發(fā)布時(shí)間：2018-08-11 21:21

【摘要】：肺癌已成為最常見的惡性腫瘤之一,其死亡率是成年人所有癌癥導(dǎo)致死亡的首位,嚴(yán)重威脅著人類的健康。雖然目前對(duì)于肺癌的研究以及綜合治療日益深入,但針對(duì)肺癌的療效仍不容樂觀,因此肺癌的研究與治療是我國惡性腫瘤防控的重點(diǎn)。非小細(xì)胞肺癌(NSCLC)是肺癌的主要類型。在人體中NSCLC的增殖、侵襲、轉(zhuǎn)移能力是非常復(fù)雜的生物學(xué)過程,直接影響肺癌患者的生存質(zhì)量和生存期。尋求診斷NSCLC的特異性標(biāo)志物和新的治療靶點(diǎn),完善個(gè)體化治療,成為當(dāng)今肺癌研究的當(dāng)務(wù)之急。在分子機(jī)制層面研究NSCLC的發(fā)生及發(fā)展,更好地開發(fā)新的診斷和治療方法,從而提高肺癌患者的有效生存率和生活質(zhì)量,將成為未來本學(xué)科的重要課題。Micro RNA(Mi RNA)是一種小片段非編碼單鏈的RNA,因其具有較高的保守性,是普遍存在于動(dòng)植物體內(nèi)的小型基因調(diào)控因子,與細(xì)胞的生長、發(fā)育、凋亡等生理過程相關(guān),尤其是與腫瘤發(fā)生和發(fā)展過程有著密切的相關(guān)性。Mi RNA表達(dá)失調(diào)和功能障礙可以通過不同的信號(hào)通路抑制腫瘤的生長及進(jìn)展。對(duì)NSCLC而言,Mi RNA對(duì)肺癌的分子機(jī)制具有重要的理論和臨床意義,成為NSCLC的潛在的生物學(xué)標(biāo)志以及新的治療方式,這有待進(jìn)一步的深入研究。Micro RNA-154(Mi R-154)是Mi RNA家族中與致癌相關(guān)的重要成員之一,是哺乳動(dòng)物一個(gè)非常保守的Mi RNA的序列。近期已有報(bào)道Mi R-154的低表達(dá)與許多腫瘤的發(fā)生、發(fā)展、侵襲及轉(zhuǎn)移有關(guān),其失調(diào)可以影響多種類型的癌癥,但在肺癌的研究較少。目前研究發(fā)現(xiàn),在肺癌與正常癌旁組織對(duì)比檢測(cè)Mi R-154呈現(xiàn)低表達(dá),但在NSCLC中Mi R-154表達(dá)失調(diào)和臨床組織病理特征之間的相關(guān)性以及Mi R-154對(duì)NSCLC細(xì)胞的發(fā)生發(fā)展的機(jī)制有何影響,尚未有報(bào)道。本課題圍繞“Mi R-154如何影響NSCLC的發(fā)生和發(fā)展以及靶基因的調(diào)控”展開深入研究,為診斷肺癌和靶向藥物研究提供重要的理論基礎(chǔ)和實(shí)驗(yàn)數(shù)據(jù)。研究目的:本研究旨在探討NSCLC中Mi R-154失調(diào)和臨床病理特征之間的相關(guān)性,分析Mi R-154在肺癌發(fā)生發(fā)展中的機(jī)制,從而為未來NSCLC的臨床治療提供理論依據(jù)。研究方法:1、在臨床相關(guān)研究中收集經(jīng)手術(shù)后病理證實(shí)的NSCLC標(biāo)本40例,通過RT-PCR檢測(cè)Mi R-154的m RNA水平表達(dá)在NSCLC癌組織、癌旁正常肺組織中存在差異。統(tǒng)計(jì)分析Mi R-154表達(dá)對(duì)NSCLC患者臨床分期以及生存期的影響。2、體外細(xì)胞實(shí)驗(yàn),利用q RT-PCR檢測(cè)Mi R-154的表達(dá)水平,篩選出具有代表特異性的細(xì)胞。Mi R-154轉(zhuǎn)染A549細(xì)胞,應(yīng)用q RT-PCR驗(yàn)證轉(zhuǎn)染效率;細(xì)胞增值實(shí)驗(yàn)、CCK-8實(shí)驗(yàn)、集落形成實(shí)驗(yàn)、劃痕試驗(yàn)和細(xì)胞侵襲試驗(yàn)檢測(cè)Mi R-154對(duì)NSCLC細(xì)胞的增殖、侵襲、遷移等能力的影響;流式細(xì)胞實(shí)驗(yàn)分析A549細(xì)胞周期;Western Blot檢測(cè)細(xì)胞凋亡與上皮間質(zhì)細(xì)胞轉(zhuǎn)化(EMT)標(biāo)記物。體內(nèi)裸鼠成瘤實(shí)驗(yàn)驗(yàn)證Mi R-154抑制NSCLC在體內(nèi)的生長。3、進(jìn)一步對(duì)Mi R-154抑制NSCLC靶基因以及分子機(jī)制的研究。使用公共數(shù)據(jù)庫(Mi Randa,Pic Tar和Target Scan S 6.2)篩選ZEB2作為Mi R-154的靶基因。q RT-PCR、Western Blot、細(xì)胞侵襲實(shí)驗(yàn)驗(yàn)證si-ZEB2(小干擾RNA)轉(zhuǎn)染調(diào)節(jié)ZEB2影響A549細(xì)胞的侵襲和轉(zhuǎn)移能力。構(gòu)建pc DNA3.0-ZEB2質(zhì)粒后,q RT-PCR、Western Blot驗(yàn)證ZEB2表達(dá)。然后建立:Mi R-154+ZEB2組(將pc DNA3.0-ZEB2質(zhì)粒和Mi R-154共同轉(zhuǎn)染A549細(xì)胞)、Mi R-154組(單獨(dú)轉(zhuǎn)染Mi R-154組)和對(duì)照組(pc DNA3.0空載體轉(zhuǎn)染),利用細(xì)胞侵襲實(shí)驗(yàn)檢測(cè)侵襲能力。研究結(jié)果:1、臨床樣本RT-PCR檢測(cè)結(jié)果證實(shí)了Mi R-154的表達(dá)在NSCLC腫瘤中顯著降低(P0.05)。統(tǒng)計(jì)分析發(fā)現(xiàn)了腫瘤體積的大小,腫瘤的術(shù)后TNM病理學(xué)分期以及淋巴結(jié)轉(zhuǎn)移的增加程度,NSCLC患者生存期均與Mi R-154表達(dá)降低相關(guān)(P0.05)。