胃癌中转录因子与miRNA共调控网络的构建及let-7i与其靶基因在胃癌侵袭转移中的作用

发布时间：2019-06-21 13:40

【摘要】：胃癌是世界范围内常见的恶性肿瘤,其病死率仅次于肺癌位居恶性肿瘤第二,严重威胁了人们的生命。迄今为止,有70%以上的胃癌病例发生在发展中国家,在中国,胃癌的病死率占居各种恶性肿瘤中的首位。尽管目前早期胃癌的治疗已经取得了很大的进步,大大提升了患者的生存率,但对于处于晚期阶段的胃癌病例来说,其五年生存率仍然很低。这不仅仅由于早期胃癌的诊断率较低,还由于肿瘤细胞强大的增殖、侵袭、迁移能力。因此,进一步探究胃癌细胞浸润生长及胃癌发生转移的分子机制,筛选出胃癌治疗中的关键干预靶点,对于胃癌的预防和治疗都具有十分重要的意义。转录因子(Transcriptionfactor)是一类在转录过程中起到决定性作用的调控原件,通过特异性结合在基因特定序列上从而调控基因的转录。有很多转录因子在胃癌的发生、发展和转移过程中起到重要作用。microRNA(miRNA)是一类单链非编码小分子RNA,能够通过与目标mRNA结合而抑制其翻译或导致其降解。转录因子和miRNA各自参与调控了基因的转录水平以及转录后水平。与此同时,转录因子和miRNA之间也存在着共同调控的关系。它们之间的相互合作与相互调控形成了一个复杂的调控网络。目前,以转录因子和miRNA为核心建立调控网络已经成为一种分析生物调控机制的有效手段,这种方法为研究疾病发生的机理提供了重要的线索。miRNA的异常表达可以调控和影响细胞中基因的异常表达,继而促进肿瘤的发生。在第一部分研究中,应用80对Affymetrix外显子芯片筛选出胃癌组织中的差异基因表达谱,得到在胃癌组织中一共有2540个差异表达基因(foldchange1.5),其中715个显著差异表达基因(foldchange2),这些基因大多数在癌组织中为表达上调,只有约五分之一为表达下调。同时应用5对miRNA芯片筛选出胃癌中差异表达的miRNA,共得到93个显著差异表达的miRNA,其中27个呈现表达上调,66个呈现表达下调。对2540个差异表达基因做出详尽系统的基因功能分析和信号通路分析,而这些分析都为今后更加系统地研究胃癌相关基因以及这些差异基因如何参与胃癌的发生、参与了哪些关键的信号通路等奠定了坚实的基础。通过整理芯片数据并整合TRED、Targetscan等数据库中预测出的转录因子和miRNA靶基因的信息,建立了胃癌中转录因子和miRNA共调控网络,并对调控网中络的基因进行分析和注释。结果揭示了这些基因分别隶属于“细胞外基质-受体相互作用”和“焦点粘连”这两个信号通路,提示这些基因与肿瘤的浸润生长关系密切,可能是参与胃癌侵袭和转移过程的关键基因。通过对调控网中的关键基因进行节点分析,发现COL1A1和NCAM1这两个基因受到最多的调控关系,因此选取这两个基因进行着重研究。通过qRT-PCR和westernblot的实验手段分别在mRNA和蛋白水平上验证了COL1A1的过表达和NCAM1的低表达,与芯片和调控网的预测结果相符。从TF-miRNA共调控网络中可见,COL1A1受到了let-7i的直接调控作用。为了研究和证实let-7i在癌症发生中所发挥的生物学功能,通过qPCR和westernblot检测到胃癌组织及胃癌细胞中let-7i的显著低表达,且胃癌组织中let-7i的表达与患者肿瘤浸润和转移程度都具有一定相关性。选取胃癌细胞株SGC-7901和MGC-803进行后续试验,通过在细胞中转染let-7imimic,人为升高let-7i在细胞中的表达水平后,发现这两种胃癌细胞的增殖能力、迁移能力及侵袭能力都发生了显著降低;同时,通过建立裸鼠体内移植瘤模型进一步证实了过表达let-7i能够有效抑制肿瘤生长。为了验证let-7i是通过调控COL1A1的表达参与胃癌的发生发展,在胃癌细胞SGC-7901和MGC-803中转染let-7imimic后,检测到COL1A1的mRNA以及蛋白水平都发生了显著下调;通过荧光素酶活性实验进一步证实了let-7i通过靶向调节COL1A1的3’UTR区域参与调控COL1A1的表达。此外,通过在胃癌细胞内转染siRNA-COL1A1人为敲减COL1A1的表达之后,两种胃癌细胞的增殖、迁移及侵袭能力都发生了显著降低,这与人为提高细胞内let-7i表达后对胃癌细胞的作用结果一致。以上结果全部说明COL1A1就是let-7i的直接靶基因。综上所述,通过建立胃癌中TF-miRNA共调控网络,预测了let-7i与COL1A1的调控关系,并通过实验验证let-7i能够减弱胃癌细胞的增殖能力、抑制细胞的迁移和侵袭能力,并在体内抑制肿瘤的生长;减少COL1A1的表达能够抑制胃癌细胞的增殖能力并削减胃癌细胞迁移与侵袭能力。Let-7i是通过上调COL1A1的表达水平促进胃癌的发展进程。提示let-7i可能作为胃癌诊断、治疗的潜在靶点。
[Abstract]:Gastric cancer is a common malignant tumor in the world. The mortality rate after lung cancer is the second of the malignant tumor, which is a serious threat to people's life. So far, more than 70% of the cases of gastric cancer have occurred in developing countries. In China, the mortality of gastric cancer is the first in all kinds of malignant tumors. Although much progress has been made in the treatment of early gastric cancer, the survival rate of patients is greatly improved, but for gastric cancer cases in the advanced stage, the five-year survival rate is still low. This is not only due to the low diagnostic rate of early gastric cancer, but also due to the strong proliferation, invasion and migration ability of the tumor cells. Therefore, it is very important for the prevention and treatment of gastric cancer to explore the molecular mechanism of gastric cancer cell infiltration and metastasis, and to screen out the key intervention target in the treatment of gastric cancer. Transcriptional factor is a kind of regulatory original which plays a decisive role in the transcription process and regulates the transcription of the gene by specifically binding to the specific sequence of the gene. Many transcription factors play an important role in the development, development and transfer of gastric cancer. MicroRNA (miRNA) is a single-chain non-coding small-molecule RNA capable of inhibiting its translation or leading to its degradation by binding to the target mRNA. The transcription factor and the miRNA are each involved in regulating the transcription level of the gene and the post-transcriptional level. At the same time, there is a common regulation between transcription factor and miRNA. The mutual cooperation and mutual control between them form a complex control network. At present, the establishment of the control network with the transcription factor and the miRNA as the core has become an effective means of analyzing the biological regulation mechanism, which provides an important clue for studying the mechanism of the disease. The abnormal expression of the miRNA can regulate and influence the abnormal expression of the gene in the cell, and then promote the occurrence of the tumor. In the first part of the study, the differential gene expression profile in the