ZFAS1在结直肠癌中的表达及其促进肿瘤转移的研究

发布时间：2019-02-16 00:22

【摘要】：第一部分ZFAS1在结直肠癌组织中的表达及其临床意义目的:检测ZFAS1在结直肠癌组织中的表达水平,并分析其与临床病理参数及预后的关系。方法:应用q RT-PCR方法检测159对结直肠癌组织及癌旁组织的ZFAS1表达量,比较两者ZFAS1表达量的差异,同时检测了12例结直肠癌肝转移患者原发肿瘤及转移灶组织ZFAS1的表达。卡方检验分析ZFAS1表达水平与临床病理参数的相关性;采用Kaplan-Meier方法绘制生存曲线,Log-Rank方法比较ZFAS1高表达组和低表达组之间的生存率;采用Cox比例风险模型进行多因素分析,以确定校正多因素作用后ZFAS1对预后的影响。结果:159例结直肠癌组织及其配对正常组织ZFAS1的表达检测结果显示,ZFAS1在结直肠癌中表达显著上调(P0.0001),其中有76例患者的ZFAS1表达在结直肠癌组织中较正常上调2倍以上;且肝转移组织中ZFAS1的表达量明显高于原发部位(P0.05)。结直肠癌组织中ZFAS1的表达与淋巴结转移(P=0.045)及TNM分期(P=0.003)呈正相关。与低表达ZFAS1的患者相比,ZFAS1高表达的患者表现出更差的预后,其无瘤生存时间(P=0.012)及总生存时间均更短(P=0.003)。多因素分析显示,ZFAS1是结直肠癌患者无瘤生存时间(HR=2.132,95%CI=1.015-4.546,P=0.048)与总生存时间(HR=1.878,95%CI=1.006-3.529,P=0.0492)的独立预后因素。结论:ZFAS1在结直肠癌组织中高表达,并与淋巴结转移及高的TNM分期相关。ZFAS1是结直肠癌患者肿瘤复发和死亡的独立预后因素。第二部分ZFAS1对结直肠癌细胞迁移与侵袭的影响目的:研究ZFAS1对结直肠癌细胞增殖、迁移与侵袭的影响。方法:检测结直肠癌细胞株中ZFAS1的表达,选择两株表达量较高的细胞系,应用si RNA技术干扰ZFAS1的表达;采用CCK-8方法研究ZFAS1基因沉默后结直肠癌细胞增殖能力的变化;应用细胞划痕实验与Transwell侵袭小室方法研究ZFAS1基因沉默对结直肠癌细胞迁移和侵袭能力的影响;采用分子克隆技术构建ZFAS1sh RNA慢病毒表达载体,包装病毒后感染结直肠癌细胞,构建ZFAS1稳定敲降细胞株;采用裸鼠尾静脉注射肺转移实验,研究ZFAS1基因沉默对结直肠癌细胞体内转移能力的影响。结果:ZFAS1基因沉默对结直肠癌细胞增殖没有明显影响;沉默ZFAS1能够抑制结肠癌细胞的体外迁移与侵袭能力及裸鼠肺转移。结论:ZFAS1能够抑制结直肠癌细胞的迁移、侵袭和转移。第三部分ZFAS1影响结直肠癌转移机制的初步研究目的:初步研究ZFAS1影响结直肠癌细胞转移的作用机制。方法:应用q RT-PCR法及Western blot法检测在ZFAS1稳定敲降的结直肠癌细胞中EMT相关基因的表达;应用si RNA技术干扰ZEB1的表达,验证ZEB1与E-钙粘素(E-cad)及波形蛋白(VIM)的关系;双荧光素酶实验验证ZEB1是否是mi R-150-5p的靶基因;应用细胞划痕实验与Transwell侵袭小室方法研究mi R-150-5p与ZEB1对结直肠癌细胞迁移和侵袭能力的影响;通过荧光素酶实验、细胞划痕实验及Transwell侵袭小室方法研究ZFAS1与mi R-150-5p的关系。结果:ZFAS1敲降后,结直肠癌细胞中上皮细胞标志E-cad表达显著上升,而间质细胞标志VIM和ZEB1表达显著下降;ZEB1能够调节E-cad及VIM的表达;mi R-150-5p通过靶向作用于ZEB1抑制结直肠癌细胞的迁移与侵袭;ZFAS1与mi R-150-5p结合,封闭了mi R-150-5p的抑癌作用。结论:ZFAS1与mi R-150-5p结合,封闭了mi R-150-5p对其靶基因ZEB1的抑制作用,引起ZEB1表达上调,加速EMT的进程,从而促进肿瘤的转移。
[Abstract]:The expression of the first part of the ZFAS1 in the colorectal cancer tissue and its clinical significance are to detect the expression level of the ZFAS1 in the colorectal cancer tissue and to analyze its relationship with the clinicopathological parameters and the prognosis. Methods: The expression of ZFAS1 in the tissues of colorectal cancer and the paracancerous tissues was detected by q-RT-PCR. The difference of the expression of ZFAS1 was compared, and the expression of ZFAS1 in the primary and metastatic lesions of 12 patients with colorectal cancer was detected. The relationship between the expression level of the ZFAS1 and the clinical pathological parameters was analyzed by the card-side test, the survival curve was drawn by the Kaplan-Meier method, the survival rate between the high-expression group of the ZFAS1 and the low-expression group was compared by the Log-Rank method, and the multi-factor analysis was carried out using the Cox proportional risk model. To determine the effect of the ZFAS1 on the prognosis after multi-factor correction. Results: The expression of ZFAS1 in 159 patients with colorectal cancer and its paired normal tissues showed that the expression of ZFAS1 in colorectal cancer was up-regulated (P <0001), of which 76 patients had more than 2-fold higher expression of ZF1in colorectal cancer. The expression of ZFAS1 in the liver metastasis was significantly higher than that of the primary site (P0.05). The expression of ZFAS1 in colorectal cancer was positively correlated with lymph node metastasis (P = 0.045) and TNM stage (P = 0. 