本文選題：非小細(xì)胞肺癌 + mi�。� 參考：《鄭州大學(xué)》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(xì)胞肺癌占幾乎80%。盡管診治水平不斷進(jìn)步,仍有30%-40%的非小細(xì)胞肺癌患者因腫瘤的轉(zhuǎn)移擴(kuò)散預(yù)后極差。而對(duì)腫瘤發(fā)生發(fā)展的潛在機(jī)制研究有助于我們進(jìn)一步認(rèn)識(shí)了解腫瘤的發(fā)病機(jī)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(gè)左右的核糖核苷酸),在物種之間及進(jìn)化上都具有高度的保守性,所有的真核細(xì)胞生物幾乎都可以有表達(dá)。mi RNA與其靶基因m RNA 3’端非編碼區(qū)(3’UTR)結(jié)合,主要結(jié)合的方式為完全或者不完全的互補(bǔ)結(jié)合,調(diào)控著靶基因的轉(zhuǎn)錄或者翻譯水平,進(jìn)而影響細(xì)胞的生物學(xué)行為的變化,包括增殖、分化、細(xì)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(jìn)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(dá),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(xì)胞肺癌組織中mi R-495的表達(dá)水平及具體的生物學(xué)作用和調(diào)控機(jī)制需進(jìn)一步探討。轉(zhuǎn)移相關(guān)蛋白3(metastasisassociated protein 3,MTA3)是轉(zhuǎn)移相關(guān)蛋白家族中一種關(guān)鍵的共調(diào)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(nèi)的多種癌癥中MTA3表達(dá)上調(diào),并能夠通過作用于多種細(xì)胞信號(hào)通路,影響腫瘤的發(fā)展與轉(zhuǎn)移。在Agilent mi RNA芯片檢測(cè)發(fā)現(xiàn)mi R-495低表達(dá)的基礎(chǔ)上,通過生物信息學(xué)分析,我們預(yù)測(cè)MTA3基因?yàn)閙i 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(yàn)證mi R-495在非小細(xì)胞肺癌中的表達(dá),同時(shí)研究MTA3在非小細(xì)胞肺癌中的表達(dá)情況,然后觀察調(diào)控mi R-495表達(dá)對(duì)非小細(xì)胞肺癌細(xì)胞Calu-3的生長、侵襲和細(xì)胞周期的影響,并同時(shí)初步探討mi R-495調(diào)控細(xì)胞生物學(xué)行為改變的分子機(jī)制。本課題包括以下兩部分:第一部分:非小細(xì)胞肺癌組織中mi RNA和MTA3異常表達(dá)及其與臨床病理特征的相關(guān)性分析;第二部分:體內(nèi)、體外調(diào)控mi R-495和表達(dá)對(duì)非小細(xì)胞肺癌細(xì)胞系Calu-3的生長、侵襲和細(xì)胞周期的影響,并對(duì)其腫瘤抑制作用機(jī)制進(jìn)行初步研究。第一部分非小細(xì)胞肺癌組織中mi R-495和MAT3表達(dá)及與臨床病理學(xué)特征的相關(guān)性分析方法 1.收集標(biāo)本,72例非小細(xì)胞肺癌組織與其向?qū)?yīng)的72例癌旁正常組織標(biāo)本。2.運(yùn)用Agilent mi RNA芯片的方法檢測(cè)3例非小細(xì)胞肺癌組織與與其向?qū)?yīng)的72例癌旁正常組織標(biāo)本中mi RNA表達(dá)情況。3.運(yùn)用q RT-PCR檢測(cè)72例標(biāo)本中mi R-495的表達(dá)水平,同時(shí)運(yùn)用q RT-PCR和Western blot方法檢測(cè)72例標(biāo)本中MTA3的表達(dá)水平,并應(yīng)用Spearman相關(guān)分析二者的相關(guān)性。4.