Role And Methylation Status Of MiR-1250 And Its Host Gene AATK In Human Esophageal Squamous Cell Carcinoma

Part one Role and methylation status of miR-1250 in esophageal squamous cell carcinomaObjectives: To detect the expression and methylation status of miR-1250 and AATK in esophageal cancer cell lines and esophageal squamous cell carcinoma(ESCC)tissues,and to discuss clinicopathologic and prognostic significance of it in ESCC tissues.To investigate the role of miR-1250 and its host gene AATK in the occurrence and development of ESCC tissues.Methods:1 Quantitative real-time RT-PCR(q RT-PCP)method was used to detect the expression of miR-1250 and AATK in esophageal cancer cell lines and the cell lines treated with 5-Aza-dC,ESCC tissues and corresponding normal tissues.2 Western Blot method was used to detect the protein expression of AATK in esophageal cancer cell lines and the cell lines treated with 5-Aza-dC.3 Immunohistochemistry method was used to examine the protein expression of AATK in ESCC tissues and corresponding normal tissues.4 MSP method was used to detect the methylation status of AATK in esophageal cancer cell lines and the cell lines treated with 5-Aza-dC,ESCC tissues and corresponding normal tissues.Result:1.The expression and methylation status of miR-1250 and AATK in esophageal cancer cell lines: The expression of miR-1250 and AATK in four treated cell lines(Eca109,TE1,Yes-2,T.Tn)was increased.The hypermethylation status of AATK in four untreated cell lines was revesed after treated with 5-Aza-dC(P<0.05).2.The expression of miR-1250 and AATK mRNA in ESCC tissues andcorresponding normal tissues: The expression of mi R-1250 and AATK mRNA in ESCC tissues was obviously lower than that in corresponding normal tissues(P<0.05).It was closely correlated with lymph node metastasis,pathological differentiation,and TNM stages(all P<0.05),it was not associated with age and gender(both P>0.05).3.The protein expression of AATK in ESCC tissues and corresponding normal tissues: The protein expression rate of AATK in ESCC tissues was obviously lower than that in corresponding normal tissues(P<0.05).It was closely correlated with pathological differentiation and TNM stages(both P<0.05),it was not associated with lymph node metastasis,age,and gender(all P>0.05).4.The methylation status of AATK in ESCC tissues and corresponding normal tissues: Methylation frequency of AATK was significantly higher in ESCC tissues than that in corresponding normal tissues(P<0.05),it was closely correlated with lymph node metastasis,pathological differentiation,and TNM stages(all P<0.05),it was not associated with age and gender(both P>0.05).5.Correlation analysis of the expression of miR-1250,AATK and methylation:1)There was a significant positive correlation between mi R-1250 and the mRNA expression of AATK in ESCC tissues(r=0.62,P<0.05).2)The mRNA expression of AATK was significantly higher in the ESCC tissues with positive protein expression than that in the ESCC tissues with negative protein expression(P<0.05).3)The expression of mi R-1250 in the ESCC tissues with methylation was significantly lower than that in ESCC tissues without methylation(P<0.05).4)The mRNA expression of AATK in the ESCC tissues with methylation was significantly lower than that in ESCC tissues without methylation(P<0.05).5)The protein expression of AATK in the ESCC tissues with methylation was significantly lower than that in ESCC tissues without methylation(P<0.05).Part two Effect of miR-1250 on the biological characteristics of esophageal cancer cellsObjectives: To detect the effect of miR-1250 over-expression on proliferation,migration,and invasion ability of esophageal cancer cell lines.Methods:1 Mimics of miR-1250 was transfected in Eca109 cells.Transfection efficiency was evaluated by qRT-PCR.2 MTS and clone formation experiment were respectively applied to detect the proliferation ability of esophageal cancer cell lines with miR-1250over-expression.3 Scratch test was applied to detect the migration ability of esophageal cancer cell lines with miR-1250 over-expression.4 Transwell chamber invasion assay was applied to detect the invasion ability of esophageal cancer cell lines with miR-1250 over-expression.Result:1.Transfection of miR-1250 after over-expression in Eca109 cells:successful over-expression of mi R-1250 in Eca109 cells.qRT-PCR analysis revealed that expression level of miR-1250 was increased significantly in transfected Eca109 cells than that in negative control cells(P<0.05).2.The effect of mi R-1250 over-expression on cell proliferation abilities of esophageal cancer cell lines: MTS and clone formation experiment demonstrated that the over-expression of miR-1250 gene could inhibit the proliferation ability of Eca109 cells in vitro(P<0.05).3.The effect of mi R-1250 gene over-expression on cell migration abilities of esophageal cancer cell lines: Scratch test demonstrated that the over-expression of miR-1250 gene could inhibit the migration ability of Eca109 cells in vitro(P<0.05).4.The effect of miR-1250 gene over-expression on cell invasion abilities of esophageal cancer cell lines: Transwell chamber invasion assaydemonstrated that the over-expression of miR-1250 gene could inhibit the invasion ability of Eca109 cells in vitro(P<0.05).Conclusion:1.The expression of miR-1250 and its host gene AATK in esophageal squamous cell carcinoma were consistent,and their decreased expression is closely related to the pathological differentiation and TNM stage in patients with esophageal squamous cell carcinoma,suggesting that AATK may be the host gene of miR-1250 and both of them may act as tumor suppressors in esophageal squamous cell carcinoma.Their inactivation may be related to the invasion and metastasis of esophageal squamous cell carcinoma.2.Aberrant hypermethylation in the promoter region may be one of the important mechanisms of AATK and mi R-1250 silencing in esophageal squamous cell carcinoma.3.Over-expression of mi R-1250 inhibited the proliferation,migration and invasion of esophageal cancer cells in vitro,further suggesting that miR-1250 is associated with the invasion and metastasis of esophageal squamous cell carcinoma.