miR-23b靶向SET8在肺腺癌中的研究

发布时间：2018-06-01 12:56

  本文选题：肺腺癌 + miR-23b　； 参考：《天津医科大学》2016年硕士论文

【摘要】：目的肺癌是世界范围内常见的恶性肿瘤,其发病率死亡率都居于首位。目前肺癌患者的5年生存率仍较低,仅为15%左右。肺癌的病因机制复杂,既有遗传因素的参与,又具有表观遗传因素的调控。microRNA是一类小分子RNA,虽不能编码蛋白,却可以参与基因表达的转录后调控。对microRNA及其调控的靶基因的研究有助于我们探寻肺癌新的诊断及治疗靶点。miR-23b是我们前期通过microRNA表达谱筛选出来的在女性非吸烟肺腺癌血浆中差异表达的miRNA,我们推测其在肺腺癌的发生发展中发挥着一定的作用。本研究通过生物信息学预测并经双荧光素酶报告基因验证发现mi R-23b的作用靶点SET8;细胞功能实验来初步探索miR23b对肺腺癌发生发展影响的作用机制;在人群水平上研究靶点SET8的表达及对肺腺癌生存及预后影响,以期筛选肺腺癌诊断及治疗的靶点,为探索肺腺癌靶向治疗提供依据。方法1、通过生物信息学筛选miR-23b的作用靶点,并通过荧光素酶报告基因实验验证SET8为miR-23b的作用靶点及作用关系;选取肺腺癌细胞系A549、H1299进行细胞功能实验,通过MTT细胞增殖实验、细胞周期实验、细胞迁移实验及细胞侵袭实验来验证过表达miR-23b对肺腺癌细胞增殖、迁移、侵袭能力的影响,证实miR-23b靶向SET8对肺腺癌的调控作用。2、选取天津医科大学肿瘤医院收集的手术切除的肺腺癌患者140例及80例配对的正常组织,并制作成组织芯片(TMA),通过免疫组织化学染色研究miR-23b的作用靶点SET8在肺腺癌中的表达情况。通过查阅病历收集患者临床信息,电话随访获得生存情况,分析SET8的表达对肺腺癌患者的发病及预后影响。SET8在肺腺癌中的表达采用Wlicoxon配对检验分析,生存分析采用Log-rank检验及多因素Cox回归分析。结果1、miR-23b在肺腺癌肿瘤组织中的表达显著低于配对的癌旁组织,差别具有统计学意义(P≤0.001)。与我们前期观察到的miR-23b在女性非吸烟肺腺癌血浆中表达趋势一致,因此我们认为其可能是肺腺癌的抑制基因。2、双荧光素酶报告基因结果显示,在肺腺癌中miR-23b与SET8具有两段结合序列,miR-23b能够从mRNA及蛋白水平上负调控SET8在肺腺癌中的表达。miR-23b能够抑制肺腺癌细胞增殖、调控细胞周期、抑制细胞迁移与侵袭。3、在肺腺癌中SET8蛋白的表达水平上调(P≤0.001),是潜在的促癌基因。SET8的表达与各临床病理特征未见相关性。生存分析结果显示,肺腺癌中SET8高表达者预后不良,差异具有统计学意义(OS:P=0.025;DFS:P=0.020)。分层结果显示,SET8蛋白高表达在男性、无肺部疾病史、无肿瘤家族史、肿瘤浸润早期、无淋巴结转移、无远处转移患者中预后较差。Cox多因素回归分析发现在肺腺癌中SET8高表达可能是独立的危险因素。结论miR-23b在肺腺癌中靶向调控基因SET8的表达,发挥着抑癌基因的作用。SET8作为受mi R-23b调控的促癌基因,其在肺腺癌中的表达水平与肺腺癌的发病及预后相关,其高表达可能为独立的预后危险因素。其作用机制仍需进一步探索。
[Abstract]:Objective lung cancer is the most common malignant tumor in the world, and its morbidity and mortality are the first. The 5 year survival rate of lung cancer patients is still low, only about 15%. The pathogenesis of lung cancer is complex, with the participation of hereditary factors and the regulation of epigenetic factors,.MicroRNA is a class of small molecule RNA, although it can not encode protein, but The study of microRNA and its regulated target genes helps us to explore new diagnostic and therapeutic targets for lung cancer,.MiR-23b, which is the differential expression of miRNA in the plasma of female non-smoking lung adenocarcinoma which we screened by microRNA expression profiles. We speculate that it is in the occurrence of adenocarcinoma of the lung. In this study, the purpose of this study was to find the target SET8 of MI R-23b through bioinformatics prediction and double luciferase reporter gene verification. Cell function experiment was used to explore the mechanism of miR23b effect on the development of lung adenocarcinoma. The expression of target SET8 and the survival and prognosis of lung adenocarcinoma were studied at the population level. Effect, in order to screen the target of diagnosis and treatment of lung adenocarcinoma, to provide the basis for exploring the target treatment of lung adenocarcinoma. Method 1, screening the target of miR-23b through bioinformatics, and using luciferase reporter gene test to verify the target and function of SET8 as miR-23b, and select the lung adenocarcinoma cell line A549, H1299 to carry out the cell function