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目的低氧诱导因子-1α(hypoxia inducible factor-1alpha,HIF-1α)在鼻咽癌组织中存在高表达,与淋巴结转移和临床分期偏晚密切相关,是鼻咽癌患者独立的不良预后因子。前期研究显示,沉默HIF-1α可有效降低鼻咽癌CNE-1细胞的黏附和侵袭能力。本研究通过探讨沉默HIF-1α对鼻咽癌CNE-1细胞移植瘤生长和转移能力的影响,进一步为临床提供参考和实验依据。方法采用RNA干扰(RNA interference,RNAi)技术沉默鼻咽癌CNE-1细胞中HIF-1α表达,RT-PCR检测其沉默效果;15只雄性裸鼠随机分为未转染组、阴性对照组和RNAi组,每组5只。将CNE-1细胞分别接种于裸鼠右大腿外侧皮下,建立鼻咽癌CNE-1细胞裸鼠移植瘤模型,观察各组成瘤时间并记录其生长情况,待分别形成肉眼可见的肿瘤后再培养20d。收集移植瘤组织及裸鼠两侧腹股沟淋巴结。测量移植瘤大小并计算其体积。采用HE染色,计算各组淋巴结转移率及阳性率;蛋白质印迹法检测3组移植瘤中HIF-1α、E-钙粘蛋白(E-cadherin)及CXCR4蛋白的表达。结果 RNAi可有效沉默鼻咽癌CNE-1细胞中HIF-1α表达;成功构建了裸鼠移植瘤模型;未转染组、阴性对照组和RNAi组裸鼠成瘤时间分别为(8.3±1.2)、(7.8±1.5)和(15.6±2.1)d,差异有统计学意义,F=21.181,P=0.002;最终成瘤体积分别为(1184.4±145.8)、(1254.0±130.5)和(322.7±118.2)mm3,F=82.081,P<0.001。淋巴结转移率及阳性率均明显降低;与未转染组及阴性对照组比较,HIF-1α(P<0.001)和CXCR4(P=0.001)蛋白在RNAi组中表达明显降低,而E-cadherin则明显增强,P=0.003。结论 RNAi沉默HIF-1α可有效抑制鼻咽癌CNE-1细胞移植瘤的生长和转移,其机制可能与上调E-cadherin、下调CXCR4蛋白表达有关。
Objective Hypoxia inducible factor-1alpha (HIF-1alpha) is highly expressed in nasopharyngeal carcinoma and is closely related to lymph node metastasis and late clinical stage, which is an independent adverse prognostic factor in patients with nasopharyngeal carcinoma. Previous studies have shown that silencing HIF-1α can effectively reduce the CNE-1 cell adhesion and invasion ability. In this study, the effect of silencing HIF-1α on the growth and metastasis of nasopharyngeal carcinoma CNE-1 cell xenografts was further explored to provide reference and experimental evidence for clinical practice. Methods RNA interference (RNAi) was used to silence the expression of HIF-1α in nasopharyngeal carcinoma CNE-1 cells. RT-PCR was used to detect the silencing effect. 15 male nude mice were randomly divided into untransfected group, RNAi group, 5 in each group. CNE-1 cells were inoculated subcutaneously in the lateral of the right thigh of nude mice respectively to establish a model of CNE-1 cell xenotransplanted in nude mice. The tumorigenic time of each group was observed and the growth of CNE-1 cells was observed. 20d. Transplanted tumor tissues and inguinal lymph nodes on both sides of nude mice were collected. The size of the xenograft was measured and its volume calculated. The expression of HIF-1α, E-cadherin and CXCR4 protein in three groups of transplanted tumors were detected by Western blotting. The HE staining was used to calculate the lymph node metastasis rate and positive rate in each group. Results RNAi could effectively silence the expression of HIF-1α in nasopharyngeal carcinoma CNE-1 cells. The nude mouse xenograft model was successfully constructed. The time of tumorigenesis in untransfected, negative control and RNAi nude mice was (8.3 ± 1.2) , (7.8 ± 1.5) and (15.6 ± 2.1) d respectively, the difference was statistically significant (F = 21.181, P = 0.002); the final tumor volume was (1184.4 ± 145.8), (1254.0 ± 130.5) and ) mm3, F = 82.081, P <0.001. The expression of HIF-1α (P <0.001) and CXCR4 (P = 0.001) in RNAi group was significantly lower than that in untransfected group and negative control group, while the expression of E-cadherin Significantly increased, P = 0.003. Conclusion Silencing of HIF-1α by RNAi can effectively inhibit the growth and metastasis of CNE-1 cell xenografts in nasopharyngeal carcinoma. The mechanism may be related to up-regulating E-cadherin and down-regulating the expression of CXCR4.