胃癌中轉(zhuǎn)錄因子與miRNA共調(diào)控網(wǎng)絡(luò)的構(gòu)建及l(fā)et-7i與其靶基因在胃癌侵襲轉(zhuǎn)移中的作用
發(fā)布時(shí)間：2019-06-21 13:40
【摘要】：胃癌是世界范圍內(nèi)常見(jiàn)的惡性腫瘤,其病死率僅次于肺癌位居惡性腫瘤第二,嚴(yán)重威脅了人們的生命。迄今為止,有70%以上的胃癌病例發(fā)生在發(fā)展中國(guó)家,在中國(guó),胃癌的病死率占居各種惡性腫瘤中的首位。盡管目前早期胃癌的治療已經(jīng)取得了很大的進(jìn)步,大大提升了患者的生存率,但對(duì)于處于晚期階段的胃癌病例來(lái)說(shuō),其五年生存率仍然很低。這不僅僅由于早期胃癌的診斷率較低,還由于腫瘤細(xì)胞強(qiáng)大的增殖、侵襲、遷移能力。因此,進(jìn)一步探究胃癌細(xì)胞浸潤(rùn)生長(zhǎng)及胃癌發(fā)生轉(zhuǎn)移的分子機(jī)制,篩選出胃癌治療中的關(guān)鍵干預(yù)靶點(diǎn),對(duì)于胃癌的預(yù)防和治療都具有十分重要的意義。轉(zhuǎn)錄因子(Transcriptionfactor)是一類在轉(zhuǎn)錄過(guò)程中起到?jīng)Q定性作用的調(diào)控原件,通過(guò)特異性結(jié)合在基因特定序列上從而調(diào)控基因的轉(zhuǎn)錄。有很多轉(zhuǎn)錄因子在胃癌的發(fā)生、發(fā)展和轉(zhuǎn)移過(guò)程中起到重要作用。microRNA(miRNA)是一類單鏈非編碼小分子RNA,能夠通過(guò)與目標(biāo)mRNA結(jié)合而抑制其翻譯或?qū)е缕浣到�。轉(zhuǎn)錄因子和miRNA各自參與調(diào)控了基因的轉(zhuǎn)錄水平以及轉(zhuǎn)錄后水平。與此同時(shí),轉(zhuǎn)錄因子和miRNA之間也存在著共同調(diào)控的關(guān)系。它們之間的相互合作與相互調(diào)控形成了一個(gè)復(fù)雜的調(diào)控網(wǎng)絡(luò)。目前,以轉(zhuǎn)錄因子和miRNA為核心建立調(diào)控網(wǎng)絡(luò)已經(jīng)成為一種分析生物調(diào)控機(jī)制的有效手段,這種方法為研究疾病發(fā)生的機(jī)理提供了重要的線索。miRNA的異常表達(dá)可以調(diào)控和影響細(xì)胞中基因的異常表達(dá),繼而促進(jìn)腫瘤的發(fā)生。在第一部分研究中,應(yīng)用80對(duì)Affymetrix外顯子芯片篩選出胃癌組織中的差異基因表達(dá)譜,得到在胃癌組織中一共有2540個(gè)差異表達(dá)基因(foldchange1.5),其中715個(gè)顯著差異表達(dá)基因(foldchange2),這些基因大多數(shù)在癌組織中為表達(dá)上調(diào),只有約五分之一為表達(dá)下調(diào)。同時(shí)應(yīng)用5對(duì)miRNA芯片篩選出胃癌中差異表達(dá)的miRNA,共得到93個(gè)顯著差異表達(dá)的miRNA,其中27個(gè)呈現(xiàn)表達(dá)上調(diào),66個(gè)呈現(xiàn)表達(dá)下調(diào)。對(duì)2540個(gè)差異表達(dá)基因做出詳盡系統(tǒng)的基因功能分析和信號(hào)通路分析,而這些分析都為今后更加系統(tǒng)地研究胃癌相關(guān)基因以及這些差異基因如何參與胃癌的發(fā)生、參與了哪些關(guān)鍵的信號(hào)通路等奠定了堅(jiān)實(shí)的基礎(chǔ)。通過(guò)整理芯片數(shù)據(jù)并整合TRED、Targetscan等數(shù)據(jù)庫(kù)中預(yù)測(cè)出的轉(zhuǎn)錄因子和miRNA靶基因的信息,建立了胃癌中轉(zhuǎn)錄因子和miRNA共調(diào)控網(wǎng)絡(luò),并對(duì)調(diào)控網(wǎng)中絡(luò)的基因進(jìn)行分析和注釋。