本文選題：非小細胞肺癌 + mi��； 參考：《鄭州大學》2015年博士論文

【摘要】：背景和目的肺癌是死亡率較高的腫瘤之一,其中非小細胞肺癌占幾乎80%。盡管診治水平不斷進步,仍有30%-40%的非小細胞肺癌患者因腫瘤的轉移擴散預后極差。而對腫瘤發(fā)生發(fā)展的潛在機制研究有助于我們進一步認識了解腫瘤的發(fā)病機制,并開發(fā)新的治療手段。微小RNA,又名micro RNA(mi RNA),是非編碼、單鏈、小分子RNA(22個左右的核糖核苷酸),在物種之間及進化上都具有高度的保守性,所有的真核細胞生物幾乎都可以有表達。mi RNA與其靶基因m RNA 3’端非編碼區(qū)(3’UTR)結合,主要結合的方式為完全或者不完全的互補結合,調控著靶基因的轉錄或者翻譯水平,進而影響細胞的生物學行為的變化,包括增殖、分化、細胞周期和凋亡。從而也使得mi RNA在腫瘤發(fā)生發(fā)展的過程中扮演著癌基因或者抑癌基因的重要角色。隨著研究進展,在多種惡性腫瘤中越來越多的mi RNA被發(fā)現(xiàn)存在異常表達,包括大腸癌、白血病、乳腺癌、肺癌、前列腺癌及腦癌等。在非小細胞肺癌組織中mi R-495的表達水平及具體的生物學作用和調控機制需進一步探討。轉移相關蛋白3(metastasisassociated protein 3,MTA3)是轉移相關蛋白家族中一種關鍵的共調節(jié)蛋白,該家族還包括MTA1和MTA2。證據(jù)顯示在包括食管、肺、乳腺、子宮和鼻咽等在內的多種癌癥中MTA3表達上調,并能夠通過作用于多種細胞信號通路,影響腫瘤的發(fā)展與轉移。在Agilent mi RNA芯片檢測發(fā)現(xiàn)mi R-495低表達的基礎上,通過生物信息學分析,我們預測MTA3基因為mi R-495的靶基因,根據(jù)靶基因在肺癌的研究,本課題擬首先驗證mi R-495在非小細胞肺癌中的表達,同時研究MTA3在非小細胞肺癌中的表達情況,然后觀察調控mi R-495表達對非小細胞肺癌細胞Calu-3的生長、侵襲和細胞周期的影響,并同時初步探討mi R-495調控細胞生物學行為改變的分子機制。本課題包括以下兩部分:第一部分:非小細胞肺癌組織中mi RNA和MTA3異常表達及其與臨床病理特征的相關性分析;第二部分:體內、體外調控mi R-495和表達對非小細胞肺癌細胞系Calu-3的生長、侵襲和細胞周期的影響,并對其腫瘤抑制作用機制進行初步研究。第一部分非小細胞肺癌組織中mi R-495和MAT3表達及與臨床病理學特征的相關性分析方法 1.收集標本,72例非小細胞肺癌組織與其向對應的72例癌旁正常組織標本。2.運用Agilent mi RNA芯片的方法檢測3例非小細胞肺癌組織與與其向對應的72例癌旁正常組織標本中mi RNA表達情況。3.運用q RT-PCR檢測72例標本中mi R-495的表達水平,同時運用q RT-PCR和Western blot方法檢測72例標本中MTA3的表達水平,并應用Spearman相關分析二者的相關性。4.依據(jù)mi R-495和MTA3的表達水平,運用雙變量相關性分別分析二者與非小細胞肺癌患者性別、年齡、病理類型、腫瘤分化程度、TNM分期及其有無淋巴結轉移之間的相關性。5.統(tǒng)計學分析:采用SPSS 21.0統(tǒng)計軟件進行數(shù)據(jù)分析,單因素方差用于分析多項指標間的比較,LSD-t檢驗用于比較兩項指標間的比較;t檢驗用于計量資料;Spearman相關分析應用于相關性檢驗,以α=0.05為顯著性水準。結果 1.Agilent mi RNA芯片檢測分析得到56個差異表達3倍以上的mi RNAs,其中有26個mi RNAs表達為上調,30個mi RNAs表達下調。2.mi R-495在非小細胞肺癌組織中低表達,MTA3呈高表達水平,且二者呈負相關(P0.05)。3.通過雙變量相關性的分析結果發(fā)現(xiàn)非小細胞肺癌組織中mi R-495和MTA3的表達水平均與有無淋巴結的轉移、分化程度及TNM分期相關(P0.05),與患者的年齡、性別和病理類型無相關性(P0.05)。第二部分調控mi R-495的表達對非小細胞肺癌細胞系Calu-3生物學行為的影響及其相關機制的初步分析方法 1.運用熒光定量PCR和Western-blot檢測非小細胞肺癌細胞系A549,Calu-3,H460,H1299,H1650和SPC-A-1中mi R-495和MTA3的表達水平。2.分別合成mi R-495agomir和mi R-495scramble,選擇對數(shù)生長期的Calu-3細胞株,根據(jù)實驗分組分別使用LipofectamineTM2000進行脂質體的轉染。3.熒光定量PCR檢測各實驗組細胞中mi R-495的表達變化。4.運用CCK8方法和克隆形成實驗檢測各實驗組Calu-3細胞增殖和生長情況變化。5.裸鼠移植瘤實驗檢測各實驗組移植瘤生長及MTA3蛋白表達的情況。6.流式細胞儀檢測各實驗組Calu-3細胞周期的變化及Western blot檢測相關周期蛋白表達的情況。7.應用Transwell侵襲實驗和劃痕實驗檢測各實驗組的Calu-3細胞侵襲轉移能力的變化。8.采用生物信息學分析軟件預測mi R-495的作用靶標。構建MTA3 3’UTR區(qū)的野生型及突變型重組雙熒光報告基因載體,分別將其與mi R-495 agomir或者mi R-495scramble共轉染入Calu-3細胞中,通過雙熒光素酶報告實驗證實mi R-495的作用靶標。9.構建無3’UTR區(qū)MTA3的表達載體,單獨轉入或與mi R-495 agomir共轉染入Calu-3細胞中,通過restore方法分析mi R-495靶向調控MTA3表達的作用機制。10.比較Calu-3細胞中轉染si RNA-MTA3與過表達mi R-495對Calu-3細胞增殖、侵襲和細胞周期的影響。11.統(tǒng)計學分析:采用SPSS 21.0軟件進行數(shù)據(jù)分析。結果 1.非小細胞肺癌細胞系中,mi R-495低表達水平見于A549,Calu-3,H460,H1650以及SPC-A-1細胞,但在H1299細胞中相對高表達(P0.05)。MTA3在A549,Calu-3,H460,H1650以及SPC-A-1細胞呈現(xiàn)高表達水平,但在H1299細胞中相對低表達(P0.05)。2.mi R-495組非小細胞肺癌細胞Calu-3的mi R-495表達水平明顯高于NC組和Mock組(P0.05)。提示轉染mi R-495后,Calu-3細胞的mi R-495表達水平上調。3.CCK-8實驗和克隆形成實驗結果表明,與NC組和Mock組相比,mi R-495組Calu-3細胞的增殖能力在轉染后2天開始出現(xiàn)顯著抑制(P0.05),并且隨時間延長日益顯著。4.mi R-495組裸鼠的生長在轉染后2周后出現(xiàn)抑制,并且隨時間的延長而日益顯著(P0.05)。4周后處死小鼠,剝離腫瘤組織,計算裸鼠移植瘤體積,結果顯示mi R-495組腫瘤體積顯著小于NC組和Mock組腫瘤體積(P0.05)。各組裸鼠移植瘤中MTA3的表達在mi R-495組的表達水平與相對于NC組和Mock組中的表達水平明顯升高(P0.05)。5.流式細胞儀分析結果顯示,相對于NC組和Mock組mi R-495組的細胞處于G0/G1期比例顯著升高,而處于S期比例降低(P0.05)。Western blot的方法分析細胞周期相關蛋白的表達變化,相比NC組和Mock組,mi R-495組周期蛋白A,周期蛋白D1,磷酸化Rb的表達水平均顯著降低(P0.05)。6.Transwell和劃痕實驗檢測結果顯示mi R-495組的細胞侵襲能力顯著低于NC組和Mock組(P0.05)。說明mi R-495過表達可抑制Calu-3細胞的侵襲能力。7.生物信息學分析和雙熒光素酶報告實驗結果提示,MTA3是mi R-495的作用靶標。8.Restore實驗結果提示,無3'UTR區(qū)的MTA3的表達不受mi R-495的調控,且其作用可覆蓋mi R-495上調對非小細胞肺癌細胞生物學行為的作用。9.Calu-3細胞中si RNA-MTA3轉染與過表達mi R-495的作用比較結果進一步提示了mi R-495通過作用于MTA3的3'UTR區(qū),負向調控MTA3蛋白的表達,導致非小細胞肺癌細胞的增殖、侵襲能力受到抑制,細胞周期受到阻滯。結論 1.非小細胞肺癌組織中檢測到56個差異表達的mi RNAs,其中有26個mi RNAs表達為上調,30個mi RNAs表達下調。非小細胞肺癌組織中,mi R-495呈低表達,其表達的水平均與有無淋巴結的轉移、分化程度以及TNM分期相關,且與MTA3的表達水平具有負相關性。2.在非小細胞肺癌細胞Calu-3中上調mi R-495表達通過作用于MTA3的3'UTR區(qū),負向調控MTA3的表達,導致Calu-3細胞生長和侵襲抑制,細胞周期阻滯。mi R-495有望成為非小細胞肺癌治療的新靶點。
