 97℃,1 min extension at 72℃for 40. The METHYLSCREEN method was used to detect the relative percentage of PUMA aberrant methylation. In lung cancer tissues medians of PUMA hypermethylation, unmethylation and intermediate methylation were 0.058166,0.923567,0.000000. In lung benign lesion tissues medians of PUMA hypermethylation, unmethylation and intermediate methylation were 0.077580,0.922420,0.000000. Noneparametric statistics analysis showed that there were no difference in hypermethylation, unmethylation and intermediate methylation of PUMA between lung cancer tissues and lung benign lesion tissues(P>0.05). There were no difference in in hypermethylation, unmethylation and intermediate methylation of PUMA between different age and gender(P>0.05). There were no difference in hypermethylation, unmethylation and intermediate methylation of PUMA among squamous carcinoma, adenoeareinoma and adenosquamous carcinoma(P>0.05).Conclusions1. The structure of PUMA BS1, BS2 site DNA squence which bound with p53 were quite stable in lung cancer tissues without mutation, insert or deletion.2. There were no relationship between PUMA promoter methylation and lung cancer. There were no relationship between PUMA promoter methylation and gendar or age of lung cancer patients or lung cancer pathologic types. |
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