MicroRNA-154抑制肺癌细胞增殖、侵袭和转移生物学作用研究
发布时间：2018-08-11 21:21
【摘要】：肺癌已成为最常见的恶性肿瘤之一,其死亡率是成年人所有癌症导致死亡的首位,严重威胁着人类的健康。虽然目前对于肺癌的研究以及综合治疗日益深入,但针对肺癌的疗效仍不容乐观,因此肺癌的研究与治疗是我国恶性肿瘤防控的重点。非小细胞肺癌(NSCLC)是肺癌的主要类型。在人体中NSCLC的增殖、侵袭、转移能力是非常复杂的生物学过程,直接影响肺癌患者的生存质量和生存期。寻求诊断NSCLC的特异性标志物和新的治疗靶点,完善个体化治疗,成为当今肺癌研究的当务之急。在分子机制层面研究NSCLC的发生及发展,更好地开发新的诊断和治疗方法,从而提高肺癌患者的有效生存率和生活质量,将成为未来本学科的重要课题。Micro RNA(Mi RNA)是一种小片段非编码单链的RNA,因其具有较高的保守性,是普遍存在于动植物体内的小型基因调控因子,与细胞的生长、发育、凋亡等生理过程相关,尤其是与肿瘤发生和发展过程有着密切的相关性。Mi RNA表达失调和功能障碍可以通过不同的信号通路抑制肿瘤的生长及进展。对NSCLC而言,Mi RNA对肺癌的分子机制具有重要的理论和临床意义,成为NSCLC的潜在的生物学标志以及新的治疗方式,这有待进一步的深入研究。Micro RNA-154(Mi R-154)是Mi RNA家族中与致癌相关的重要成员之一,是哺乳动物一个非常保守的Mi RNA的序列。近期已有报道Mi R-154的低表达与许多肿瘤的发生、发展、侵袭及转移有关,其失调可以影响多种类型的癌症,但在肺癌的研究较少。目前研究发现,在肺癌与正常癌旁组织对比检测Mi R-154呈现低表达,但在NSCLC中Mi R-154表达失调和临床组织病理特征之间的相关性以及Mi R-154对NSCLC细胞的发生发展的机制有何影响,尚未有报道。本课题围绕“Mi R-154如何影响NSCLC的发生和发展以及靶基因的调控”展开深入研究,为诊断肺癌和靶向药物研究提供重要的理论基础和实验数据。研究目的:本研究旨在探讨NSCLC中Mi R-154失调和临床病理特征之间的相关性,分析Mi R-154在肺癌发生发展中的机制,从而为未来NSCLC的临床治疗提供理论依据。研究方法:1、在临床相关研究中收集经手术后病理证实的NSCLC标本40例,通过RT-PCR检测Mi R-154的m RNA水平表达在NSCLC癌组织、癌旁正常肺组织中存在差异。统计分析Mi R-154表达对NSCLC患者临床分期以及生存期的影响。2、体外细胞实验,利用q RT-PCR检测Mi R-154的表达水平,筛选出具有代表特异性的细胞。Mi R-154转染A549细胞,应用q RT-PCR验证转染效率;细胞增值实验、CCK-8实验、集落形成实验、划痕试验和细胞侵袭试验检测Mi R-154对NSCLC细胞的增殖、侵袭、迁移等能力的影响;流式细胞实验分析A549细胞周期;Western Blot检测细胞凋亡与上皮间质细胞转化(EMT)标记物。体内裸鼠成瘤实验验证Mi R-154抑制NSCLC在体内的生长。3、进一步对Mi R-154抑制NSCLC靶基因以及分子机制的研究。使用公共数据库(Mi Randa,Pic Tar和Target Scan S 6.2)筛选ZEB2作为Mi R-154的靶基因。q RT-PCR、Western Blot、细胞侵袭实验验证si-ZEB2(小干扰RNA)转染调节ZEB2影响A549细胞的侵袭和转移能力。构建pc DNA3.0-ZEB2质粒后,q RT-PCR、Western Blot验证ZEB2表达。然后建立:Mi R-154+ZEB2组(将pc DNA3.0-ZEB2质粒和Mi R-154共同转染A549细胞)、Mi R-154组(单独转染Mi R-154组)和对照组(pc DNA3.0空载体转染),利用细胞侵袭实验检测侵袭能力。研究结果:1、临床样本RT-PCR检测结果证实了Mi R-154的表达在NSCLC肿瘤中显著降低(P0.05)。统计分析发现了肿瘤体积的大小,肿瘤的术后TNM病理学分期以及淋巴结转移的增加程度,NSCLC患者生存期均与Mi R-154表达降低相关(P0.05)。2、体外细胞实验:RT-PCR进行检测Mi R-154在NSCLC细胞系A549、H1299、SPCA1、H358和正常肺细胞的表达水平,A549中Mi R-154的表达下降最为显著,其特异性最高,可以被选定为特异性代表进一步研究。