【摘要】：第一部分mi R-124在胃癌中表達(dá)降低胃癌在全世界屬常見(jiàn)的惡性腫瘤之一,在我國(guó)發(fā)病率及死亡率均居惡性腫瘤的前列。但胃癌發(fā)病的分子機(jī)制至今尚未完全明了,新近大量腫瘤表觀遺傳學(xué)研究資料表明,在人類(lèi)多種腫瘤的發(fā)生發(fā)展過(guò)程中存在表觀遺傳學(xué)的改變。表觀遺傳調(diào)控機(jī)制包括:DNA甲基化、組蛋白修飾、染色質(zhì)重構(gòu)及非編碼RNA等。非編碼RNA是近年來(lái)的腫瘤研究新熱點(diǎn)之一,mi RNA參與了胃癌的發(fā)生發(fā)展及轉(zhuǎn)移等過(guò)程。研究表明mi R-124在人類(lèi)多種腫瘤中發(fā)揮著抑瘤基因的作用,其在胃癌中的表達(dá)及意義尚不清楚。目的:檢測(cè)分析mi R-124在胃癌細(xì)胞及組織中的表達(dá)。方法:本研究采用q RT-PCR,檢測(cè)了mi R-124在永生化胃粘膜上皮細(xì)胞GES1及胃癌細(xì)胞系MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS中的表達(dá);采用原位雜交,檢測(cè)了mi R-124在胃癌及癌旁正常組織中的表達(dá),分析mi R-124表達(dá)與胃癌患者臨床病理意義及預(yù)后的關(guān)系。結(jié)果:mi R-124在MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS細(xì)胞中的表達(dá),均低于GES-1細(xì)胞;mi R-124在胃癌組織表達(dá)降低或缺失,而在癌旁胃粘膜中的表達(dá)呈強(qiáng)陽(yáng)性,mi R-124表達(dá)與胃癌病理組織學(xué)分級(jí)、TNM分期及淋巴結(jié)轉(zhuǎn)移密切相關(guān),而與患者的年齡、性別及腫瘤大小無(wú)關(guān),mi R-124低表達(dá)與患者預(yù)后及生存時(shí)間有關(guān)。結(jié)論:mi R-124在胃癌組織及細(xì)胞系中均存在mi R-124的表達(dá)下降,其表達(dá)與胃癌分級(jí)、分期及淋巴結(jié)轉(zhuǎn)移及患者預(yù)后相關(guān)。第二部分mi R-124調(diào)控EZH2表達(dá)對(duì)胃癌細(xì)胞系增殖及藥物增敏性mi RNA在腫瘤細(xì)胞的生長(zhǎng)增殖、周期調(diào)控、凋亡、侵襲轉(zhuǎn)移、血管形成、上皮間質(zhì)樣變及干細(xì)胞特性維持等過(guò)程發(fā)揮重要作用;mi R-124具有抑制腫瘤生長(zhǎng)、增殖及化療藥物增敏作用。目的:探討初步mi R-124在胃癌中的作用及分子機(jī)制。方法:本研究采用MTT法,檢測(cè)mi R-124的轉(zhuǎn)入,對(duì)胃癌細(xì)胞系生長(zhǎng)增殖及5-Fu藥物增敏作用的影響;采用軟瓊脂克隆形成實(shí)驗(yàn),檢測(cè)mi R-124轉(zhuǎn)入MKN-45、AGS細(xì)胞系,對(duì)MKN-45、AGS細(xì)胞克隆形成能力的影響;施用裸鼠成瘤實(shí)驗(yàn),探討mi R-124對(duì)裸鼠荷瘤生長(zhǎng)增殖的影響,體內(nèi)實(shí)驗(yàn)驗(yàn)證mi R-124在胃癌細(xì)胞的作用。通過(guò)在線軟件預(yù)測(cè)分析mi R-124的靶基因,EZH2基因可能是mi R-124所調(diào)控的靶基因,運(yùn)用熒光素酶報(bào)告分析、q RT-PCR及Western blot等方法,實(shí)驗(yàn)證實(shí)EZH2基因是mi R-124所調(diào)控的靶基因;采用免疫組織化學(xué)及原位雜交方法,分析mi R-124與EZH2在胃癌組織中表達(dá)、相關(guān)性及臨床病理意義;MTT法,檢測(cè)EZH2對(duì)AGS細(xì)胞系增殖影響,分析mi R-124增強(qiáng)AGS細(xì)胞對(duì)5-Fu的敏感性。結(jié)果:轉(zhuǎn)染mi R-124 mimics后,GES-1、MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS細(xì)胞的存活率分別為:91.6±9.3%、68.4±5.7%、54.7±3.5%、64.2±6.1%、38.9±3.7%、43.6±5.2%及63.1±4.8%;與GES-1比,MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS細(xì)胞的存活率均顯著降低。轉(zhuǎn)染mi R-124 mimics后,MKN-45細(xì)胞克隆形成率(22.3±2.1%)與AGS細(xì)胞克隆形成率(59.1±4.7%)均較對(duì)照組降低。裸鼠成瘤實(shí)驗(yàn)表明,mi R-124 mimics治療組移植瘤大小及重量明顯小于對(duì)照組。mi R-124可增加5-Fu對(duì)AGS細(xì)胞的抑制作用。mi R-124可以與EZH2 3’UTR結(jié)合,并在胃癌細(xì)胞中抑制其表達(dá),并且EZH2在胃癌組織中的表達(dá)與mi R-124呈負(fù)相關(guān)。EZH2的表達(dá)與胃癌患者性別、年齡、腫瘤大小、TNM分期及淋巴結(jié)轉(zhuǎn)移無(wú)關(guān),但是與腫瘤分化程度密切相關(guān)。EZH2 si RNA轉(zhuǎn)染AGS細(xì)胞后24 h的增殖率與對(duì)照組無(wú)顯著差異,而48 h及72 h的細(xì)胞增殖率明顯低于對(duì)照組。mi R-124 inhibitor與si-control組的細(xì)胞增殖率大于inhibitor control與si-control組,EZH2 si RNA與inhibitor control組細(xì)胞增殖率低于inhibitor control與si-control組,而mi R-124 inhibitor與EZH2 si RNA組的細(xì)胞增殖率較對(duì)照組無(wú)明顯變化。結(jié)論:mi R-124可體外抑制胃癌細(xì)胞增殖,體內(nèi)抑制成瘤,并增加胃癌細(xì)胞對(duì)5-Fu的藥物敏感性,mi R-124的直接靶基因EZH2在胃癌組織中表達(dá)與腫瘤分化程度相關(guān),參與了mi R-124對(duì)胃癌細(xì)胞5-Fu敏感性的調(diào)控。