結(jié)果揭示了這些基因分別隸屬于“細(xì)胞外基質(zhì)-受體相互作用”和“焦點(diǎn)粘連”這兩個(gè)信號(hào)通路,提示這些基因與腫瘤的浸潤(rùn)生長(zhǎng)關(guān)系密切,可能是參與胃癌侵襲和轉(zhuǎn)移過(guò)程的關(guān)鍵基因。通過(guò)對(duì)調(diào)控網(wǎng)中的關(guān)鍵基因進(jìn)行節(jié)點(diǎn)分析,發(fā)現(xiàn)COL1A1和NCAM1這兩個(gè)基因受到最多的調(diào)控關(guān)系,因此選取這兩個(gè)基因進(jìn)行著重研究。通過(guò)qRT-PCR和westernblot的實(shí)驗(yàn)手段分別在mRNA和蛋白水平上驗(yàn)證了COL1A1的過(guò)表達(dá)和NCAM1的低表達(dá),與芯片和調(diào)控網(wǎng)的預(yù)測(cè)結(jié)果相符。從TF-miRNA共調(diào)控網(wǎng)絡(luò)中可見(jiàn),COL1A1受到了let-7i的直接調(diào)控作用。為了研究和證實(shí)let-7i在癌癥發(fā)生中所發(fā)揮的生物學(xué)功能,通過(guò)qPCR和westernblot檢測(cè)到胃癌組織及胃癌細(xì)胞中l(wèi)et-7i的顯著低表達(dá),且胃癌組織中l(wèi)et-7i的表達(dá)與患者腫瘤浸潤(rùn)和轉(zhuǎn)移程度都具有一定相關(guān)性。選取胃癌細(xì)胞株SGC-7901和MGC-803進(jìn)行后續(xù)試驗(yàn),通過(guò)在細(xì)胞中轉(zhuǎn)染let-7imimic,人為升高let-7i在細(xì)胞中的表達(dá)水平后,發(fā)現(xiàn)這兩種胃癌細(xì)胞的增殖能力、遷移能力及侵襲能力都發(fā)生了顯著降低;同時(shí),通過(guò)建立裸鼠體內(nèi)移植瘤模型進(jìn)一步證實(shí)了過(guò)表達(dá)let-7i能夠有效抑制腫瘤生長(zhǎng)。為了驗(yàn)證let-7i是通過(guò)調(diào)控COL1A1的表達(dá)參與胃癌的發(fā)生發(fā)展,在胃癌細(xì)胞SGC-7901和MGC-803中轉(zhuǎn)染let-7imimic后,檢測(cè)到COL1A1的mRNA以及蛋白水平都發(fā)生了顯著下調(diào);通過(guò)熒光素酶活性實(shí)驗(yàn)進(jìn)一步證實(shí)了let-7i通過(guò)靶向調(diào)節(jié)COL1A1的3’UTR區(qū)域參與調(diào)控COL1A1的表達(dá)。此外,通過(guò)在胃癌細(xì)胞內(nèi)轉(zhuǎn)染siRNA-COL1A1人為敲減COL1A1的表達(dá)之后,兩種胃癌細(xì)胞的增殖、遷移及侵襲能力都發(fā)生了顯著降低,這與人為提高細(xì)胞內(nèi)let-7i表達(dá)后對(duì)胃癌細(xì)胞的作用結(jié)果一致。以上結(jié)果全部說(shuō)明COL1A1就是let-7i的直接靶基因。綜上所述,通過(guò)建立胃癌中TF-miRNA共調(diào)控網(wǎng)絡(luò),預(yù)測(cè)了let-7i與COL1A1的調(diào)控關(guān)系,并通過(guò)實(shí)驗(yàn)驗(yàn)證let-7i能夠減弱胃癌細(xì)胞的增殖能力、抑制細(xì)胞的遷移和侵襲能力,并在體內(nèi)抑制腫瘤的生長(zhǎng);減少COL1A1的表達(dá)能夠抑制胃癌細(xì)胞的增殖能力并削減胃癌細(xì)胞遷移與侵襲能力。Let-7i是通過(guò)上調(diào)COL1A1的表達(dá)水平促進(jìn)胃癌的發(fā)展進(jìn)程。提示let-7i可能作為胃癌診斷、治療的潛在靶點(diǎn)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2
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3 石s，
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