[Abstract]:Background and objective lung cancer is one of the higher mortality rates, in which non small cell lung cancer accounts for almost 80%., although the level of diagnosis and treatment is progressing, the prognosis of non small cell lung cancer patients with 30%-40% is still very poor because of the metastasis and diffusion of tumor. The small RNA, also known as micro RNA (MI RNA), is a non coding, single chain, small molecule RNA (about 22 ribonucleotides), and is highly conserved between species and evolution, and all eukaryotic cells can almost have a combination of.Mi RNA with its target gene m RNA 3 'end non coding region (3' UTR). The main combination is a complete or incomplete complementary combination that regulates the transcriptional or translation level of the target gene, thereby affecting the changes in biological behavior of the cells, including proliferation, differentiation, cell cycle and apoptosis, thus making mi RNA an important corner of the oncogene or tumor suppressor gene in the progression of tumor development. As the research progresses, more and more mi RNA in a variety of malignant tumors have been found to have abnormal expression, including colorectal cancer, leukemia, breast cancer, lung cancer, prostate cancer and brain cancer. The expression level of MI R-495 in non-small cell lung cancer and the specific biological use and regulation mechanism need to be further discussed. Transfer related protein 3 (M Etastasisassociated protein 3, MTA3) is a key co regulated protein in the metastasis related protein family. The family also includes MTA1 and MTA2. evidence that the expression of MTA3 is up-regulated in a variety of cancers, including the esophagus, lungs, breast, uterus and nasopharynx, and can affect the development of cancer by acting on a variety of cell signaling pathways. On the basis of the detection of the low expression of MI R-495 by Agilent mi RNA chip, we predict that the MTA3 gene is the target gene of MI R-495 through bioinformatics analysis. According to the target gene in the study of lung cancer, this topic is to first verify the expression of MI R-495 in non-small cell lung cancer and to study the expression of MTA3 in non-small cell lung cancer. And then observe the effects of MI R-495 expression on the growth, invasion and cell cycle of non small cell lung cancer cell Calu-3, and discuss the molecular mechanism of MI R-495 regulating cell biological behavior changes. This topic includes the following two parts: the first part: the abnormal expression of MI RNA and MTA3 in non-small cell lung cancer and its clinical significance. The correlation analysis of pathological features; the second part: in vitro, the effect of MI R-495 and expression on the growth, invasion and cell cycle of non small cell lung cancer cell line Calu-3 in vitro, and the mechanism of its tumor inhibition are preliminarily studied. The expression of MI R-495 and MAT3 in the first part of non small cell lung cancer and its clinicopathological features Correlation analysis method 1. specimens were collected, 72 non-small cell