real The effects of miR-23b on the proliferation, migration and invasion of lung adenocarcinoma cells were verified by MTT cell proliferation experiment, cell cycle experiment, cell migration experiment and cell invasion experiment. The effect of miR-23b targeting SET8 on lung adenocarcinoma was confirmed by miR-23b, and 14 patients with surgical resection of lung adenocarcinoma collected by the Cancer Hospital of Medical University Of Tianjin were selected. 0 cases and 80 matched normal tissues were made into tissue chip (TMA), and the expression of the target SET8 in the lung adenocarcinoma was studied by immunohistochemical staining. The clinical information of the patients was collected by consulting the medical records and the survival of the miR-23b was collected by telephone follow-up. The influence of the expression of SET8 on the incidence and prognosis of lung adenocarcinoma was analyzed by.SET8. The expression in lung adenocarcinoma was analyzed by Wlicoxon paired test. The survival analysis was analyzed by Log-rank test and multiple factor Cox regression analysis. Results 1, the expression of miR-23b in the tumor tissues of lung adenocarcinoma was significantly lower than that of the paired paracancerous tissues (P < 0.001). The expression trend in cancer plasma is consistent, so we think that it may be an inhibitory gene.2 of lung adenocarcinoma. The results of double luciferase reporter gene show that miR-23b and SET8 have two segment binding sequences in lung adenocarcinoma, and miR-23b can negatively regulate the expression of SET8 in lung adenocarcinoma from mRNA and protein levels and can inhibit the proliferation of lung adenocarcinoma cells. Regulation of cell cycle, inhibition of cell migration and invasion of.3, the expression of SET8 protein in lung adenocarcinoma was up regulated (P < 0.001). The expression of the potential oncogene.SET8 was not related to the clinicopathological features. Survival analysis showed that the SET8 high expression of SET8 in lung adenocarcinoma was bad, and the difference was statistically significant (OS:P=0.025; DFS:P=0.0). 20). The stratified results showed that SET8 protein was highly expressed in male, no history of lung disease, no family history of tumor, early tumor invasion, no lymph node metastasis, and no distant metastasis in patients with poor prognosis of.Cox multifactor regression analysis found that high expression of SET8 in lung adenocarcinoma may be an independent risk factor. Conclusion miR-23b in lung adenocarcinoma target regulator The expression of SET8 plays a role in the role of tumor suppressor gene.SET8 as a oncogene regulated by Mi R-23b. The expression level in lung adenocarcinoma is related to the incidence and prognosis of lung adenocarcinoma, and its high expression may be an independent prognostic factor. The mechanism of its action still needs to be further explored.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2
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本文编号：1964258