003). Compared with patients with low expression of ZFAS1, the patients with high expression of ZFAS1 showed a worse prognosis with no tumor survival time (P = 0.012) and shorter overall survival time (P = 0.003). The multi-factor analysis showed that ZFAS1 was an independent prognostic factor for non-tumor survival time in patients with colorectal cancer (HR = 2.132, 95% CI = 1.015-4.546, P = 0.048) and total survival time (HR = 1.878, 95% CI = 1.006-3.529, P = 0.0492). Conclusion: ZFAS1 is highly expressed in colorectal cancer and is associated with lymph node metastasis and high TNM stage. ZFAS1 is an independent prognostic factor for tumor recurrence and death in patients with colorectal cancer. The effect of the second part of ZFAS1 on the migration and invasion of colorectal cancer cells is to study the effects of ZFAS1 on the proliferation, migration and invasion of colorectal cancer cells. Methods: The expression of ZFAS1 in colorectal cancer cell line was detected, two cell lines with higher expression were selected, and the expression of ZFAS1 was disturbed by the technique of si RNA. The effect of ZF1gene silencing on the cell migration and invasion ability of colorectal cancer cells was studied by using the cell scratch test and the Transwell invasion cell method. The lentiviral expression vector of the ZFAS1sh RNA was constructed by the molecular cloning technique. The effect of ZFAS1 gene silencing on the in vivo transfer of colorectal cancer cells was studied by using the experiment of lung transfer in the tail vein of nude mice. Results: The silencing of ZFAS1 gene has no significant effect on the proliferation of colorectal cancer cells, and the silencing of ZFAS1 can inhibit the in vitro migration and invasion ability of colon cancer cells and the lung metastasis of nude mice. Conclusion: ZFAS1 can inhibit the migration, invasion and metastasis of colorectal cancer cells. The third part of the study on the effect of ZFAS1 on the transfer mechanism of colorectal cancer is to study the mechanism of the effect of ZFAS1 on the transfer of colorectal cancer cells. Methods: The expression of EMT-related gene in colorectal cancer cells stabilized by ZFAS1 was detected by the method of q-RT-PCR and Western blot. The expression of ZEB1 was disturbed by the technique of si-RNA, and the relationship between ZEB1 and E-cadherin (E-cad) and VIM was verified. The results showed that ZEB1 is the target gene of mi R-150-5p, and the effect of mi R-150-5p and ZEB1 on the cell migration and invasion ability of colorectal cancer is studied by using cell scratch test and Transwell's invasion cell method. The relationship between ZFAS1 and mi R-150-5p was studied by cell scratch test and Transwell's invasion cell method. Results: The expression of E-cad in the cells of colorectal cancer was significantly increased after the knock-down of the ZFAS1, and the expression of VIM and ZEB1 in the interstitial cell markers decreased significantly; the expression of E-cad and VIM was regulated by ZEB1; and the expression of mi R-150-5p in the inhibition of colorectal cancer cells was inhibited by targeting the ZEB1; and the ZFAS1 was combined with mi R-150-5p. The anti-cancer effect of mi R-150-5p was closed. Conclusion: The binding of ZFl to mi R-150-5p and the inhibition of the expression of ZEB1 by mi R-150-5p, and the up-regulation of the expression of ZEB1, accelerate the process of EMT and promote the metastasis of the tumor.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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