Effects Of Celecoxib On The Methylation Status,mRNA Expression Of P16^ink4a Gene Promotor And Proliferation In Squamous Cell Carcinoma Of The Esophagus

Objective: Esophageal carcinoma is a common malignant tumor.It can be divided into five histological types.,Esophageal squamous cell carcinoma（ESCC）is the most common in the five types and accounting for about 90 percent.The long-term survival of the patients with esophageal cancer is still disappointing;the 5-year survival rate is about 30 after surgery and less than10% after radiochemotherapy..Therefore,the early diagnosis and treatment and looking for effective molecular markers for treatment of patients is of great significance.A large number of clinical studies and experiments show that epigenetics plays a important role in the development of tumor,DNA methylation is one of the important mechanisms of epigenetics,whcih makes the research of demethylated anti-tumor drugs become possible.With the studying of demethylated drugs,nonsteroidal anti-inflammatory drugs（NSAIDs）such as aspirin and celecoxib were found to have demethylation effects,which has been demonstrated in gastric cancer,colon cancer and rectal cancer.But the research about the effect of NSAIDs on the methylation of ESCC is rare.This study observed the methylation status of P16^ink4a gene promoter in ESCC and its adjacent normal esophageal squamous tissues（NESTs）,analyzed the relationship between methylation status of P16^ink4a gene promoter and ESCC clinicopathological features and evaluated the effect of celecoxib on the methylation status of P16^ink4a gene promoter and proliferation of ESCC cell Eca109.Methods:1 ESCC: fifty patients who underwent ESCC were recruited in the Fourth Hospital of Hebei Medical University from March 2016 to october 2016.All of them were pathologically confirmed ESCC preoperatively andpostoperatively.There were no history of radiotherapy and chemotherapy,long-term use of aspirin and other NSAIDs.Methylation status of ESCC and its adjacent NESTs were detected by methylation-specific PCR（MSP）.2 ESCC cell Eca109: The experimental group was treated by celecoxib for 48 hours and the control group was treated by DMSO.The methylation status of experimental and control group was analyzed by MSP.The P16^ink4a m RNA expression was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.Cell proliferation was evaluated using the Cell Counting Kit-8（CCK-8）.3 SPSS software package was used for statistical analysis of the experimental data.Rusults:1 The relationship between P16^ink4a gene promoter methylation status and the clinical and pathological characteristicsMSP showed that the methylation rate of P16^ink4a gene promoter in ESCC was 48%（24/50）and 12%（6/50）in adjacent NESTs,the difference was statistically significant（P<0.05）.The methylation status of P16^ink4a gene promoter was not related to the sex and smoking history of patients（P>0.05）,but related to the history of drinking,lymph node metastasis,vascular thrombosis,nerve invasion and TNM staging（P<0.05）.2 Effects of celecoxib on methylation status of P16^ink4a gene promoter in ESCC cell Eca109MSP showed that the extant of methylation was decreased and the extant of unmethylation was gradually increased after treatment with celecoxib.But the extant of methylation was not changed in the control group.3 Effects of celecoxib on m RNA expression of P16^ink4a gene in ESCC cell Eca109In the experimental group,when the concentration was 40μmol/L,60μmol/L,80μmol/L and 100μmol/L,q RT-PCR showed that the expression of m RNA in Eca109 was significantly higher than those without treated by celecoxib（P = 0.037;P = 0.014;P = 0.002;P = 0.043）.But in the controlgroup,there was no significant difference between with or without treatment by DMSO.4 Effects of celecoxib on proliferation in ESCC cell Eca109The results of CCK-8 test showed that,the proliferation rate of cells in experimental group were significantly decreased than those cells without treatment（P<0.05）.There were no significant differences in proliferation rate of cells between control group were no treatment group（P>0.05）.Conclusions:1 The methylation status of P16^ink4a gene promoter is not associated with sex and smoking history,but related to the history of drinking,lymph node metastasis,vascular thrombosis,nerve invasion and TNM staging.2 Celecoxib can reduce the degree of methylation of P16^ink4a gene promoter.3 Celecoxib can improve the expression of P16^ink4a gene in ESCC cell Eca109.4 Celecoxib can inhibit the proliferation of ESCC cell Eca109.