miR-339表达对肺腺癌A549细胞生物学行为的影响

发布时间：2018-06-18 03:03

本文选题：miR-339 + VEGF　；参考：《郑州大学》2016年博士论文

【摘要】：背景和目的肺癌是目前是全世界肿瘤性疾病死亡率的主要原因,每年可造成多于150万人死亡。目前多数肺癌患者明确诊断时已是晚期,临床症状不明显、早期诊断困难,组织学亚型不同、对肿瘤生物学特征的认识不足导致了肺癌的预后不良及高死亡率。虽然新的分子靶向治疗对于某些类型的肺癌的疗效表现出非常可观的前景,但目前还没有适用于大规模的肺癌病人的靶向治疗。总体来说,肺癌的总体生存率依然不容乐观,很多患者明确诊断后生存期仅仅短短数月。因此,寻找新型肺癌诊断相关的肿瘤标记物或新的治疗策略对于肺癌的控制显得至关重要。miRNA是一段大约由19到25个核苷酸组成的非编码小RNA,通过对其靶m RNA的翻译抑制或者降解在动植物体内发挥重要的调节作用。miRNA是多细胞生物体内众多基因调控分子的重要组成部分之一,同时很可能影响着许多蛋白编码基因的表达输出。miRNA按照其在肿瘤的发生发展中的调控作用可分为致癌miRNA和抑癌miRNA,调控癌细胞的增殖、侵袭、凋亡及血管生成情况。miRNA的表达模式可能与肺癌亚型中临床病理特征有关,提示了miRNA作为生物标志物在肿瘤起源、组织学类型、侵袭性和化学敏感性等分型中的应用潜力。总的来说,涉及肺癌的miRNA范围广泛,let-7家族、miR-34、miR-200、miR-126等miRNAs在肺癌中的作用已被广泛研究。通过查阅相关文献及搜索miRBase数据库,发现miR-339在甲状腺癌、肺癌、卵巢癌、胃癌等多种肿瘤生长及侵袭中起到负向调控作用。miR-339如何调控肿瘤的生物学行为,其具体作用机制尚未阐明,且miR-339在肺癌尤其是临床肺癌组织中的表达情况亦较少研究。目前多数研究者认为肿瘤的生长必须依靠新生血管的生成来提供其足够的氧气和营养物质,因此以抗肿瘤血管为主的治疗成为肿瘤临床治疗的热点,血管内皮生长因子(vascular endothelial growth factor,VEGF)被认为可作用于血管内皮细胞,诱导体内新生血管生成。抑制VEGF的作用也成为了抑制肿瘤生长的临床研究基础。当VEGF与特异性的VEGF受体结合后,会发生一系列的生物效应,能促进血管内皮细胞增殖诱导新血管的生成;增加血管通透性;改变血管内皮细胞的基因及其表达,利于新血管的形成。本研究通过miRNA的靶基因预测数据库对miR-339的靶基因进行预测,对3种靶基因预测数据库所预测的靶基因交集进行功能分析,最终筛选出VEGF作为miR-339的靶基因进行研究。本课题分为三个部分:第一部分通过qRT-PCR及Western blot方法分析41例肺腺癌组织中miR-339和VEGF的表达水平;第二部分采用慢病毒载体技术,建立稳定高表达miR-339的肺腺癌A549细胞株,研究体内外上调miR-339表达对肺腺癌A549细胞增殖、侵袭、化疗敏感性及血管生成的影响;第三部分通过双荧光素酶报告实验及Restore实验进一步研究miR-339对VEGF转录后的调控机制。第一部分41例肺腺癌组织中miR-339和VEGF的表达水平分析方法1.收集41例肺腺癌组织与相应配对的癌旁正常组织标本。2.运用qRT-PCR技术检测41例标本中miR-339及VEGFmRNA的表达水平,Spearman相关分析对二者的相关性进行分析;Western blot方法检测41例标本中VEGF蛋白表达水平。3.分析miR-339与VEGFm RNA在不同年龄、性别、淋巴结转移情况、分化程度、TNM分期病例之间是否存在差异。4.统计学方法:本研究数据应用SPSS 21.0软件进行统计分析,符合正态分布的数据以表示;单因素方差分析用于同时具备正态性及方差齐性数据的多项指标间的比较,LSD-t检验用于多组数据的两两比较;t检验用于两独立样本计量资料的比较,配对t检验用于两配对样本计量资料的比较;Spearman相关分析用于两组数据资料的相关性分析,检验水准α=0.05。结果1.miR-339在肺癌组织中的表达约为癌旁正常组织的1/3,而VEGFm RNA的表达水平是癌旁正常组织的3倍,二者的表达水平之间存在负相关性(r=-0.419,P=0.006)。2.miR-339及VEGFmRNA在不同TNM分期及淋巴结转移情况的肺腺癌组织中表达水平差异有统计学意义(P0.05)。第二部分上调miR-339表达对肺腺癌A549细胞增殖、侵袭、化疗敏感性及血管生成的影响方法1.制备和包装miR-339及无关序列慢病毒表达载体,感染肺腺癌A549细胞,建立稳定高表达miR-339的肺腺癌A549细胞株,实验分miR-339组,NC组,Blank组。2.qRT-PCR检测三组细胞miR-339及VEGFmRNA表达,Western blot检测VEGF蛋白及侵袭相关蛋白MMP2、MMP9表达。3.CCK-8、克隆形成实验检测三组细胞的增殖能力。4.Transwell小室检测三组细胞的侵袭能力。5.CCK-8检测不同浓度顺铂作用下三组细胞的增殖能力。6.建立裸鼠移植瘤模型,应用小动物活体成像仪检测三组裸鼠瘤体的增殖情况。7.小动物活体成像仪检测顺铂作用下裸鼠瘤体的增殖情况。8.免疫组化技术检测移植瘤标本的微血管密度变化。9.采用SPSS 21.0统计软件进行数据分析,检验水准α=0.05。结果1.成功制备miR-339及无关序列重组慢病毒载体,二者重组慢病毒滴度分别为1.6×108TU/m L及2.2×108 TU/m L。miR-339在miR-339组表达水平明显高于NC组及Blank组(P0.05)。2.miR-339组VEGFm RNA表达水平明显低于NC组及Blank组,Westren blot显示miR-339组VEGF、MMP2、MMP9蛋白表达较NC组、Blank组明显降低(P0.05)。3.CCK-8结果显示,miR-339组细胞的吸光度值在24h,48h,72h较Blank组及NC组明显降低,且随时间延长,其差异呈增大趋势(P0.05)。4.克隆形成实验显示,miR-339组克隆形成数较NC组及Blank组明显减少(P0.05)。5.Transwell小室结果显示,miR-339组细胞transwell穿膜细胞数较NC组及Blank组明显减少(P0.05),Blank组及NC组之间穿膜细胞数无明显差异(P0.05)。6.CCK-8检测0μg/m L,4μg/m L,8μg/m L,12μg/m L,16μg/m L,20μg/m L浓度顺铂作用24小时的吸光度值,结果显示,三组细胞的细胞活力随顺铂浓度升高而降低(P0.05);miR-339组细胞在顺铂作用下细胞活力降低程度较NC组及Blank组细胞更大,差异具有统计学意义(P0.05)。7.小动物活体成像系统检测裸鼠移植瘤体内增殖结果显示,从第2周起,miR-339组裸鼠移植瘤荧光信号较Blank组及NC组均明显降低(P0.05),且随时间延长,miR-339组瘤体与Blank组及NC组荧光信号差异愈加显著(P0.05)。8.顺铂作用下miR-339组裸鼠移植瘤荧光信号较Blank组及NC组均明显降低(P0.05),三组裸鼠移植瘤顺铂组的荧光信号均较无顺铂组降低(P0.05),其中miR-339组在顺铂作用下荧光信号下降程度较Blank组及NC组更加显著(P0.05)。9.miR-339组裸鼠移植瘤标本微血管密度较Blank组及NC组相比均明显降低(P0.05)。第三部分miR-339对VEGF转录后调控机理的研究方法1.运用miRNA的靶基因预测数据库对miR-339的靶基因进行预测。2.构建pmir GLO-Wt(野生型)及pmir GLO-Mt(seed region突变型)荧光素酶双报告基因载体,分组转染A549细胞,双报告基因验证miR-339的靶基因。3.