lung cancer tissues and 72 normal paracancerous tissue specimens of corresponding.2. were used to detect the expression of MI RNA in 3 non small cell lung cancer tissues and 72 cases of normal tissues adjacent to cancer by Agilent mi RNA chip. The MI R-4 in 72 cases of Q RT-PCR detected by Q RT-PCR was used for MI R-4. The expression level of 95 was 95, and the expression level of MTA3 in 72 specimens was detected by Q RT-PCR and Western blot, and the correlation.4. of the Spearman correlation analysis was based on the expression level of MI R-495 and MTA3. The sex, age, pathological type and tumor differentiation of the two patients with non-small cell lung cancer were analyzed by bivariate correlation. The correlation of degree, TNM staging and the correlation between lymph node metastasis and.5. statistical analysis: using SPSS 21 statistical software for data analysis, single factor variance used to compare the comparison among multiple indexes, LSD-t test was used to compare the comparison among the two indexes; t test was used to measure the material; Spearman correlation analysis was applied to correlation test, to alpha Results =0.05 was a significant level. Results 1.Agilent mi RNA chip detection and analysis obtained 56 differential expressions of more than 3 times of MI RNAs, of which 26 mi RNAs were expressed as up, 30 mi RNAs expressed downregulation of.2.mi R-495 in non small cell lung cancer tissues and high expression, and the two were negatively correlated by bivariate correlation. The results of the analysis showed that the expression level of MI R-495 and MTA3 in non small cell lung cancer tissues was related to the metastasis of lymph node, the degree of differentiation and the TNM staging (P0.05), and no correlation with the age, sex and pathological type of the patients (P0.05). The second part regulated the expression of MI R-495 in the expression of Calu-3 biological behavior of non-small cell lung cancer cell lines. Preliminary analysis of its related mechanisms and its related mechanisms 1. using fluorescent quantitative PCR and Western-blot to detect non small cell lung cancer cell lines A549, Calu-3, H460, H1299, MI R-495 and MTA3 expression level.2. in H1650 and SPC-A-1, respectively. The transfection of liposomes using LipofectamineTM2000 to detect the expression of MI R-495 in the cells of each experimental group by transfection of.3. PCR and.4. using CCK8 method and clone formation test to detect the proliferation and growth of Calu-3 cells in each experimental group. The growth of the transplanted tumor and the expression of MTA3 protein in the experimental group were detected by the experimental group of.5. nude mice. Detection of the changes of Calu-3 cell cycle in each experimental group by.6. flow cytometry and the expression of periodic protein by Western blot detection.7. application of Transwell invasion experiment and scratch test to detect the change of Calu-3 cell invasion and metastasis ability of each experimental group.8. using bioinformatics analysis soft ware to predict the function target of MI R-495. The wild type and mutant recombinant double fluorescent reporter gene vector of 3 'UTR region was transfected into Calu-3 cells with MI R-495 agomir or MI R-495scramble respectively. The target.9. of MI R-495 was confirmed by the double Luciferase Report experiment, and the target.9. of MI R-495 was constructed without 3' UTR MTA3. In lu-3 cells, the restore method was used to