组织因子及其剪切体在胃癌组织中表达的临床意义及作用机制研究

发布时间：2018-07-07 13:02

  本文选题：组织因子 + 剪切体　； 参考：《苏州大学》2016年博士论文

【摘要】：组织因子(tissue factor,TF)是一种分子量为47kDa的膜性糖蛋白,不仅作为凝血激活因子,在多种生物学过程尤其在肿瘤生长与转移中具有重要意义。TF表达为两种自然发生的蛋白亚型,膜结合的全长TF(flTF)和选择性剪切亚型组织因子(asTF)。实验和临床证据均表明,在肿瘤中有异常的TF表达,并促进肿瘤生长与转移。TF及其剪切亚型(剪切体)的表达和功能的调节受多种因素影响。肿瘤相关刺激信号可调节TF的表达。作为TF转录后调节机制,microRNA直接或间接控制TF的表达和功能。胃癌是最常见的恶性肿瘤之一。目前证实胃癌中有TF表达异常,并且其表达水平与肿瘤的多种生物学行为有关。但asTF在胃癌中的临床意义及调节机制尚不完全明了。本实验通过自行设计asTF的引物和探针应用qRT-PCR方法检测人胃癌组织中flTF、asTF mRNA的表达水平并分析与临床病理的相关性,应用Cox模型探讨其在患者生存时间等预后因素中的价值。体外研究了促炎因子TNF-α刺激胃癌MKN28细胞后flTF、asTF mRNA表达水平的变化。由于长链非编码RNA(lncRNAs)可以作为一种竞争性内源性RNA(ceRNA)与miRNA相互作用,参与靶基因的表达调控,并在肿瘤的发生发展中发挥重要的作用。我们通过构建慢病毒pLVX-H19 lncRNA-shRNA下调载体系统,TF过表达载体,qRT-PCR法检测基因表达水平,WesternBlot检测TF蛋白表达水平,CCK8法测定细胞生长活力,Transwell检测细胞侵袭能力,探讨lncRNA H19与miRNA19a(miR-19a)相互作用通路在胃癌细胞中对flTF、asTF基因表达的调控机制,为TF的转录后调节途径的靶向治疗提供理论依据。本课题结果提示:(1)meta分析结果表明,TF阳性表达与肿瘤区域淋巴结转移之间存在一定的相关性(P=0.001),单变量与多变量相关生存分析TF阳性表达与肿瘤生存、预后之间均存在明显的相关性(P值均0.0001);我们未发现TF阳性表达与肿瘤瘤体大小、生长之间有相关性;关于asTF在肿瘤中表达及临床意义的研究报道较少,检索到的文献原始数据不足,无法完成meta分析。(2)本研究通过自行设计的检测astf的引物和taqman探针将上游引物设计在第四、第六外显子的连接处,达到了避免同时扩增出fltf基因片段的目的,可高效、准确地检测fltf及astf的基因表达。(3)胃癌组织中fltfmrna相对定量[7.45(0.34~33.68)]显著高于正常胃组织[3.00(1.36~5.02)],胃癌组织中astfmrna相对定量[0.88(0.07~26.00)]显著高于正常胃组织[0.33(0.03~0.97)],差异均有统计学意义(p0.01)。fltf与astf基因表达水平与性别、年龄、tnm分期、肿瘤大小、组织学分型和化疗敏感性等相关因素的关系差异均无统计学意义(p0.05)。log-rank生存分析显示fltfmrna低表达组[33.83(25.24~42.43)]比高表达组[18.18(13.32~23.04)]平均术后生存时间显著延长15.66个月(χ2=6.185,p=0.013)。astfmrna低表达组[25.32(21.19~29.45)]比高表达组[20.50(12.51~28.48)]平均术后生存时间显著延长4.82个月(χ2=4.604,p=0.032)。fltfmrna与astfmrna都是低表达组与fltfmrna或astfmrna有一组高表达组比较,平均生存时间(月)及95%ci分别为28.96(24.90~33.00)、23.40(16.27~30.51),患者术后平均生存时间显著延长5.56个月(χ2=4.548,p=0.033)。fltfmrna与astfmrna同时低表达组与fltfmrna与astfmrna同时高表达组比较,平均生存时间(月)及95%ci分别为31.07(23.21~38.92)、11.13(6.90~15.36),患者术后平均生存时间显著延长19.94个月(χ2=13.005,p=0.000)。fltfmrna与astfmrna有一组高表达组较同时高表达组胃癌患者平均生存时间显著延长16.92个月(χ2=6.346,p=0.012)。多因素cox模型分析显示,fltf及astf均是胃癌独立的预后风险因素(hr=6.03,95%ci=1.02~35.71,p0.05)及(hr=10.74,95%ci=2.32~49.59,p0.01)。(4)体外实验发现tnf-α刺激下胃癌mkn28细胞的lncrnah19和fltf及astfmrna表达水平增高而mir-19a表达水平降低。mir-19a可与tf3’utr结合抑制其表达,mir-19a可抑制胃癌细胞的tf蛋白水平的表达,fltf/astf可促进胃癌细胞的增殖和侵袭能力,而mir-19a可抑制其促增殖和侵袭作用。下调lncrnah19可促进mir-19a表达水平增高,降低astf/fltf表达水平,抑制胃癌细胞的增殖和侵袭能力。综上所述,我们自行设计的astf引物和taqman探针可较准确地检测astf的基因表达。研究发现astfmrna的表达与fltfmrna一样,在胃癌组织中高表达,且与临床病理无相关性,但与预后密切相关,其表达水平与胃癌患者术后生存时间负相关,联合fltf的基因表达水平预测患者生存时间更有意义。证实tnf-α可体外促进胃癌MKN28细胞TF表达增加,且受lncRNA H19与miR-19a通路的转录后调控,参与促进胃癌的发生与发展。asTF与flTF以及lncRNA H19可作为胃癌预后判断的重要生物学指标。本研究不仅发现了胃癌发生发展过程中TF相关的lncRNA H19与mi R-19a的转录后调节通路,而且为asTF与fl TF以及lncRNA H19在胃癌的靶向免疫治疗提供了实验基础,具有一定的临床应用价值。
[Abstract]:Tissue factor (TF) is a membranous glycoprotein with a molecular weight of 47kDa, not only as a coagulation activator, but also in a variety of biological processes, especially in tumor growth and metastasis,.TF is expressed as two naturally occurring protein subtypes, full long TF (flTF) and selective shear subtype tissue factor (asTF) in membrane binding. Both the clinical and clinical evidence show that abnormal TF expression in the tumor and the regulation of the expression and function of tumor growth and metastasis.TF and its shear subtype (shear body) are influenced by many factors. The tumor related