Investigation Of Bin1 Methylation Status And The Effect On Development In Esophageal Squamous Cell Cancer

Part one Investigation of Bin1 Methylation status and clinical significance in esophageal squamous cell cancer patientsObjective: To study the expression and methylation status of Bin1 in patients with esophageal squamous cell cancer(ESCC)tissues and to investigate the association of status of Bin1 and patients clinical features.Methods:1 Tumor and para-carcinoma tissue specimens were collected from 58 cases of ESCC patients who underwent surgical resection in the Department of Chest Surgery,the Fourth Hospital of Hebei Medical University between June,2014 and June,2015.Each specimen was divided into 3 pieces and treated as follows for different uses: fixed in 4% formaldehyde for making paraffin embedded blocks,and put in liquid nitrogen for extracting RNA and DNA.2 Bin1 protein and mRNA expression in the carcinoma and paracarcinoma specimens was assessed by immunohistochemistry and reverse transcription polymerase(qRT-PCR)respectively.The methylation status of Bin1 was assessed by methylation-specific PCR(MSP).The association between the methylation status and the expression of Bin1 with ESCC tissues and patient clinical features were analyzed.Results:1 In 58 ESCC patients,the incidence rate of Bin1 protein low expression was significant higher than that in the corresponding para-cancerous normal tissues [37/58(63.79%)vs.13/58(22.41%),P<0.01].We further analyzed Bin1 expression at gene level using qRT-PCR,compared with para-carcinoma tissue specimens,the expression of Bin1 mRNA in carcinoma tissue specimens was lower significantly(0.78±0.05)vs(1.03±0.03)(t=9.643,P<0.01).The expression of Bin1 mRNA in ESCC tissues with methylation status was significant lower than that in ESCC tissues without methylation(0.68±0.04)vs(0.85±0.07)(t=2.476,P<0.05).35 cases of ESCC tissues exhibited a low expression of Bin1 mRNA,for 94.59% low Bin1 protein expression,which indicated that Bin1 expression were consistent at gene and protein level.2 Among the 35 tumor tissues with low Bin1 mRNA expression,the hypermethylation of Bin1 could be observed in 32 cases(91.43%),accounting for 89.19% low Bin1 protein expression.3 The methylation frequency of Bin1 was correlated with TNM stage,tumor invasion depth,tumor differentiation and lymph node metastasis.Conclusions: Bin1 was hypermethylation state in ESCC tissue and it might be closely associated with TNM stage,tumor invasion depth,tumor differentiation and lymph node metastasis Part two The effect on development in esophageal squamous cell cancer of Bin1 Methylation statusObjective: To detect the expression and the methylation status of Bin1 in YES-2,TE13,TE1,KYSE30 and EC109 cells.YES-2 and TE13 cells were selected and treated by DNA methylation inhibitor 5-Aza-2’deoxycytidine(5-Aza-dC)to investigate ESCC cell biological activity and EMT-associated protein expression.Methods:1 Bin1 mRNA expression level in five ESCC cells: YES-2,TE13,TE1,KYSE30,EC109 were evaluated by qRT-PCR.To detect the methylation status of these 5 ESCC cell lines.YES-2 and TE13 cells were selected for treated with DNA methylation inhibitor 5-Aza-2’ deoxycytidine(5-Aza-d C).The change of Bin1 expression was assessed by Western-blotting and the methylation status was assessed by MSP.2 MTT and colony formation assays were used to detect the cell viability of ESCC.3 Flow cytometry(FCM)was used to detect the cycle distribution and apoptosis level of ESCC cells.4 Scratch test and transwell chamber experiment were used to detect the cell migration and invasion ability of ESCC cells.5 Western-blotting was used to detect the expression of epithelial mesenchymal transition(EMT)associated proteins E-cadherin(E-cad),Ncadherin(N-cad),Snail,Matrix Metalloproteinase-2(MMP-2)and MMP-9.Results:1 qRT-PCR analysis showed significant lower expression in these 5 ESCC cells: TE13,TE1,KYSE30,EC109 and YES-2(P<0.01).YES-2 and TE1 cells was both in a state of complete methylation.YES-2 and TE13 cells were selected for treated with DNA methylation inhibitor 5-Aza-2’-deoxycytidine(5-Aza-dC).The expression of Bin1 were increased with 30,60,and 90 μM 5-Aza-dC treated in YES-2 and TE13 cells(P<0.01).2 Colony formation assay showed that treated with 30,60,and 90 μM 5-Aza-d C in YES-2,the colonies of YES-2 cells was(233±29),(189±23)and(105±16),which was significant lower than the control group(71±11)(P<0.01);Treated with 30,60,and 90 μM 5-Aza-dC in TE13,the colonies of TE13 cells was(281±33),(235±28)and(139±19),which was significant lower than the control group(83±12)(P<0.01).MTT and Clone assays showed that treated with 30,60,and 90 μM 5-Aza-dC in YES-2 and TE13 cells,the cell viability was significantly decreased(P<0.01).3 FCM showed that treated with 30,60,and 90 μM 5-Aza-dC increased the number of cells in G0/G1 phase and decreased the number of cells in S phase in YES-2 and TE13 cells,which indicated that cell cycle was arrested at G1 phase(P<0.01).Compared with the control group apoptosis was increased of YES-2 and TE13 cells treated with 30,60,and 90 μM 5-Aza-dC(P<0.01).4 The scratch test results show that treated with 5-Aza-dC,YES-2 and TE13 cell migration distance was significant shorter(57.7±4.5 vs.122.4±9.4,p<0.01)(64.2±4.8 vs.133.4±8.1,P<0.01),which showed that cell migration and invasion ability significantly decreased YES-2 and TE13 cells after treated with 5-Aza-dC(P<0.01).5 Western-blotting assay showed that EMT-associated protein N-cad,Snail,MMp-2,MMp-9 decreased significantly(P<0.01),and E-cad increased significantly compared with the control(P<0.01).After treated with 5-Aza-dC,the expression of Bin1 increased and EMT were suppressed in YES-2 and TE13 cells.Conclusions: In vitro experiments confirmed that Bin1 methylation could affect ESCC cell proliferation,migration,invasion and apoptosis ability and promote the development of esophageal cancer by promoting the epithelial mesenchymal transition of esophageal cancer cells.