依據(jù)mi R-495和MTA3的表達(dá)水平,運(yùn)用雙變量相關(guān)性分別分析二者與非小細(xì)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(jié)轉(zhuǎn)移之間的相關(guān)性。5.統(tǒng)計(jì)學(xué)分析:采用SPSS 21.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析,單因素方差用于分析多項(xiàng)指標(biāo)間的比較,LSD-t檢驗(yàn)用于比較兩項(xiàng)指標(biāo)間的比較;t檢驗(yàn)用于計(jì)量資料;Spearman相關(guān)分析應(yīng)用于相關(guān)性檢驗(yàn),以α=0.05為顯著性水準(zhǔn)。結(jié)果 1.Agilent mi RNA芯片檢測(cè)分析得到56個(gè)差異表達(dá)3倍以上的mi RNAs,其中有26個(gè)mi RNAs表達(dá)為上調(diào),30個(gè)mi RNAs表達(dá)下調(diào)。2.mi R-495在非小細(xì)胞肺癌組織中低表達(dá),MTA3呈高表達(dá)水平,且二者呈負(fù)相關(guān)(P0.05)。3.通過雙變量相關(guān)性的分析結(jié)果發(fā)現(xiàn)非小細(xì)胞肺癌組織中mi R-495和MTA3的表達(dá)水平均與有無淋巴結(jié)的轉(zhuǎn)移、分化程度及TNM分期相關(guān)(P0.05),與患者的年齡、性別和病理類型無相關(guān)性(P0.05)。第二部分調(diào)控mi R-495的表達(dá)對(duì)非小細(xì)胞肺癌細(xì)胞系Calu-3生物學(xué)行為的影響及其相關(guān)機(jī)制的初步分析方法 1.運(yùn)用熒光定量PCR和Western-blot檢測(cè)非小細(xì)胞肺癌細(xì)胞系A(chǔ)549,Calu-3,H460,H1299,H1650和SPC-A-1中mi R-495和MTA3的表達(dá)水平。2.分別合成mi R-495agomir和mi R-495scramble,選擇對(duì)數(shù)生長期的Calu-3細(xì)胞株,根據(jù)實(shí)驗(yàn)分組分別使用LipofectamineTM2000進(jìn)行脂質(zhì)體的轉(zhuǎn)染。3.熒光定量PCR檢測(cè)各實(shí)驗(yàn)組細(xì)胞中mi R-495的表達(dá)變化。4.運(yùn)用CCK8方法和克隆形成實(shí)驗(yàn)檢測(cè)各實(shí)驗(yàn)組Calu-3細(xì)胞增殖和生長情況變化。5.裸鼠移植瘤實(shí)驗(yàn)檢測(cè)各實(shí)驗(yàn)組移植瘤生長及MTA3蛋白表達(dá)的情況。6.流式細(xì)胞儀檢測(cè)各實(shí)驗(yàn)組Calu-3細(xì)胞周期的變化及Western blot檢測(cè)相關(guān)周期蛋白表達(dá)的情況。7.應(yīng)用Transwell侵襲實(shí)驗(yàn)和劃痕實(shí)驗(yàn)檢測(cè)各實(shí)驗(yàn)組的Calu-3細(xì)胞侵襲轉(zhuǎn)移能力的變化。8.采用生物信息學(xué)分析軟件預(yù)測(cè)mi R-495的作用靶標(biāo)。構(gòu)建MTA3 3’UTR區(qū)的野生型及突變型重組雙熒光報(bào)告基因載體,分別將其與mi R-495 agomir或者mi R-495scramble共轉(zhuǎn)染入Calu-3細(xì)胞中,通過雙熒光素酶報(bào)告實(shí)驗(yàn)證實(shí)mi R-495的作用靶標(biāo)。9.構(gòu)建無3’UTR區(qū)MTA3的表達(dá)載體,單獨(dú)轉(zhuǎn)入或與mi R-495 agomir共轉(zhuǎn)染入Calu-3細(xì)胞中,通過restore方法分析mi R-495靶向調(diào)控MTA3表達(dá)的作用機(jī)制。10.比較Calu-3細(xì)胞中轉(zhuǎn)染si RNA-MTA3與過表達(dá)mi R-495對(duì)Calu-3細(xì)胞增殖、侵襲和細(xì)胞周期的影響。11.統(tǒng)計(jì)學(xué)分析:采用SPSS 21.0軟件進(jìn)行數(shù)據(jù)分析。結(jié)果 1.