本文選題：肺腺癌 + miR-23b��； 參考：《天津醫(yī)科大學》2016年碩士論文

【摘要】：目的肺癌是世界范圍內常見的惡性腫瘤,其發(fā)病率死亡率都居于首位。目前肺癌患者的5年生存率仍較低,僅為15%左右。肺癌的病因機制復雜,既有遺傳因素的參與,又具有表觀遺傳因素的調控。microRNA是一類小分子RNA,雖不能編碼蛋白,卻可以參與基因表達的轉錄后調控。對microRNA及其調控的靶基因的研究有助于我們探尋肺癌新的診斷及治療靶點。miR-23b是我們前期通過microRNA表達譜篩選出來的在女性非吸煙肺腺癌血漿中差異表達的miRNA,我們推測其在肺腺癌的發(fā)生發(fā)展中發(fā)揮著一定的作用。本研究通過生物信息學預測并經雙熒光素酶報告基因驗證發(fā)現(xiàn)mi R-23b的作用靶點SET8;細胞功能實驗來初步探索miR23b對肺腺癌發(fā)生發(fā)展影響的作用機制;在人群水平上研究靶點SET8的表達及對肺腺癌生存及預后影響,以期篩選肺腺癌診斷及治療的靶點,為探索肺腺癌靶向治療提供依據。方法1、通過生物信息學篩選miR-23b的作用靶點,并通過熒光素酶報告基因實驗驗證SET8為miR-23b的作用靶點及作用關系;選取肺腺癌細胞系A549、H1299進行細胞功能實驗,通過MTT細胞增殖實驗、細胞周期實驗、細胞遷移實驗及細胞侵襲實驗來驗證過表達miR-23b對肺腺癌細胞增殖、遷移、侵襲能力的影響,證實miR-23b靶向SET8對肺腺癌的調控作用。2、選取天津醫(yī)科大學腫瘤醫(yī)院收集的手術切除的肺腺癌患者140例及80例配對的正常組織,并制作成組織芯片(TMA),通過免疫組織化學染色研究miR-23b的作用靶點SET8在肺腺癌中的表達情況。通過查閱病歷收集患者臨床信息,電話隨訪獲得生存情況,分析SET8的表達對肺腺癌患者的發(fā)病及預后影響。SET8在肺腺癌中的表達采用Wlicoxon配對檢驗分析,生存分析采用Log-rank檢驗及多因素Cox回歸分析。結果1、miR-23b在肺腺癌腫瘤組織中的表達顯著低于配對的癌旁組織,差別具有統(tǒng)計學意義(P≤0.001)。與我們前期觀察到的miR-23b在女性非吸煙肺腺癌血漿中表達趨勢一致,因此我們認為其可能是肺腺癌的抑制基因。2、雙熒光素酶報告基因結果顯示,在肺腺癌中miR-23b與SET8具有兩段結合序列,miR-23b能夠從mRNA及蛋白水平上負調控SET8在肺腺癌中的表達。miR-23b能夠抑制肺腺癌細胞增殖、調控細胞周期、抑制細胞遷移與侵襲。3、在肺腺癌中SET8蛋白的表達水平上調(P≤0.001),是潛在的促癌基因。SET8的表達與各臨床病理特征未見相關性。生存分析結果顯示,肺腺癌中SET8高表達者預后不良,差異具有統(tǒng)計學意義(OS:P=0.025;DFS:P=0.020)。分層結果顯示,SET8蛋白高表達在男性、無肺部疾病史、無腫瘤家族史、腫瘤浸潤早期、無淋巴結轉移、無遠處轉移患者中預后較差。Cox多因素回歸分析發(fā)現(xiàn)在肺腺癌中SET8高表達可能是獨立的危險因素。結論miR-23b在肺腺癌中靶向調控基因SET8的表達,發(fā)揮著抑癌基因的作用。SET8作為受mi R-23b調控的促癌基因,其在肺腺癌中的表達水平與肺腺癌的發(fā)病及預后相關,其高表達可能為獨立的預后危險因素。其作用機制仍需進一步探索。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R734.2
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本文編號：1964258