DNA Methylation Detection Of The Promoter Region Of The Human Telomerase Reverse Transcriptase Gene In Gastric Cancer Cell Line

Objective: Gastric cancer was imalignant tumor which Severityinfluence human health.But the etiological factor of gastric cancer was notclear and curative effect of gastric cancer was far to satisfaction up to now.Now the human telomerase reverse transcriptase (hTERT)gene is aimportant target for tumor's diagnosis and therapy .But the regulation ofhTERT was extremely complicated. Using methylation-specificpolymerase chain reaction (MSP), we analyse methylation status of thepromoter region of hTERT gene of different differentiated gastric cancercell line. We discussed whether the region exists methylation and whetherthe degree of methylation correlated with cancer malignancy in order toexplain the mechanism of gastric canceration farther. Furthermore, throughdetecting the normal human immortalized cells with hTERT-expressingand lymphocytic cell with weak hTERT-expressing, We discussed thevalue of DNA methylation regulating hTERT and offered a new idea forcancer gene therapy by means of hTERT. Methods: We choosed gastric moderately differentiatedadenocarcinoma cell line SGC7901 and poorly differentiatedadenocarcinoma cell line BGC823, breast cancer cell line MCF-7 ,humanumbilical vein endothelial cells line ECV 304, normal human lymphocyticcell as positive contrast, human fetal lung fibroblast cell line WI-38 asnegative ontrast. Logarithmic phase cells operating high salt methodextracted cells' genome DNA .Using ultraviolet spectrophotometerdefinited nucleic acid purity and fixed quantify. High purity genome DNAoperated hot-start and touch-down PCR with wild primer to examin part ofinitial rank of hTERT promoter in order to determine whether existed rankalteration in the core of the promoter region of hTERT gene in gastriccancer cell line .Genome DNA (1-2ug) through alkali denaturation,sulfuration, deammonia , purification, desulfuration, neutralize andprecipitation complete bisulfite treatment of DNA. Modified DNA operatedmethylation-specific polymerase chain reaction (MSP) with methylationprimer and unmethylation primer to analyse methylation status of thepromoter region of hTERT gene. Each PCR products were analyzed onethidium bromide-stained 2%-1.5% agarose gels in TAE buffer. Resultswere observed with ultraviolet transmission reflection Instrument . PurifiedMSP products fixed quantify ultraviolet spectrophotometer and clonesequencing analyzed methylation of the CpG site of hTERT promoter. Results: We detected the core of the promoter region of hTERT genein different differentiated gastric cancer cell, breast cancer cell ,humanumbilical vein endothelial cells , normal human lymphocytic cell normalhuman fetal lung fibroblast cell line. The core of the promoter region ofhTERT gene in gastric cancer cell were not found gene mutation, deletionand rearrangement et al which could affect hTERT expression. Not onlygastric cancer cell ,breast cancer cell ,human umbilical vein endothelialcells with hTERT-expressing but also normal human lymphocytic cell withweak hTERT-expressing could get object fragment which amplified byMSP with methylation primer other than unmethylation primier, in contrastto hTERT-negative human fetal lung fibroblast cell. Examining by clonesequencing, PCR products of three pair of primer were correct and foundthat differentiated gastric cancer cell exist different methylation at CpGpoint of promoter region of hTERT gene. Conclusion: The promoter region of hTERT gene existed methylationin different differentiated gastric cancer cell line.But the degree ofmethylation was different and that might be dependent of degree ofdifferentiation in gastric cancer cell. We detected the promoter region ofhTERT gene exist methylation in hTERT-expressing cancer cell ,thenormal human immortalized cells and weak hTERT-expressing normalhuman lymphocytic cell. most hTERT-expressing tissues and cells mightexist DNA methylation regulation that protected or promoted hTERTexpression through some mechanisms. the promoter region of hTERT genein hTERT-negative human fibroblast cell did not exist methylation. ThathTERT-ne...