stimulus signals can regulate the expression of TF. As a post transcriptional regulation mechanism of TF, microRNA directly or indirectly controls the expression and function of TF. Gastric cancer is one of the most common malignant tumors. It is confirmed that the expression of TF is abnormal in gastric cancer and its expression level is related to a variety of biological behavior of the tumor. But the clinical significance and regulation mechanism of asTF in gastric cancer are not completely clear. This experiment was used to detect human gastric cancer tissue by using qRT-PCR method by self designed asTF primers and probes. The expression level of flTF, asTF mRNA and the correlation with clinicopathological analysis and the value of the Cox model in the prognosis factors such as the patient's survival time. In vitro, the changes of flTF, asTF mRNA expression level after stimulating TNF- alpha in gastric cancer cells were studied. Long chain non coded RNA (lncRNAs) could be a competitive factor. Endogenous RNA (ceRNA) interacts with miRNA, participates in the regulation of target gene expression, and plays an important role in the development of tumor. We constructed the lentivirus pLVX-H19 lncRNA-shRNA down regulation system, TF overexpression vector, qRT-PCR method to detect the gene expression level, WesternBlot to detect the expression level of TF protein, CCK8 method for the determination of fine. Cell growth activity, Transwell detection of cell invasiveness, and explore the regulatory mechanism of lncRNA H19 and miRNA19a (miR-19a) interaction pathway in the expression of flTF and asTF in gastric cancer cells, providing a theoretical basis for the targeting therapy of TF's post transcriptional regulation pathway. The results of this study show that (1) meta analysis results show that TF positive expression and tumor area There was a certain correlation between regional lymph node metastasis (P=0.001). There was a significant correlation between the positive expression of TF and the survival of the tumor and the prognosis (P value 0.0001). We did not find the correlation between the positive expression of TF and the tumor size and growth, and the expression and clinical meaning of asTF in the tumor. The original data of the retrieved literature are insufficient and the meta analysis can not be completed. (2) this study designed the primers of the ASTF and the TaqMan probe to design the upstream primers at the junction of fourth and sixth exons, thus achieving the purpose of avoiding the simultaneous amplification of the fltf gene fragment, which can detect fltf efficiently and accurately. The gene expression of ASTF. (3) fltfmrna relative quantitative [7.45 (0.34~33.68) in gastric carcinoma was significantly higher than that of normal gastric tissue [3.00 (1.36~5.02)], astfmrna relative quantitative [0.88 (0.07~26.00) in gastric cancer tissues was significantly higher than normal gastric tissue [0.33 (0.03~0.97)], and the difference was statistically significant (P0.01) and sex, age and age. There was no significant difference in the relationship between TNM staging, tumor size, histological type and chemosensitivity (P0.05).Log-rank survival analysis