H2-calponin在食管鳞癌中的表达及意义

目的: 探讨H2 钙调理蛋白(H2 calponin)在食管鳞癌中的表达及意义。方法: 免疫组化技术检测30例食管鳞癌患者癌组织及癌旁组织芯片中H2 calponin的表达水平；体外培养人食管鳞癌ECA109、TE 1细胞株和正常食管上皮Het 1a细胞；荧光定量PCR和蛋白质印迹法检测食管鳞癌细胞株中H2 calponin的表达；将食管鳞癌TE 1细胞分为空白对照组（常规培养）、阴性对照组（转染空载体siRNA）和H2 calponin siRNA组（转染H2 calponin siRNA），采用蛋白质印迹法测定细胞中增殖细胞核抗原(PCNA)、E 钙黏蛋白(E cadherin)，波形蛋白以及基质金属蛋白酶 2(MMP 2)、MMP 9、内皮生长因子 A(VEGF A)、血管内皮生长因子 C(VEGF C)蛋白的表达；ELISA法测定各组细胞培养上清液中MMP 2，MMP 9和VEGF C含量；荧光定量PCR检测NF κB通路抑制剂PDTC处理前（0 h）及处理后0.5, 1, 2, 4, 8, 12 h H2 calponin mRNA的表达；蛋白质印迹技术检测H2 calponin及NF κB通路蛋白的表达。结果： 免疫芯片结果显示，食管鳞癌组织中H2 calponin表达量低于癌旁组织， H2 calponin阳性表达率与食管鳞癌患者性别、年龄、肿瘤部位及病理分级之间无明显相关性(P均>0.05)；荧光定量PCR和蛋白质印迹结果显示，食管鳞癌细胞中H2 calponin表达明显低于正常食管细胞(P<0.01)；下调H2 calponin表达后，PCNA、E 钙黏蛋白和VEGF A表达无明显变化，而波形蛋白、MMP 2、MMP 9和VEGF C表达明显增加；ELISA结果显示，MMP 2、MMP 9和VEGF C分泌量明显增加(P<0.05)；PDTC处理后可显著增加IκBα的蛋白表达进而恢复并上调H2 calponin的表达(P<0.01)。 结论： NF κB通路通过抑制H2 calponin表达保证波形蛋白，MMP 2，MMP 9，VEGF C持续表达，促进食管鳞癌细胞侵袭、转移发生。

Expression of H2-calponin in esophageal squamous cell carcinoma and its clinical significance

Objective: To investigate the expression and significance of H2 calponin in esophageal squamous cell carcinoma(ESCC). Methods: The expression of H2 calponin in a tissue microarray consisted of 30 patients with ESCC and adjacent tumor tissues were detected by immunohistochemistry. The expression of H2 calponin in ESCC cell line (ECA109 and TE 1) and normal human esophageal epithelial cell(Het 1a) were detected by RT PCR and Western blotting. TE 1 cells were divided into blank control (routinely cultured), negative control (transfected with negative siRNA) and siRNA H2 calponin group (transfected with H2 calponin siRNA). The expression of PCNA, E cadherin, Vimentin, MMP 2, MMP 9, VEGF A and VEGF C in each group was detected by Western blotting. The secretion of MMP 2, MMP 9 and VEGF C were detected by ELISA. The expression changes of H2 calponin before(0 h) and after PDTC treatment (0.5, 1, 2, 4, 8, 12 h) were detected by RT PCR. Western blotting was used to determine the expresion of H2 calponin and NF κB pathway related protein. Results: H2 calponin were weakly expressed in ESCC tissues compared with adjacent normal tissues; expression of H2 calponin was not significantly correlated with gender, age, tumor location and pathological grade in esophageal squamous cell carcinoma(P>0.05). ESCC cell lines showed higher H2 calponin expression levels than that in Het 1a cells(P<0.01). There were no significant change of PCNA, E cadherin and VEGF A expression, while the expression of MMP 2, MMP 9 and VEGF C were up regulated after siRNA H2 calponin treatment(P<0.05). PDTC treatment could rapidly restored the expression of H2 calponin(P<0.01) by increasing IκBα expression. Conclusion: NF κB pathway promotes the invasion and metastasis of ESCC by restraining H2 calponin through enhancing continuous expression of Vimentin, MMP 2, MMP 9 and VEGF C.