胃癌中miR-124表达的意义及作用机制研究

发布时间：2018-07-15 11:28

【摘要】：第一部分mi R-124在胃癌中表达降低胃癌在全世界属常见的恶性肿瘤之一,在我国发病率及死亡率均居恶性肿瘤的前列。但胃癌发病的分子机制至今尚未完全明了,新近大量肿瘤表观遗传学研究资料表明,在人类多种肿瘤的发生发展过程中存在表观遗传学的改变。表观遗传调控机制包括:DNA甲基化、组蛋白修饰、染色质重构及非编码RNA等。非编码RNA是近年来的肿瘤研究新热点之一,mi RNA参与了胃癌的发生发展及转移等过程。研究表明mi R-124在人类多种肿瘤中发挥着抑瘤基因的作用,其在胃癌中的表达及意义尚不清楚。目的:检测分析mi R-124在胃癌细胞及组织中的表达。方法:本研究采用q RT-PCR,检测了mi R-124在永生化胃粘膜上皮细胞GES1及胃癌细胞系MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS中的表达;采用原位杂交,检测了mi R-124在胃癌及癌旁正常组织中的表达,分析mi R-124表达与胃癌患者临床病理意义及预后的关系。结果:mi R-124在MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS细胞中的表达,均低于GES-1细胞;mi R-124在胃癌组织表达降低或缺失,而在癌旁胃粘膜中的表达呈强阳性,mi R-124表达与胃癌病理组织学分级、TNM分期及淋巴结转移密切相关,而与患者的年龄、性别及肿瘤大小无关,mi R-124低表达与患者预后及生存时间有关。结论:mi R-124在胃癌组织及细胞系中均存在mi R-124的表达下降,其表达与胃癌分级、分期及淋巴结转移及患者预后相关。第二部分mi R-124调控EZH2表达对胃癌细胞系增殖及药物增敏性mi RNA在肿瘤细胞的生长增殖、周期调控、凋亡、侵袭转移、血管形成、上皮间质样变及干细胞特性维持等过程发挥重要作用;mi R-124具有抑制肿瘤生长、增殖及化疗药物增敏作用。目的:探讨初步mi R-124在胃癌中的作用及分子机制。方法:本研究采用MTT法,检测mi R-124的转入,对胃癌细胞系生长增殖及5-Fu药物增敏作用的影响;采用软琼脂克隆形成实验,检测mi R-124转入MKN-45、AGS细胞系,对MKN-45、AGS细胞克隆形成能力的影响;施用裸鼠成瘤实验,探讨mi R-124对裸鼠荷瘤生长增殖的影响,体内实验验证mi R-124在胃癌细胞的作用。通过在线软件预测分析mi R-124的靶基因,EZH2基因可能是mi R-124所调控的靶基因,运用荧光素酶报告分析、q RT-PCR及Western blot等方法,实验证实EZH2基因是mi R-124所调控的靶基因;采用免疫组织化学及原位杂交方法,分析mi R-124与EZH2在胃癌组织中表达、相关性及临床病理意义;MTT法,检测EZH2对AGS细胞系增殖影响,分析mi R-124增强AGS细胞对5-Fu的敏感性。结果:转染mi R-124 mimics后,GES-1、MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS细胞的存活率分别为: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%;与GES-1比,MKN-74、MKN-28、MKN-45、MGC-803、SGC-7901及AGS细胞的存活率均显著降低。转染mi R-124 mimics后,MKN-45细胞克隆形成率(22.3±2.1%)与AGS细胞克隆形成率(59.1±4.7%)均较对照组降低。裸鼠成瘤实验表明,mi R-124 mimics治疗组移植瘤大小及重量明显小于对照组。mi R-124可增加5-Fu对AGS细胞的抑制作用。mi R-124可以与EZH2 3’UTR结合,并在胃癌细胞中抑制其表达,并且EZH2在胃癌组织中的表达与mi R-124呈负相关。EZH2的表达与胃癌患者性别、年龄、肿瘤大小、TNM分期及淋巴结转移无关,但是与肿瘤分化程度密切相关。EZH2 si RNA转染AGS细胞后24 h的增殖率与对照组无显著差异,而48 h及72 h的细胞增殖率明显低于对照组。mi R-124 inhibitor与si-control组的细胞增殖率大于inhibitor control与si-control组,EZH2 si RNA与inhibitor control组细胞增殖率低于inhibitor control与si-control组,而mi R-124 inhibitor与EZH2 si RNA组的细胞增殖率较对照组无明显变化。结论:mi R-124可体外抑制胃癌细胞增殖,体内抑制成瘤,并增加胃癌细胞对5-Fu的药物敏感性,mi R-124的直接靶基因EZH2在胃癌组织中表达与肿瘤分化程度相关,参与了mi R-124对胃癌细胞5-Fu敏感性的调控。
[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.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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