摘要
目的:探讨RNAi沉默N-cadherin表达对人食管鳞状细胞癌EC9706细胞体内生物学行为的影响。方法:通过逆转录病毒介导的RNAi技术将N-cadherin基因的RNA干扰载体pMSCVneo/N-cadherin质粒及对照质粒pEGFP-MSCVneo质粒分别转染至人食管癌细胞系EC9706,运用G418进行抗性克隆的筛选和扩增,进而得到稳定转染的人食管癌细胞系EC9706(分别命名为干扰载体组和空载体组),将正常对照组、空载体组和干扰载体组EC9706细胞通过皮下注射的方式接种于裸鼠背部肩胛区,比较各组裸鼠成瘤期、成瘤大小、瘤体终重量及终体积等情况。运用免疫组织化学方法和Western blotting方法检测3组裸鼠移植瘤组织中E-cadherin、N-cad-herin和MMP-9蛋白的表达情况。TUNEL法检测3组裸鼠移植瘤组织中细胞的凋亡水平。结果:(1)与正常对照组和空载体组相比较,干扰载体组裸鼠移植瘤的生长速率、成瘤大小、瘤体终重量和终体积明显降低(P<0.05)。(2)与正常对照组和空载体组相比,干扰载体组裸鼠移植瘤组织中N-cadherin和MMP-9的蛋白表达均下调(P<0.05),而E-cadherin的蛋白表达则无明显变化(P>0.05)。(3)与正常对照组(51.55±4.68)和空载体组(54.17±5.26)相比,干扰载体组(106.81±6.47)裸鼠移植瘤组织中凋亡的阳性细胞数明显增加(P<0.05)。结论:RNAi沉默N-cadherin表达可明显抑制EC9706细胞裸鼠体内移植瘤的生长,其作用机制可能是通过下调MMP-9的表达,降低细胞对细胞外基质的降解能力,进而降低EC9706细胞的体内侵袭力。RNAi沉默N-cadher-in表达具有体内抑制食管鳞状细胞癌细胞生长的作用。N-cadherin可望成为抗食管鳞状细胞癌治疗的潜在分子靶点。
AIM： To explore the effect of N - cadherin knock - down on the biological behavior of EC9706 ceils in vivo. METHODS： The control vector pEGFP - MSCVneo and recombinant retroviral vector pMSCVneo/N - cadherin plasmids were transfected into esophageal squamous cell carcinoma（ESCC） cell line EC9706 according to the manufacturer＇s instructions. Stable EC9706 cell clones were selected using selection medium containing G418. Untreated EC9706 cells, control vector - transfected EC9706 cells and N - cadherin RNAi - transfected EC9706 cells were inoculated subcutaneously into the right flank of BALB/c mice （5 for each group）, respectively. When tumors became palpable, the diameters of the tumors were measured with a caliper each week after subcutaneous implantation, and the volume （ nun3 ） and weight （g） of the tumors were also calculated. Immunohistochemistry and Western blotting were employed to examine the expression levels of E - cadberin, N - cadherin and MMP - 9 in the tumor tissues. The cell apoptosis was analyzed by TUNEL method. RESULTS ： Compared with untreated group and control vector group, there was an obvious decrease in the volumes and weights of the tumors in N - cadherin RNAi group （P 〈 0. 05 ）. No difference of E - cadherin expression in the 3 groups was observed. However, the expression of N - cadherin and MMP - 9 in N - cadherin RNAi group was apparently reduced, and the positive number of cell apoptosis was obviously increased in N -cadherin RNAi group （106. 81 ± 6. 47） as compared with that in untreated group （51.55 ±4. 68） and control vector group （54. 17.±5.26）. CONCLUSION ： N - cadherin knock - down inhibits the tumor formation of EC9706 cells in nude mice by decreasing MMP - 9 expression, resulting in less degradation of ECM and less aggression of the cancer cells. N - eadherin is an important factor in the progression and metastasis of ESCC, and may serve as a potential molecular target for biotherapy of ESCC.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2011年第8期1537-1543,共7页
Chinese Journal of Pathophysiology
基金
河南省科技厅科技攻关资助项目(No.072102310054)