2、體外細(xì)胞實(shí)驗(yàn):RT-PCR進(jìn)行檢測(cè)Mi R-154在NSCLC細(xì)胞系A(chǔ)549、H1299、SPCA1、H358和正常肺細(xì)胞的表達(dá)水平,A549中Mi R-154的表達(dá)下降最為顯著,其特異性最高,可以被選定為特異性代表進(jìn)一步研究。3、轉(zhuǎn)染Mi R-154到A549細(xì)胞,RT-PCR結(jié)果顯示Mi R-154轉(zhuǎn)染到A549后Mi R-154的m RNA表達(dá)水平顯著增高(P0.05),證實(shí)了轉(zhuǎn)染的有效性。4、細(xì)胞增值實(shí)驗(yàn)和集落形成實(shí)驗(yàn)結(jié)果發(fā)現(xiàn):與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞組增殖指數(shù)、集落形成效率顯著降低(P0.05)。5、流式細(xì)胞檢測(cè)結(jié)果發(fā)現(xiàn):與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,Mi R-154轉(zhuǎn)染的A549細(xì)胞組G0/G1期階段百分比顯著增高(P0.05)。6、Western Blot檢測(cè)結(jié)果顯示:與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞組細(xì)胞凋亡因子caspase3和caspase8顯著增加(P0.05)。7、劃痕試驗(yàn)和細(xì)胞侵襲試驗(yàn)結(jié)果顯示:與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞組遷移和侵襲能力明顯下降(P0.05)。8、Western Blot檢測(cè)結(jié)果顯示:與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞組上皮間質(zhì)轉(zhuǎn)化(EMT)標(biāo)志物顯著升高(P0.05)。9、在體內(nèi)裸鼠成瘤實(shí)驗(yàn)結(jié)果顯示:與轉(zhuǎn)染Mi R-control和陰性對(duì)照組相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞明顯降低體內(nèi)移植物的腫瘤體積和腫瘤重量(P0.05)。10、利用公共數(shù)據(jù)庫(mi Randa,Pic Tar和Target Scan S 6.2)分析結(jié)果和生物信息學(xué)檢測(cè)驗(yàn)證篩選出ZEB2可以作為Mi R-154的靶基因。11、q RT-PCR和Western Blot檢測(cè)結(jié)果顯示:與轉(zhuǎn)染Mi R-control相比,轉(zhuǎn)染Mi R-154的A549細(xì)胞組ZEB2的m RNA表達(dá)水平顯著降低(P0.05);波形蛋白(Vimentin)表達(dá)水平降低(P0.05);E-鈣粘蛋白(E-cadherin)的蛋白表達(dá)水平增加(P0.05)。12、q RT-PCR和Western Blot檢測(cè)結(jié)果顯示:si-ZEB2轉(zhuǎn)染入A549細(xì)胞ZEB2的m RNA水平和蛋白水平表達(dá)顯著降低(P0.05)。同時(shí)進(jìn)行細(xì)胞侵襲實(shí)驗(yàn)結(jié)果顯示:si-ZEB2轉(zhuǎn)染入A549細(xì)胞侵襲能力下降(P0.05),證明了降低ZEB2與Mi R-154高表達(dá)具有類似的效果。13、構(gòu)建pc DNA3.0-ZEB2質(zhì)粒轉(zhuǎn)染到肺腺癌A549細(xì)胞中,通過RT-PCR和Western Blot檢測(cè)證明pc DNA3.0-ZEB2能顯著提高ZEB2的表達(dá)。說明質(zhì)粒構(gòu)建成功以及轉(zhuǎn)入后的有效性。14、細(xì)胞侵襲實(shí)驗(yàn)結(jié)果顯示:與對(duì)照組相比,Mi R-154細(xì)胞組遷移和侵襲能力下降(P0.05),Mi R-154與ZEB2組無統(tǒng)計(jì)學(xué)意義(P0.05),證明了ZEB2高表達(dá)可逆轉(zhuǎn)Mi R-154對(duì)肺癌侵襲的抑制作用。結(jié)論:1、在臨床相關(guān)研究中證實(shí)了Mi R-154在NSCLC組織中m RNA表達(dá)水平降低,并在研究過程中發(fā)現(xiàn)了Mi R-154影響臨床病理分期及NSCLC患者生存期這一現(xiàn)象,推測(cè)Mi R-154可能抑制NSCLC生長。2、為了進(jìn)一步證明我們的推測(cè),然后我們進(jìn)行體外細(xì)胞實(shí)驗(yàn),發(fā)現(xiàn)上調(diào)A549細(xì)胞中Mi R-154表達(dá)可以抑制其細(xì)胞增殖、侵襲和轉(zhuǎn)移能力,并且上調(diào)Mi R-154表達(dá)可以誘導(dǎo)肺腺癌A549細(xì)胞周期阻滯和促進(jìn)細(xì)胞凋亡。在體內(nèi)成瘤實(shí)驗(yàn)再次證實(shí)Mi R-154高表達(dá)可以有效抑制NSCLC的生長。3、深入機(jī)制研究尋找到ZEB2是Mi RNA-154的靶基因。Mi R-154高表達(dá)直接與3'-UTR結(jié)合下調(diào)ZEB2基因的表達(dá)來抑制EMT轉(zhuǎn)化,從而抑制肺腺癌A549細(xì)胞的增殖、侵襲和轉(zhuǎn)移能力。綜上所述,我們的研究一方面證實(shí)了Mi R-154與NSCLC生長的相關(guān)性;另一方面找到Mi R-154抑制肺癌的靶向基因。這將有可能為肺癌的分子靶向治療提供新的靶點(diǎn),從而為肺癌的治療提供理論依據(jù),具有一定的臨床應(yīng)用價(jià)值。