gastric cancer tissue was screened by using 80 pairs of the Affymetrix exon chip, and there were 2540 differentially expressed genes (folchange1.5) in the gastric cancer tissues, of which 715 significant difference expression genes (folchange2), Most of these genes are up-regulated in cancer tissues and only about one-fifth of these genes are down-regulated. At the same time, the miRNA of the differentially expressed miRNA in the gastric cancer was screened by the 5-pair miRNA chip, and a total of 93 miRNAs with significant difference expression were obtained, of which 27 of the miRNAs were up-regulated and 66 of the expression levels were down-regulated. The gene function analysis and signal pathway analysis of 2540 differentially expressed genes were carried out, and these analyses provide a more systematic study of gastric cancer-related genes and how these differential genes take part in the occurrence of gastric cancer in the future. The key signal paths and so on provide a solid foundation. By arranging the information of the transcription factors and the miRNA target genes predicted in the database such as TRED, Targetscan, and the like, the transcription factor and the miRNA co-regulation network in the gastric cancer are established, and the gene in the regulating net is analyzed and annotated. The results show that these genes are subordinate to the two signal pathways of "Extracellular matrix-receptor interaction" and "focal adhesion", suggesting that these genes are closely related to the invasion and growth of the tumor, and may be the key gene involved in the invasion and metastasis of the gastric cancer. Through the node analysis of the key genes in the control network, the two genes of COL1A1 and NCAM1 were found to be controlled by the most, so the two genes were selected to focus on the study. The overexpression of COL1A1 and the low expression of NCAM1 were verified by qRT-PCR and western blot. From the TF-miRNA co-regulation network, COL1A1 is directly regulated by let-7i. In order to study and confirm the biological function of let-7i in the occurrence of cancer, the expression of let-7i in gastric cancer and gastric cancer cells was detected by qPCR and western blot. The human gastric cancer cell line SGC-7901 and the MGC-803 were selected for subsequent experiments. After the expression level of the let-7imic was transfected into the cells, the proliferation ability, the migration ability and the invasion ability of the two kinds of gastric cancer cells were significantly reduced, and meanwhile, The model of transplanted tumor in nude mice further confirmed that the overexpressed let-7i can effectively inhibit the growth of the tumor. In order to verify that let-7i was involved in the development of gastric cancer by regulating the expression of COL1A1, the expression of COL1A1 and the level of protein were significantly reduced after the expression of let-7imic in gastric cancer cells SGC-7901 and MGC-803. The luciferase activity experiment further confirmed that let-7i was involved in the regulation of the expression of COL1A1 by targeting the 3 'UTR region of COL1A1. In addition, after the siRNA-COL1A1 was transfected into the gastric cancer cells, the proliferation, migration and invasion ability of the two kinds of gastric cancer cells were significantly reduced, which was consistent with the effect of the human-made expression of the let-7i on the gastric cancer cells. All of the above results indicate that COL1A1 is the direct target gene of let-7i. To sum up, the regulation and control of the let-7i and COL1A1 were predicted by establishing the TF-miRNA co-regulation network in the gastric cancer, and the proliferation ability of the gastric cancer cells can be reduced by the experiment of the let-7i, the migration and the invasion ability of the cells can be inhibited, and the growth of the tumor is inhibited in the body; The reduction of the expression of COL1A1 can inhibit the proliferation of gastric cancer cells and reduce the migration and invasion ability of gastric cancer cells. Let-7i is a process of promoting the development of gastric cancer by up-regulating the level of expression of COL1A1. It is suggested that let-7i may be a potential target for the diagnosis and treatment of gastric cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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