[Abstract]:The first part of the expression of MI R-124 in gastric cancer is one of the most common malignant tumors in the world. The incidence and mortality of the cancer are both in the forefront of malignant tumors in China. However, the molecular mechanism of gastric cancer has not been fully understood. Epigenetic changes exist in the process. Epigenetic regulation mechanisms include DNA methylation, histone modification, chromatin remodeling and non coding RNA. Non coded RNA is one of the new hot spots in cancer research in recent years. Mi RNA participates in the development and metastasis of gastric cancer. The study shows that MI R-124 plays an inhibitory role in a variety of human tumors. The expression and significance of the tumor gene in gastric cancer is not clear. Objective: to detect and analyze the expression of MI R-124 in gastric cancer cells and tissues. Methods: This study used Q RT-PCR to detect the expression of MI R-124 in the GES1 of immortalized gastric mucosal epithelial cells and the expression of MKN-74 in gastric cancer cell line, MKN-28, MKN-45, MGC-803, SGC-7901, and the expression. The expression of MI R-124 in gastric and adjacent normal tissues was detected, and the relationship between the expression of MI R-124 and the clinicopathological significance and prognosis of gastric cancer patients was analyzed. Results: the expression of MI R-124 in MKN-74, MKN-28, MKN-45, MGC-803, SGC-7901 and AGS cells were lower than those of the gastric carcinoma. The expression in paralgastric mucosa was strongly positive. The expression of MI R-124 was closely related to the histopathological classification of gastric cancer, TNM staging and lymph node metastasis, but not related to the age, sex and tumor size of the patients. The low expression of MI R-124 was related to the prognosis and survival time of the patients. Conclusion: the expression of MI R-124 in gastric cancer tissues and cell lines all have the expression of MI R-124. The expression was related to the classification, staging, lymph node metastasis and prognosis of gastric cancer. Second mi R-124 regulation of EZH2 expression on the proliferation of gastric cancer cell lines and drug sensitization mi RNA in the growth and proliferation of tumor cells, cycle regulation, apoptosis, invasion and metastasis, angiogenesis, epithelial mesenchymal change and stem cell characteristics maintenance, and so on. Mi R-124 has the effect of inhibiting tumor growth, proliferation and chemosensitivity. Objective: To explore the role and molecular mechanism of primary mi R-124 in gastric cancer. Methods: This study used MTT method to detect the effect of MI R-124 on the growth and proliferation of gastric cancer cell lines and the effect of 5-Fu drug sensitization, and the test of soft agar clone formation test. Mi R-124 was transferred into MKN-45, AGS cell line, and the effect on the cloning ability of MKN-45 and AGS cells. The effect of MI R-124 on the growth and proliferation of nude mice was investigated by the application of MI R-124 to the tumor growth and