analyze the mechanism of MI R-495 targeting the expression of MTA3,.10. compared the effect of Si RNA-MTA3 and overexpression of MI R-495 on the proliferation, invasion and cell cycle of Calu-3 cells in Calu-3 cells. The low expression level of I R-495 is found in A549, Calu-3, H460, H1650 and SPC-A-1 cells, but the relatively high expression of (P0.05).MTA3 in H1299 cells is in A549. It was higher than group NC and group Mock (P0.05). After transfection of MI R-495, the expression level of MI R-495 in Calu-3 cells was up-regulated and the experimental results of.3.CCK-8 experiment and clone formation showed that the proliferation ability of the MI cells began to appear significantly at the 2 day after the transfection. The growth of the nude mice was inhibited 2 weeks after the transfection, and the mice were sacrificed more and more significantly (P0.05).4 weeks after the transfection. The tumor tissue was stripped and the tumor volume of nude mice was calculated. The results showed that the tumor volume of the MI R-495 group was significantly smaller than that of the NC group and the Mock group (P0.05). The expression of MTA3 in the xenografts in nude mice was in the MI R-495 group. The expression level and the expression level relative to the group NC and the Mock group increased significantly (P0.05).5. flow cytometry analysis showed that the proportion of the cells in the MI R-495 group in the NC group and the Mock group was significantly higher than that in the MI R-495 group, while the reduction (P0.05) in the S phase (P0.05) was compared to the changes in the expression of the cell cycle related proteins. The expression level of A, cyclin D1, and phosphorylated Rb in group NC and Mock group decreased significantly (P0.05).6.Transwell and scratch test results showed that the invasive ability of MI R-495 group was significantly lower than that of NC and other groups. And the results of the double Luciferase Report showed that MTA3 was the target of the action target of MI R-495. The expression of MTA3 without 3'UTR region was not regulated by Mi R-495, and the effect could cover the up regulation of MI R-495 on the biological behavior of non-small cell lung cancer cells. The results further suggest that MI R-495 can regulate the expression of MTA3 in the 3'UTR region of MTA3, negative regulation of the expression of MTA3 protein, resulting in the proliferation of non small cell lung cancer cells, the inhibition of invasion, and the cell cycle arrest. Conclusion 1. non small cell lung cancer tissues have detected 56 differentially expressed mi RNAs, of which 26 mi RNAs expressions are expressed. For up regulation, the expression of 30 mi RNAs was down. The expression level of MI R-495 in non-small cell lung cancer was low, and the expression level was related to the metastasis of lymph node, the degree of differentiation and the TNM staging, and the expression level of MTA3 was negatively correlated with.2. in non small cell lung cancer cell Calu-3 up regulation of MI R-495 expression passed the MTA3 3'UTR region. Negative regulation of MTA3 expression leads to Calu-3 cell growth and invasion inhibition. Cell cycle arrest of.Mi R-495 is expected to become a new target for non-small cell lung cancer treatment.

【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R734.2

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1 楚荷瑩;miR-495表達對非小細胞肺癌細胞生物學行為的影響[D];鄭州大學;2015年

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