凋亡相关基因及其交互作用与食管鳞癌生物学特性的临床研究
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摘要
目的：1)通过研究食管鳞癌不同组织部位膜转运蛋白-166(TMEM166)在转录水平的表达与食管鳞癌不同组织部位细胞早期凋亡率,探讨TMEM166与食管鳞癌不同组织部位细胞早期凋亡率的关系；2)通过研究食管鳞癌不同组织部位中程序化细胞死亡蛋白5(PDCD5)在转录和翻译水平的表达及其相关性,探讨PDCD5与食管鳞癌病理特征的关系；3)研究食管鳞癌不同组织部位中TMEM166、PDCD5、Tip60、MDM2mRNA与p53蛋白的表达,探讨TMEM166、PDCD5、Tip60、MDM2mRNA与p53蛋白与食管鳞癌病理特征的相关性及其在食管鳞癌不同部位组织中的交互作用。方法：1)标本收集：选用新疆医科大学附属肿瘤医院2008年5月～2009年4月手术切除的85例术前未进行抗肿瘤治疗的食管鳞状细胞癌新鲜手术标本,分别留取癌组织、癌旁组织、远癌组织标本；2)按照性别、民族、年龄、临床分期匹配(2009年第七版UICC国际食管癌分期),选择Ⅰ期、Ⅱ期、Ⅲ期各12例共36例食管鳞癌、远癌组织手术标本,应用流式细胞术、RT-PCR、免疫组化的方法,分别对食管鳞癌组织、远癌组织细胞的早期凋亡率、TMEM166mRNA、TMEM166蛋白表达进行检测；RT-PCR、Western-blot检测食管鳞癌组织不同部位PDCD5mRNA、PDCD5蛋白的表达,分析PDCD5mRNA、PDCD5蛋白的相关性及与食管鳞癌的病理特征的关系；3)85例食管鳞癌及远癌组织RT-PCR检测TMEM166、PDCD5、Tip60、MDM2mRNA表达,免疫组化检测p53蛋白表达,分析上述基因表达与食管鳞癌的病理学特点及上述基因间的交互作用；4)统计学方法：用SPSS13.0软件进行统计学分析。计量资料以均数±标准差(x±s)表示,均数比较采用方差分析(ANOVA)、SNK检验(Student-Newman-Keuls)、χ2检验、Pearson积差相关分析；分类资料采用t检验、R×C列联表确切概率法、Spearman等级相关分析。检验水准α=0.05。结果：1)食管鳞癌组织TMEM166 mRNA表达(1.1214±0.2262)低于远癌组织(1.4645±0.3396)(P=0.014)；TMEM166基因表达随病变分期增加呈下降趋势(rs=-0.357,P=0.033)；2)食管鳞癌组织细胞早期凋亡率(2.1563±0.4740)高于远癌组织(1.6313±0.3121)(P<0.001),食管鳞癌组织细胞早期凋亡率与食管鳞癌临床分期负相关(rs=-0.584,P<0.001),与TMEM166基因表达正相关(r=0.310,P=0.036)；3)与远癌组织相比,食管鳞癌组织中TMEM166蛋白亚细胞定位发生改变；4)食管鳞癌组织中PDCD5蛋白表达(0.4387±0.1632)低于远癌组织(0.5659±0.2272),随临床分期增加,其表达量有进一步下调的趋势(rs=-0.678,P<0.001；rs=-0.619,P<0.001)；PDCD5mRNA的表达与蛋白表达高度相关(r=0.715,0.781)；PDCD5mRNA及蛋白表达水平与食管鳞癌的肿瘤侵润深度、淋巴结分期、分化程度的相关性不均衡；5)食管鳞癌组织细胞中,TMEM166mRNA、PDCD5mRNA表达受抑制,MDM2mRNA、p53在食管鳞癌组织中存在过表达；6)Tip60在食管鳞癌、远癌组织的表达未发现统计学差异(0.7359±0.2489 vs 0.7204±0.1512,P=0.012)；7)食管鳞癌肿瘤侵润深度与PDCD5 mRNA负相关(rs=-0.220,P=0.043),与p53蛋白、Tip60mRNA正相关(rs=0.443,0.523；P<0.001)。淋巴结分期与TMEM166、PDCD5mRNA负相关(rs=-0.593,-0.416；P=0.008,0.013)：与Tip60、MDM2 mRNA正相关(rs=0.273,0.182；P=0.038,0.009)。肿瘤细胞G分级与TMEM166mRNA负相关(rs=-0.423,P=0.029)；与MDM2 mRNA、P53蛋白正相关(rs=0.442,0.299；P=0.027,0.045)；8)食管鳞癌组织中TMEM166 mRNA与PDCD5 mRNA正相关(rs=0.516,P<0.001),与Tip60 mRNA、MDM2 mRNA、p53蛋白负相关(rs=-0.236,-0.453,-0.426；P=0.015,<0.001,<0.001);PDCD5 mRNA与Tip60、MDM2mRNA及p53蛋白负相关(rs=-0.417,-0.631,-0.589；P<0.001)；Tip60mRNA与MDM2mRNA、p53蛋白正相关(rs=0.388,0.321；P<0.001,0.001)；MDM2 mRNA与p53蛋白正相关(rs=0.440,P<0.001)；9)食管鳞癌远癌组织中TMEM166 mRNA与PDCD5 mRNA正相关(rs=0.305,P=0.002)；PDCD5 mRNA与MDM2mRNA、p53蛋白负相关(rs=-0.315,-0.210；P=0.001,0.031)；MDM2 mRNA与p53蛋白正相关(rs=0.221,P=0.023)。结论：1)食管鳞癌组织细胞早期凋亡率增高,TMEM166 mRNA在食管鳞癌的表达受抑制；TMEM166亚细胞定位的改变可能导致TMEMmRNA表达量的下降和该基因功能的变化；2)PDCD5与食管鳞状细胞癌的发生有关,PDCD5mRNA的表达与蛋白表达高度相关；3)食管鳞癌组织细胞中,TMEM166、PDCD5mRNA表达受抑制,MDM2mRNA、p53蛋白存在过表达,与食管鳞癌生物学特点有关；4)Tip60mRNA在食管鳞癌不同组织部位的表达无差异；5)Tip60在食管鳞癌细胞中活性可能与促凋亡基因PDCD5、TMEM166及癌基因MDM2的双重调节有关。
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.
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.
引文
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