构建不含3'UTR VEGF的pcDNA3.1-VEGF表达载体,分别单独转染、与miR-339mimics共转染A549细胞,Western blot方法检测各组细胞VEGF表达情况,Transwell小室检测各组细胞侵袭能力,restore方法分析miR-339对VEGF的调控机制。4.采用SPSS 21.0统计软件进行数据分析。结果1.通过数据库预测VEGF可能为miRNA的靶基因,二者之间存在互补配对的“seed region”区,成功构建pmir GLO-Wt及pmir GLO-Mt报告基因重组载体,并转染A549细胞。2.双荧光素酶报告实验显示miR-339mimics、pmirGLO-Wt共转染组细胞的荧光素酶活性较miR-339scramble、pmir GLO-Wt共转染组及miR-339mimics、pmir GLO-Mt共转染组明显降低,miR-339可作用于VEGF 3’UTR区并产生负调控作用使其表达降低。3.成功构建不含VEGF 3’UTR的pcDNA3.1-VEGF重组表达载体,Restore实验显示,将不含VEGF 3’UTR的pc DNA3.1-VEGF重组载体转染至A549细胞后,可回复上调miR-339对VEGF蛋白表达的抑制作用及miR-339对A549细胞的侵袭抑制作用。结论1.miR-339在肺腺癌组织中呈低表达,VEGF在肺癌组织中呈高表达,二者之间存在负相关,miR-339及VEGF表达水平在不同淋巴结转移情况及不同TNM分期之间存在差异。2.体外上调miR-339表达可降低肺腺癌A549细胞的增殖及侵袭能力,增加细胞对顺铂的敏感性,动物实验表明上调miR-339表达可抑制裸鼠移植瘤的增殖,增加其对顺铂的敏感性,降低瘤组织微血管密度。3.miR-339通过作用于VEGF的3’UTR区对其表达产生负调控作用。
[Abstract]:Background and objective lung cancer is the main cause of mortality in all the world's Oncology, which can cause more than 1 million 500 thousand deaths a year. At present, most patients with lung cancer are clearly diagnosed at the present time, the clinical symptoms are not obvious, the early diagnosis is difficult, the histologic subtypes are different, and the lack of understanding of the biological characteristics of the tumor leads to the prognosis of lung cancer. Poor and high mortality. Although new molecular targeting therapy has shown great prospects for the efficacy of some types of lung cancer, it is not yet suitable for targeted therapy for large scale lung cancer patients. Overall, the overall survival rate of lung cancer is still not optimistic, and many patients have a clear diagnosis of only a few months after the diagnosis. Therefore, looking for tumor markers related to new lung cancer diagnosis or new treatment strategies is important for the control of lung cancer..miRNA is a non coded small RNA, which is composed of approximately 19 to 25 nucleotides, and the.MiRNA is a multicell organism by inhibiting or degradation of the target m RNA. One of the most important components of a number of gene regulatory molecules in the body is likely to affect the expression of a number of protein encoding genes, which can be divided into carcinogenic miRNA and tumor suppressor miRNA in accordance with its regulatory role in the development of tumor, and the expression pattern of.MiRNA, which regulates the proliferation, invasion, apoptosis and angiogenesis of cancer cells, may be regulated. It is associated with the clinicopathological features of lung cancer subtypes, suggesting the potential application of miRNA as a biomarker in tumor origin, histologic type, invasive and chemosensitivity isoforms. Generally, the miRNA range involving lung cancer is wide, and the role of miRNAs in the let-7 family, miR-34, miR-200, miR-126 and other miRNAs in lung cancer has been widely studied. After consulting related literature and searching for miRBase database, it is found that miR-339 plays a negative regulatory role in the growth and invasion of thyroid cancer, lung cancer, ovarian cancer and gastric cancer, and how.MiR-339 regulates the biological behavior of the tumor. Its specific mechanism has not been elucidated, and the expression of miR-339 in lung cancer, especially in lung cancer tissues At present, most researchers believe that the growth of tumor must rely on the formation of new blood vessels