proliferation in nude mice. In vivo experiments were conducted to verify the effect of MI R-124 in gastric cancer cells. The target gene, using the method of luciferase report analysis, Q RT-PCR and Western blot, confirmed that the EZH2 gene was the target gene regulated by Mi R-124, and the expression, correlation and clinical significance of MI R-124 and EZH2 in gastric cancer tissues were analyzed by immunohistochemistry and in situ hybridization, and MTT method was used to detect the effect of EZH2 on the proliferation of cell lines. Results: Mi R-124 enhanced the sensitivity of AGS cells to 5-Fu. Results: after transfection of MI R-124 mimics, the survival rates of GES-1, MKN-74, MKN-28, MKN-45, MGC-803, 54.7 +, 54.7 + 3.5%, 64.2 + 6.1%, 38.9 + 3.7%, 43.6 + 5.2% and 63.1 + cells The survival rate decreased significantly. After transfection of MI R-124 mimics, the clone formation rate of MKN-45 cells (22.3 + 2.1%) and AGS cell clone formation rate (59.1 + 4.7%) were lower than those of the control group. The tumor size and weight of the MI R-124 mimics treatment group showed that the size and weight of the transplanted tumor in the MI R-124 mimics group were significantly smaller than that of the group.Mi R-124, which could increase the inhibitory effect of 5-Fu on AGS cells. R-124 can be combined with EZH2 3 'UTR and inhibits its expression in gastric cancer cells, and the expression of EZH2 in gastric cancer tissue is negatively correlated with MI R-124 and is not related to sex, age, tumor size, TNM staging and lymph node metastasis of gastric cancer patients, but it is closely related to the degree of tumor differentiation in.EZH2 Si RNA transfection of AGS cells after 24. The proliferation rate was not significantly different from that in the control group, but the proliferation rate of 48 h and 72 h cells was significantly lower than that of the control group.Mi R-124 inhibitor and Si-Control group, and the proliferation rate of the cells was higher than that of the inhibitor control and Si-Control group. The cell proliferation rate of the 2 Si RNA group has no obvious change compared with the control group. Conclusion: Mi R-124 can inhibit the proliferation of gastric cancer cells in vitro, inhibit the tumor in vivo, and increase the drug sensitivity of gastric cancer cells to 5-Fu. The expression of MI R-124's direct target gene EZH2 in gastric cancer tissue is related to the degree of tumor differentiation, and participates in MI R-124 for the 5-Fu sensitivity of gastric cancer cells. Regulation and control.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.2

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相關(guān)期刊論文 前1條

1 Wei-Yuan Wei;Lin-Hai Yan;Xiao-Tong Wang;Lei Li;Wen-Long Cao;Xiao-Shi Zhang;Ze-Xu Zhan;Han Yu;Yu-Bo Xie;Qiang Xiao;;E2F-1 overexpression inhibits human gastric cancer MGC-803 cell growth in vivo[J];World Journal of Gastroenterology;2015年02期

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本文編號(hào)：2123912