| ObjectivesThrough detection the methylation levels of hsa-mi R-3665, hsa-mi R-375 and hsami R-4530 gene promoter region in esophageal squamous carcinoma(ESCC) tissues and adjacent tissues, to explore the relationship between hsa-mi R-3665, hsa-mi R-375, hsa-mi R-4530 and the clinical parameters of ESCC patients and the prognosis respectively, and find out the micrornas which influence of esophageal squamous carcinoma’s occurrence, development and prognosis, provide the basis for ESCC prognosis’ diagnose.Methods1. A total of 145 ESCC patients’ fresh tissues and adjacent tissues which distance the cancer tissue more than 3 centimeters who underwent surgery between July 2012 and July 2014 from The First Hospital Affiliated of Fujian Medical University,Zhangzhou Municipal Hospital and Anxi County Hospital, the corresponding clinical parameters and follow up parameters of 145 patients were collected, and all of the patients were followed up until May 9, 2015.2. Methylation primers of hsa-mi R-3665, hsa-mi R-375 and hsa-mi R-4530 gene were designed.Methylation levels of ESCC tissues and adjacent tissues were detected by the methylation sensitivity of high resolution dissolve curve. Combined with the clinical parameters and follow up parameters, found out micro RNAs which was correlated with clinical parameters and prognosis.3. MTT method determined the growth situation of Eca109 cell after EGCG hangling concentration of 5 μM,10 μM,20 μM,50 μM and 100 μM.Then analysised the relationship between the growth situation of Eca109 cell and concentration and time by factorial analysis. Real-time quantitative RT-PCR detected micro RNA expression level which was sieved out with 20 μM EGCG handling.Testing methylation levels of 20 μM EGCG and 1 μM DAC which was a methylation inhibitor.Results1. Four pairs of corresponding methylation primers were designed, and the Correlation analysis of hsa- mi R-3665(2) methylation levels had statistical significant( c2= 14.800, P=0.022) between ESCC tissues and adjacent tissues.And the hypermethylation levels of cesophageal squamous cell carcinoma tissues higher than the adjacent tissues.2.Compared with the clinical parameters,the methylation levels of hsa-mi R-3665(2) iscorrelated with TNM stages(P=0.024),the methylation leves in most patients in TNM stage(II and III) is between 50% to 100%.The methylation levels of hsa-mi R-375 is correlatedwith residual residues(P=0.024),TNM stages(P=0.016),postoperative infection(P<0.001)and lymph node metastasis(P=0.017),compared with the patients of residual residues,postoperative infection and lymph node metastasis, the numbers and the methylation levesof no residual residues, postoperative infection and lymph node metastasis are higher.TNMstaging had the same result with hsa-mi R-3665(2).The methylation levels of hsa-mi R-4530 is correlated with degree of infiltration(P=0.026), residual residues(P=0.038) and lymphnode metastasis(P=0.015),the methylation leves in most patients in infiltration T3 isbetween 0% to 50%,residual residues and lymph node metastasis had the same results withhsa-mi R-3665(2).3. Two-stage test results showed that difference methylation groups in hsa-mi R-3665(2) and hsa-mi R-375 were significant in the survival rate(TSPV =0.014,TSPV < 0.001).COX multivariate regression model analysis showed that the methylation levels of hsa-mi R-4530 had statistical significant with prognosis(P=0.040).4. The inhibition of ESCC in different handling time and different EGCG concentrations are different.Real time quantitative RT-PCR showed that hsa-mi R-3665(2) was upregulated after EGCG and DAC handled 48 h and 72 h,and the methylation level detection results also showed that both of them were lower than control.Conclusions1. The methylation levels of hsa-mi R-3665 in esophageal squamous cell carcinoma tissues and the adjacent tissues existenced correlation.And the hypermethylation levels of cesophageal squamous cell carcinoma tissues higher than the adjacent2.The methylation levels of ESCC in hsa-mi R-375 and hsa-mi R-4530 with no lymph node metastasis is higher than metastasis. The methylation levels of hsa-mi R-3665(2) and hsami R-375 are correlated with TNM stages,the methylation leves in most patients in TNM stage(II and III) is between 50% to 100%.The methylation levels of hsa-mi R-4530 is correlated with degree of infiltration,the methylation leves in most patients in infiltration T3 is between 0% to 50%.The methylation levels of ESCC in hsa-mi R-375 and hsa-mi R-4530 with no residual residues is higher than residual residues.The methylation levels of ESCC in hsa-mi R-375 with no postoperative infection is higher than infection.3. The methylation levels of hsa-mi R- 3665(2),hsa-mi R-375 and hsa-mi R-4530 may be epigenetic factors of prognosis in ESCC.4. The inhibition of ESCC in different handling time and different EGCG concentrations are different, and its mechanism may be related to the methylation levels of hsa-mi R-3665 promoter region. |
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