to provide enough oxygen and nutrients. Therefore, the antitumor vascular therapy has become a hot spot in the clinical treatment of tumor. Vascular endothelial growth factor (VEGF) is considered to be a function of blood vessels. Endothelial cells, which induce neovascularization in the body, inhibit the role of VEGF as a basis for the inhibition of tumor growth. When VEGF is combined with a specific VEGF receptor, a series of biological effects will occur, which can promote the proliferation of vascular endothelial cells to induce the generation of new blood vessels, increase vascular permeability, and change vascular endothelial cells. Gene and its expression are beneficial to the formation of new blood vessels. In this study, the target gene of the target gene of miR-339 was predicted by the target gene prediction database of miRNA, and the target gene intersection predicted by the 3 target gene prediction database was analyzed. Finally, the target gene of VEGF was selected as the target gene of miR-339. The subject was divided into three parts: the first part The expression level of miR-339 and VEGF in 41 cases of lung adenocarcinoma was analyzed by qRT-PCR and Western blot. The second part of the lung adenocarcinoma A549 cell line with high expression of miR-339 was established by lentivirus vector technology, and the effects of the up regulation of miR-339 expression on the A549 cell proliferation, invasion, chemosensitivity and angiogenesis of lung adenocarcinoma in vivo and in vivo were studied. The third part, through the double Luciferase Report experiment and the Restore experiment, further studies the regulatory mechanism of miR-339 after VEGF transcription. Part 1: analysis of the expression level of miR-339 and VEGF in 41 cases of lung adenocarcinoma 1. to collect 41 cases of lung adenocarcinoma tissue and the corresponding paired normal tissue specimens of the paracancerous tissue,.2. using qRT-PCR technique to detect 41 cases. The expression level of miR-339 and VEGFmRNA in this study, the correlation of Spearman correlation analysis to the two, Western blot method was used to detect the VEGF protein expression level.3. analysis of miR-339 and VEGFm RNA in different age, sex, lymph node metastasis, differentiation degree, TNM staging between the existence of.4. statistical methods: SPSS 21 software is used for statistical analysis in accordance with the data of normal distribution; single factor variance analysis is used for the comparison of multiple indexes with normal and variance homogeneity data. The LSD-t test is used for 22 comparison of multiple groups of data; t test is used for comparison of two independent sample data and paired t test For comparison of two paired sample data, Spearman correlation analysis was used for correlation analysis of two groups of data. The expression of 1.miR-339 in lung cancer tissue was approximately 1/3 in the normal tissues adjacent to cancer, while the expression level of VEGFm RNA was 3 times as high as that of normal tissue adjacent to the cancer, and there was a negative correlation between the level of expression of the two groups. The expression level of r=-0.419, P=0.006.2.miR-339 and VEGFmRNA in the lung adenocarcinoma tissues of different TNM staging and lymph node metastasis was statistically significant (P0.05). Second part