[Abstract]:Lung cancer has become one of the most common malignant tumors. The mortality rate of lung cancer is the first of all cancers in adults, which seriously threatens human health. Non-small cell lung cancer (NSCLC) is the main type of lung cancer. The proliferation, invasion and metastasis of NSCLC in human body are very complex biological processes, which directly affect the quality of life and survival of lung cancer patients. It is imperative to study the genesis and development of NSCLC at the molecular level and to develop new diagnostic and therapeutic methods so as to improve the effective survival rate and quality of life of lung cancer patients. Small gene regulators, which are ubiquitous in plants and animals, are associated with physiological processes such as cell growth, development, and apoptosis, especially with tumorigenesis and development. Mi RNA dysfunction and dysfunction can inhibit tumor growth and progression through different signaling pathways. For NSCLC, Mi RNA is associated with lung Micro RNA-154 (Mi R-154) is one of the important members of the Mi RNA family related to carcinogenesis and a very conserved sequence of miRNA in mammals. The low expression of Dou Mi R-154 is related to the occurrence, development, invasion and metastasis of many tumors. The disorder of Dou Mi R-154 may affect many types of cancer, but there are few studies on lung cancer. There is no report about the relationship between them and the effect of Mi R-154 on the genesis and development of NSCLC cells. This study focuses on how Mi R-154 affects the genesis and development of NSCLC and the regulation of target genes, providing important theoretical basis and experimental data for the diagnosis of lung cancer and targeted drug research. The purpose of this study was to investigate the correlation between Mi R-154 imbalance and clinicopathological features in NSCLC, and to analyze the mechanism of Mi R-154 in the occurrence and development of lung cancer, so as to provide theoretical basis for future clinical treatment of NSCLC. Statistical analysis of the effect of Mi R-154 expression on the clinical stage and survival time of NSCLC patients. 2. In vitro cell experiment, the expression level of Mi R-154 was detected by Q RT-PCR, and representative specific cells were screened out. Mi R-154 was transfected into A549 cells, and Q RT-PCR was used to detect the expression level of Mi R-154. The effects of Mi R-154 on the proliferation, invasion and migration of NSCLC cells were examined by cell proliferation test, CCK-8 test, colony formation test, scratch test and cell invasion test; the cell cycle of A549 was analyzed by flow cytometry; apoptosis