showed that [33.83 (25.24~42.43) in fltfmrna low expression group ([18.18 (13.32~23.04)) was significantly longer than the high expression group [18.18 (13.32~23.04)] for 15.66 months (x 2=6.185, p=0.013).Astfmrna low table The average postoperative survival time of the group [25.32 (21.19~29.45) was significantly longer than the high expression group [20.50 (12.51~28.48)] (x 2=4.604, p=0.032).Fltfmrna and astfmrna were both low expression group and fltfmrna or astfmrna group with a group of high expression groups, the average survival time (month) and 95%ci were 28.96 (24.90~33.00), 23.40, patients, respectively. The average survival time was significantly longer after 5.56 months (x 2=4.548, p=0.033).Fltfmrna and astfmrna, compared with fltfmrna and astfmrna high expression groups, the average survival time (month) and 95%ci were 31.07 (23.21~38.92) and 11.13 (6.90~15.36), and the average survival time of the patients was significantly longer after 19.94 months (x 2=13.005, p=0.000). The average survival time of gastric cancer patients with high expression group of.Fltfmrna and astfmrna was significantly prolonged by 16.92 months (x 2=6.346, p=0.012). The multivariate Cox model analysis showed that fltf and ASTF were both independent prognostic risk factors of gastric cancer (hr=6.03,95%ci=1.02~35.71, P0.05) and (hr=10.74,95%ci=2.32~49.59, P0.01). (4) in vitro reality Tnf- alpha stimulated the expression of lncrnah19, fltf and astfmrna in gastric cancer MKN28 cells, while mir-19a expression level decreased.Mir-19a can inhibit the expression of.Mir-19a with TF3 'UTR. Mir-19a can inhibit the expression of TF protein level in gastric cancer cells. Fltf/astf can promote the proliferation and invasion of gastric cancer cells. Proliferation and invasion. Down regulation of lncrnah19 can increase the level of mir-19a expression, reduce the expression level of astf/fltf, inhibit the proliferation and invasion of gastric cancer cells. In summary, our self designed ASTF primers and TaqMan probes can accurately detect the gene expression of ASTF. The study found that the expression of astfmrna is like fltfmrna in gastric cancer. The high expression in the tissue is not related to the clinicopathological correlation, but it is closely related to the prognosis. The expression level is negatively related to the survival time of the patients with gastric cancer. It is more meaningful to predict the survival time of the patients with fltf gene expression. It is confirmed that tnf- alpha can promote the expression of TF in gastric cancer MKN28 cells in vitro, and be transferred to the lncRNA H19 and miR-19a pathway. .asTF and flTF, as well as lncRNA H19, are important biological indicators for predicting the prognosis of gastric cancer. This study not only found the post transcriptional regulation pathway of TF related lncRNA H19 and MI R-19a in the development of gastric cancer, but also for asTF and FL TF, as well as the targeted immunization of TF in gastric cancer. The treatment provides experimental basis and has certain clinical application value.
【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】：R735.2
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本文编号：2105044