Clinical Studies On Interactions Among Apoptosis-related Genes And Biological Characteristics In Esophageal Squamous Cell Cancer

Objective:1)To investigate the expression of Transmembrane protein-166 mRNA (TMEM166) and the rate of early apoptosis in esophageal squamous cell carcinoma (ESCC) and remote esophageal tissues, explore the relationships between TMEM166 and the rate of early apoptosis in different locations in ESCC.2) To inspect the expression in transcription and translation of Programmed Cell Death 5 (PDCD5) i n different locations, explore the correlations between PDCD5 and biological characteristics in ESCC.3)To study the expression of TMEM166,PDCD5,Tip60,MDM2mRNA and p53 protein in different locations in ESCC, analyze the correlations between the genes and biological characteristics, the interactions among TMEM166, PDCD5,Tip60,MDM2 and p53 genes in different locations in ESCC.Methods:1)Samples collection:85 fresh samples with ESCC were selected which were not undergoing any anti-tumor therapy before operation in May,2008 to April,2009 in Tumor Hospital affiliated Xin-jiang Medical University.2) Paried by gender, nationality, age, clinical staging (According to 2009 7th UICC ESCC Staging), there were 12 samples in stagingⅠ,Ⅱ,Ⅲrespectively (total 36 samples). RT-PCR, Flow cytometry, Immunohistochemistry were used to detect the mRNA expression of TMEM166, the rate of early apoptosis, the protein expression of TMEM166 in different location (cancer and remote cancer tissues) in ESCC samples and the results were compared to demonstrat the relationships between TMEM166 and the rate of early apoptosis in ESCC; 3) RT-PCR, Western-bloting were also used to detect the expression of PDCD5mRNA and protein in different location (cancer and remote cancer tissues) in ESCC tissues. The data were analysed to biological characteristics of ESCC to explore the correlations between PDCD5mRNA and PDCD5 protein, the associativity between PDCD5 and biological characteristics in ESCC;4)Finally, the interactions was anslysed using the data of the expressin of TMEM166, PDCD5,Tip60, MDM2mRNA and p53 protein detected by RT-PCR and Immunohistochemistry respectively in different location in 85 fresh samples; 5) Statistical analysis:The data was analyzed by SPSS11.0 software. In data of quantity, ANOVA, SNK (Student-Newman-Keuls) test, Chi-squared test, Pearson Correlation Analysis was used respectively. In data of quality, t-test, R×C exact probability, Spearman Correlation Analysis was used respectively. The test significant levelα=0.05.Results:1)Semiquantitative analysis, expression of TMEM166 mRNA in ESCC tissues was lower than that in the remote esophageal tissues (1.1214±0.2262 vs 1.4645±0.3396;P=0.014). The expression of TMEM166mRNA was related to clinical staging in ESCC, the higher clinical staging in ESCC, the lower expression of TMEM166 mRNA (rs=-0.357, P=0.033);2) There was positive correlation between the rate of early apoptosis and the expression of TMEM166mRNA in ESCC (r=0.310, P=0.036). The rate of early apoptosis was negatively related to clinical staging (rs=-0.584, P<0.001);3)The phenomenon was observed that the location of TMEM166 protein has changed in ESCC compared to the esophageal epithelium mucosae; 4) The expression of PDCD5 protein was lower in ESCC tissues by Western-bloting than that in the remote esophageal tissues (0.4387±0.1632 vs 0.5659±0.2272,P=0.008).There was a down-expression tend of PDCD5 followed the increase of clinical staging both in ESCC and remote esophageal tissues (r,=-0.678,P<0.001 and rs=-0.619, P<0.001). The expression of PDCD5mRNA was significantly correlated to the expression of PDCD5 protein (r=0.781,0.715;P<0.001) both in ESCC and remote esophageal tissues. There were no correlated banlance between the expression of PDCD5mRNA and invasive deepth, lymphe node staging, cell differentiation.5) The expression of TMEM166mRNA (1.1003±0.2342), PDCD5mRNA (0.6333±0.2086) were inhabited in ESCC by comparison to the remote esophageal tissues(1.4269±0.3363,0.7352±0.1963 and P< 0.001),but MDM2mRNA,p53 protein were overexpressed in ESCC (P<0.001);6) There was no statistical significance of the expression of Tip60mRNA between ESCC and remote esophageal tissues (0.7359±0.2489 vs 0.7204±0.1512, P=0.012); 7) In ESCC tissues, there was negative correlation to the expression of PDCD5mRNA and invasive deeth (rs=-0.220, P=0.043) but positive correlation to the expression of p53 protein and Tip60mRNA (rs=0.443,0.523;P<0.001).The lymph node staging were negatively correlated to the expression of TMEM-166mRNA and PDCD5mRNA (rs=-0.593, P=0.008 vs rs=-0.416, P=0.013) and positively correlated to the expression of Tip60 and MDM2mRNA (rs=0.273, P=0.038 vs rs=0.182, P=0.009). G classification of tumor cells was negatively correlated to TMEM-166mRNA (rs=-0.423,P=0.029), but positively correlated to MDM2mRNA(rs=-0.442, P=0.027) and p53 protein (rs=0.299, P=0.045).8) In ESCC tissues, there was positive correlation to the expression of TMEM166mRNA and PDCD5mRNA (rs=0.516, P<0.001) and was negative correlation to the expression of Tip60mRNA,MDM2mRNA and p53 protein (rs=-0.236,-0.453,-0.426 and P=0.015, <0.001,<0.001).There were negative correlation to the expression of PDCD5mRNA and Tip60mRNA, MDM2mRNA, p53 protein (rs=-0.417,-0.631,-0.589;P<0.001).The expression of Tip60mRNA was positively correlated to MDM2 mRNA, p53 protein (rs=0.388,0.321 and P<0.001,0.001).There was positive correlation to the expression of MDM2 mRNA and p53 protein (rs=0.440, P<0.001).9) In remote esophageal tissues, there was positive correlation between the expression of TMEM166 mRNA and PDCD5 mRNA (rs=0.305, P=0.002). The expression of PDCD5mRNA was negatively correlated to MDM2mRNA and p53 protein (rs=-0.315,-0.210 and P=0.001,0.031)and there was positive correlation between the expression of MDM2mRNA and p53 protein (rs=0.221, P=0.023).Conclusion:1) The rate of early apoptosis in ESCC was significantly higher in ESCC than that in the remote esophageal tissues. The expression of TMEM166 was inhabited in ESCC.The subcellular localization of TMEM166 has changed in ESCC by comparision to the remote esophageal epithelium mucosae and it's function has been inhibited clearly; 2) It was significantly related to PDCD5 and ocurrance of ESCC.The expression of PDCD5mRNA was significantly correlated to the expression of PDCD5 protein; 3)There was no statistical significance of the expression of Tip60mRNA between ESCC and remote esophageal tissues; 4) In ESCC tissues, the expression of TMEM166, PDCD5m RNA were downregulated, but the expression of MDM2mRNA and p53 protein were upregulated. All of TMEM166, PDCD5, Tip60, MDM2, p53 were correlated to the biological characteristics in ESCC; 5) As malignant transformation occuered in cells in esophagus mucosa,the role of PDCD5 saved as co-active factors to increase the acitivity of Tip60 was declined. The acitivy of Tip60 in ESCC might be duble-regulated by apoptosis-related gene PDCD5,TMEM166 and oncogene MDM2.