and epithelial-mesenchymal cell transformation (EMT) markers were detected by Western Blot. Mi R-154 inhibited the growth of NSCLC in vivo. 3. Further studies on the inhibitory effect of Mi R-154 on NSCLC target genes and molecular mechanisms were carried out. ZEB2 was screened as a target gene for Mi R-154 using a public database (Mi Randa, Pic Tar and Target Scan S 6.2). Q RT-PCR, Western Blot, and cell invasion assay confirmed that si-ZEB2 (small interfering RNA) transfection regulates Z. EB2 affects the invasion and metastasis of A549 cells. After pcDNA 3.0-ZEB2 plasmid was constructed, the expression of ZEB2 was verified by Q RT-PCR and Western Blot. Results: 1. The expression of Mi R-154 in NSCLC tumors was significantly decreased by RT-PCR in clinical samples (P 0.05). 2. In vitro cell experiment: RT-PCR was used to detect the expression of Mi R-154 in NSCLC cell lines A549, H1299, SPCA1, H358 and normal lung cells. The expression of Mi R-154 in A549 decreased most significantly, and its specificity was the highest. Mi R-154 could be selected as the representative of further study. 3. Mi R-154 was transfected into A549 cells. RT-PCR results showed that Mi R-154 was transfected into A54 cells. Mi R-154 m RNA expression level increased significantly after 9 (P 0.05), confirming the effectiveness of transfection. 4. Cell proliferation test and colony formation test showed that: compared with Mi R-control and negative control group, Mi R-154 transfected A549 cells proliferation index, colony formation efficiency significantly decreased (P 0.05). The percentage of G0/G1 phase in A549 cells transfected with Mi R-control was significantly higher than that in negative control group (P 0.05). Western Blot assay showed that the expression of caspase 3 and caspase 8 in A549 cells transfected with Mi R-control was significantly higher than that in Mi R-control and negative control group (P 0.05). The results of cell invasion test showed that the migration and invasion ability of A549 cells transfected with Mi R-control and negative control groups were significantly decreased (P 0.05). The Western Blot test showed that the epithelial-mesenchymal transition (EMT) markers of A549 cells transfected with Mi R-control were significantly increased compared with those of Mi R-control and negative control groups. High (P 0.05). 9 Tumor formation in nude mice in vivo showed that the A549 cells transfected with Mi R-control significantly reduced the tumor volume and weight of the grafts in vivo (P 0.05). 