非小細(xì)胞肺癌細(xì)胞系中,mi R-495低表達(dá)水平見于A549,Calu-3,H460,H1650以及SPC-A-1細(xì)胞,但在H1299細(xì)胞中相對(duì)高表達(dá)(P0.05)。MTA3在A549,Calu-3,H460,H1650以及SPC-A-1細(xì)胞呈現(xiàn)高表達(dá)水平,但在H1299細(xì)胞中相對(duì)低表達(dá)(P0.05)。2.mi R-495組非小細(xì)胞肺癌細(xì)胞Calu-3的mi R-495表達(dá)水平明顯高于NC組和Mock組(P0.05)。提示轉(zhuǎn)染mi R-495后,Calu-3細(xì)胞的mi R-495表達(dá)水平上調(diào)。3.CCK-8實(shí)驗(yàn)和克隆形成實(shí)驗(yàn)結(jié)果表明,與NC組和Mock組相比,mi R-495組Calu-3細(xì)胞的增殖能力在轉(zhuǎn)染后2天開始出現(xiàn)顯著抑制(P0.05),并且隨時(shí)間延長日益顯著。4.mi R-495組裸鼠的生長在轉(zhuǎn)染后2周后出現(xiàn)抑制,并且隨時(shí)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(jì)算裸鼠移植瘤體積,結(jié)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(dá)在mi R-495組的表達(dá)水平與相對(duì)于NC組和Mock組中的表達(dá)水平明顯升高(P0.05)。5.流式細(xì)胞儀分析結(jié)果顯示,相對(duì)于NC組和Mock組mi R-495組的細(xì)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(xì)胞周期相關(guān)蛋白的表達(dá)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(dá)水平均顯著降低(P0.05)。6.Transwell和劃痕實(shí)驗(yàn)檢測(cè)結(jié)果顯示mi R-495組的細(xì)胞侵襲能力顯著低于NC組和Mock組(P0.05)。說明mi R-495過表達(dá)可抑制Calu-3細(xì)胞的侵襲能力。7.生物信息學(xué)分析和雙熒光素酶報(bào)告實(shí)驗(yàn)結(jié)果提示,MTA3是mi R-495的作用靶標(biāo)。8.Restore實(shí)驗(yàn)結(jié)果提示,無3'UTR區(qū)的MTA3的表達(dá)不受mi R-495的調(diào)控,且其作用可覆蓋mi R-495上調(diào)對(duì)非小細(xì)胞肺癌細(xì)胞生物學(xué)行為的作用。9.Calu-3細(xì)胞中si RNA-MTA3轉(zhuǎn)染與過表達(dá)mi R-495的作用比較結(jié)果進(jìn)一步提示了mi R-495通過作用于MTA3的3'UTR區(qū),負(fù)向調(diào)控MTA3蛋白的表達(dá),導(dǎo)致非小細(xì)胞肺癌細(xì)胞的增殖、侵襲能力受到抑制,細(xì)胞周期受到阻滯。結(jié)論 1.非小細(xì)胞肺癌組織中檢測(cè)到56個(gè)差異表達(dá)的mi RNAs,其中有26個(gè)mi RNAs表達(dá)為上調(diào),30個(gè)mi RNAs表達(dá)下調(diào)。非小細(xì)胞肺癌組織中,mi R-495呈低表達(dá),其表達(dá)的水平均與有無淋巴結(jié)的轉(zhuǎn)移、分化程度以及TNM分期相關(guān),且與MTA3的表達(dá)水平具有負(fù)相關(guān)性。2.在非小細(xì)胞肺癌細(xì)胞Calu-3中上調(diào)mi R-495表達(dá)通過作用于MTA3的3'UTR區(qū),負(fù)向調(diào)控MTA3的表達(dá),導(dǎo)致Calu-3細(xì)胞生長和侵襲抑制,細(xì)胞周期阻滯。mi R-495有望成為非小細(xì)胞肺癌治療的新靶點(diǎn)。
[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(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2

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