食管鳞状细胞癌中Beclin1表达及其对增殖的影响

Expression of Beclin1 in esophageal squamous cell carcinoma and effect on proliferation of TE1 cell

目的探讨Beclin1在食管鳞状细胞癌(esophagus squamous cell carcinoma,ESCC)中的表达及意义,分析其过表达对人ESCC细胞株TE1体外生长活性的影响。方法分别采用免疫组化SP法和RT-PCR技术检测57例ESCC及癌旁正常食管黏膜组织中Beclin1蛋白及mRNA的表达;利用基因转染、RT-PCR及Western blot法观察外源性Beclin1过表达对ESCC细胞株TE1的影响;MTT法分析外源性Beclin1过表达对TE1细胞增殖的影响;流式细胞仪(flow cytometry,FCM)检测转染后肿瘤细胞的细胞周期和凋亡;在荧光显微镜下观察转染后肿瘤细胞的自噬。结果免疫组化染色结果示,在ESCC中Beclin1蛋白的阳性率为29.82%(17/57),明显低于癌旁正常食管黏膜组织100%(57/57)(P<0.00),Beclin1蛋白的表达与ESCC患者的分化程度(χ2=6.158,P=0.046)、淋巴结转移(χ2=5.664,P=0.017)有关;与患者性别、年龄、肿瘤大小、浸润深度无关;RT-PCR检测结果示,在ESCC中Beclin1 mRNA的表达量(0.168±0.038)明显低于癌旁正常食管黏膜组织(0.280±0.052)(P<0.00)。pCMV6-Entry-Beclin1脂质体法转染TE1细胞后,在mRNA和蛋白水平Beclin1的表达均高于TE1PE组及TE1组。在TE1细胞中过表达Beclin1后可抑制肿瘤细胞的体外生长,抑制率为57.21%。FCM检测pCMV6-Entry-Beclin1转染后G1期细胞明显增多,S期细胞明显减少,细胞增殖受到抑制;凋亡率为5.91%,高于TE1PE组和TE1组(P<0.05)。在荧光显微镜下见TE1Beclin1组中MDC标记的自噬囊泡数量明显增加。结论自噬相关基因Beclin1 mRNA及蛋白在ESCC中表达下调,自噬活性的降低可能与ESCC的发生、发展及侵袭转移有关;Beclin1过表达可抑制人ESCC细胞株TE1的增殖,并诱导其自噬和凋亡,利用自噬基因Beclin1对ESCC基因治疗也许具有可行性。

Purpose To investigate the expression and significance of autophagy relate gene Beclinl in esophagus squamous cell carci- noma（ESCC） and to analyzes the effect of its over-expression on the growth of human esophagus carcinoma cell TEl in vitro. Methods Immunohistochemical staining and RT-PCR were used to detect the protein and mRNA expressions of Beclinl in 57 specimens ESCC and normal esophageal mucosa tissues. Gene over-expression, RT-PCR and Western blot were used to investigate the influence of exog- enous Beclinl on ESCC TEl cells. MTT assay was used to evaluate the effect of Beclinl over-expression on the proliferation and growth of the transfected cells. Cell cycle and apoptosis were measured by flow cytometry （FCM）. Autophagy phenomenon was observed under fluorescence microscope. Results The immunohistochemical staining results showed that the expression of Beclinl protein in ESCC were 29. 82% （ 17/57 ） , which was significantly lower than those in normal esophageal mucosa tissues （57/57） （P 〈 0. 00）. Beclinl expression was significantly correlated with differentiation （ X2 = 6. 158, P = 0. 046 ） and lymph node metastasis （ X2 = 5. 664, P = 0. 017 ） , but not significantly correlated with sex, age, tumor size and invasion depth of the ESCC. RT-PCR analyses showed that the expression of Beclinl mRNA in ESCC were 0. 168 ± 0. 038, which was significantly lower than those in normal esophageal mucosa tis- sues 0. 280 ± 0. 052 （P 〈 0. 00）. The expressions of Beclinl mRNA and protein in TEl cells after transfected with pCMV6-Entry-Bec- linl were significantly higher than those after transfected with blank plasmid （TEl PE group） and non- transfection group （TEl group）. MTF assay showed that the growth of TEl cells with pCMV6-Entry-Beclinl transfection was significantly inhibited compared with the TEl PE group and the TEl group, the suppression rate was 57. 21%. The results of FCM showed that after over-expression of Beclinl in TEl cells, the proportion of cells at Gl phase increased significantly, the proportion of cells at S stage decreased significantly, cell pro- liferation was inhibited, the apoptosis rate was 5.91% , which was significantly higher than those in TEl rE group and TEl group （P 〈 0. 05 ）. The number of autophagy vesicles labeled by MDC in TEl Beciiol group increased significantly under fluorescence microscope. Conclusion Autophagy gene Beclinl expression is down-regulated in ESCC, which may relate to tumorigenesis and development of ESCC. Beclinl over-expression can inhibit proliferation of ESCC TEl ceils, and induce autophagy and apoptosis of tumor cells. Gene therapy targeting to autophagy gene Beclinl may be feasible in treatment of ESCC.