10. The results were analyzed using public databases (mi Randa, Pic Tar and Target Scan S 6.2) and bioinformatics test validation screening. ZeB2 was selected as the target gene of Mi R-154. The results of Q RT-PCR and Western Blot showed that the expression of M RNA, vimentin and E-cadherin in the A549 cells transfected with Mi R-154 decreased significantly (P 0.05), and the expression of E-cadherin increased (P 0.05). 12, q-RT-PCR and Western Blot assays showed that the expression of M RNA and protein in ZEB2 transfected with si-ZEB2 significantly decreased (P 0.05). Meanwhile, the invasion ability of A549 cells transfected with si-ZEB2 decreased (P 0.05), which proved that the effect of reducing the high expression of ZEB2 and Mi R-154 was similar. NA3.0-ZEB2 plasmid was transfected into lung adenocarcinoma cell line A549. RT-PCR and Western Blot assay showed that PC DNA 3.0-ZEB2 could significantly increase the expression of ZEB2. These results indicated that the plasmid was successfully constructed and effective after transfection. There was no significant difference between EB2 group and EB2 group (P 0.05), indicating that the high expression of ZEB2 could reverse the inhibitory effect of Mi R-154 on the invasion of lung cancer. Conclusion: 1. Mi R-154 expression in NSCLC tissue was decreased in clinical related studies, and Mi R-154 was found to affect the clinical pathological stage and the survival of NSCLC patients. R-154 may inhibit the growth of NSCLC. 2. To further prove our hypothesis, we then conducted in vitro cell experiments and found that up-regulation of Mi R-154 expression in A549 cells can inhibit their proliferation, invasion and metastasis, and up-regulation of Mi R-154 expression can induce cell cycle arrest and promote cell apoptosis in lung adenocarcinoma A549 cells. The high expression of Mi R-154 can effectively inhibit the growth of NSCLC. 3. In-depth study of the mechanism found that ZEB2 is the target gene of Mi RNA-154. Mi R-154 overexpression directly binds to 3'-UTR and down-regulates the expression of ZEB2 gene to inhibit EMT transformation, thereby inhibiting the proliferation, invasion and metastasis of lung adenocarcinoma A549 cells. On the one hand, the study confirmed the correlation between Mi R-154 and NSCLC growth; on the other hand, we found the targeted gene of Mi R-154 inhibiting lung cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Wan-Qing Chen;Rong-Shou Zheng;Si-Wei Zhang;Hong-Mei Zeng;Xiao-Nong Zou;;The incidences and mortalities of major cancers in China, 2010[J];Chinese Journal of Cancer;2014年08期

2 ;Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non-small cell lung cancer[J];癌癥;2011年06期

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本文編號(hào)：2178268