3、转染Mi R-154到A549细胞,RT-PCR结果显示Mi R-154转染到A549后Mi R-154的m RNA表达水平显著增高(P0.05),证实了转染的有效性。4、细胞增值实验和集落形成实验结果发现:与转染Mi R-control和阴性对照组相比,转染Mi R-154的A549细胞组增殖指数、集落形成效率显著降低(P0.05)。5、流式细胞检测结果发现:与转染Mi R-control和阴性对照组相比,Mi R-154转染的A549细胞组G0/G1期阶段百分比显著增高(P0.05)。6、Western Blot检测结果显示:与转染Mi R-control和阴性对照组相比,转染Mi R-154的A549细胞组细胞凋亡因子caspase3和caspase8显著增加(P0.05)。7、划痕试验和细胞侵袭试验结果显示:与转染Mi R-control和阴性对照组相比,转染Mi R-154的A549细胞组迁移和侵袭能力明显下降(P0.05)。8、Western Blot检测结果显示:与转染Mi R-control和阴性对照组相比,转染Mi R-154的A549细胞组上皮间质转化(EMT)标志物显著升高(P0.05)。9、在体内裸鼠成瘤实验结果显示:与转染Mi R-control和阴性对照组相比,转染Mi R-154的A549细胞明显降低体内移植物的肿瘤体积和肿瘤重量(P0.05)。10、利用公共数据库(mi Randa,Pic Tar和Target Scan S 6.2)分析结果和生物信息学检测验证筛选出ZEB2可以作为Mi R-154的靶基因。11、q RT-PCR和Western Blot检测结果显示:与转染Mi R-control相比,转染Mi R-154的A549细胞组ZEB2的m RNA表达水平显著降低(P0.05);波形蛋白(Vimentin)表达水平降低(P0.05);E-钙粘蛋白(E-cadherin)的蛋白表达水平增加(P0.05)。12、q RT-PCR和Western Blot检测结果显示:si-ZEB2转染入A549细胞ZEB2的m RNA水平和蛋白水平表达显著降低(P0.05)。同时进行细胞侵袭实验结果显示:si-ZEB2转染入A549细胞侵袭能力下降(P0.05),证明了降低ZEB2与Mi R-154高表达具有类似的效果。13、构建pc DNA3.0-ZEB2质粒转染到肺腺癌A549细胞中,通过RT-PCR和Western Blot检测证明pc DNA3.0-ZEB2能显著提高ZEB2的表达。说明质粒构建成功以及转入后的有效性。14、细胞侵袭实验结果显示:与对照组相比,Mi R-154细胞组迁移和侵袭能力下降(P0.05),Mi R-154与ZEB2组无统计学意义(P0.05),证明了ZEB2高表达可逆转Mi R-154对肺癌侵袭的抑制作用。结论:1、在临床相关研究中证实了Mi R-154在NSCLC组织中m RNA表达水平降低,并在研究过程中发现了Mi R-154影响临床病理分期及NSCLC患者生存期这一现象,推测Mi R-154可能抑制NSCLC生长。2、为了进一步证明我们的推测,然后我们进行体外细胞实验,发现上调A549细胞中Mi R-154表达可以抑制其细胞增殖、侵袭和转移能力,并且上调Mi R-154表达可以诱导肺腺癌A549细胞周期阻滞和促进细胞凋亡。在体内成瘤实验再次证实Mi R-154高表达可以有效抑制NSCLC的生长。3、深入机制研究寻找到ZEB2是Mi RNA-154的靶基因。Mi R-154高表达直接与3'-UTR结合下调ZEB2基因的表达来抑制EMT转化,从而抑制肺腺癌A549细胞的增殖、侵袭和转移能力。综上所述,我们的研究一方面证实了Mi R-154与NSCLC生长的相关性;另一方面找到Mi R-154抑制肺癌的靶向基因。这将有可能为肺癌的分子靶向治疗提供新的靶点,从而为肺癌的治疗提供理论依据,具有一定的临床应用价值。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2178268
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
【参考文献】
相关期刊论文 前2条
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];癌症;2011年06期
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