up regulation of miR-339 expression on the proliferation, invasion, chemosensitivity and angiogenesis of A549 cells of lung adenocarcinoma 1. preparation and packaging miR-339 and unrelated order The expression vector of lentivirus was used to infect A549 cells of lung adenocarcinoma and to establish a A549 cell line with high expression of miR-339 in lung adenocarcinoma. The expression of miR-339 and VEGFmRNA in three groups was detected in group miR-339, NC and Blank, and Western blot was used to detect the VEGF protein and invasion related protein. Cell proliferation ability.4.Transwell cell detection of three groups of cell invasiveness.5.CCK-8 detection of the proliferation ability of three groups of cells under the action of cisplatin and.6. to establish a nude mouse transplanted tumor model. The proliferation of three groups of nude mice was detected by small animal living imaging apparatus and.7. small animal living body imager was used to detect nude mice under cisplatin The proliferation of.8. immunohistochemical technique was used to detect the microvascular density change of the transplanted tumor specimens..9. was analyzed by SPSS 21 software. The level of alpha =0.05. results 1. was tested to produce miR-339 and unrelated recombinant lentivirus vector successfully. The two of the recombinant lentivirus titers were 1.6 * 108TU/m L and 2.2 x 108 TU/m L.miR-339 respectively in miR-33. The expression level of 9 groups was significantly higher than that of group NC and group Blank (P0.05).2.miR-339 group VEGFm RNA expression level was significantly lower than that of NC group and Blank group. Westren blot showed miR-339 VEGF. The difference showed an increasing trend (P0.05).4. clone formation experiment showed that the number of clone formation in miR-339 group was significantly lower than that of NC group and Blank group (P0.05).5.Transwell chamber results showed that the number of Transwell transmembrane cells in miR-339 group was lower than that of NC and Blank group (P0.05), and the number of membrane cells between the group and the group was not clear. The significant difference (P0.05).6.CCK-8 detected 0 mu g/m L, 4 mu g/m L, 8 mu g/m L, 12 micron L, 16 micron g/m L, and 20 micron concentration of cisplatin for 24 hours. The results showed that the cell viability in the three groups decreased with the increase of cisplatin concentration. P0.05.7. small animal living imaging system detected the proliferation of xenograft in nude mice. From second weeks, the fluorescence signal of miR-339 group was significantly lower than that of group Blank and NC group (P0.05), and the difference of fluorescence signal between miR-339 group and Blank group and NC group was more significant (P0.05).8. along with time. The fluorescence signal of miR-339 group nude mice was significantly lower than that of group Blank and NC group (P0.05). The fluorescence signal of cisplatin group in three groups of nude mice was lower than that in non cisplatin group (P0.05). The decrease of fluorescence signal in miR-339 group was more significant than that in Blank group and NC group (P0.05).9.miR-339 group nude mice transplantation tumor specimen The microvascular density was significantly lower than that of the Blank group and the NC group (P0.05). Third a study on the mechanism of post transcriptional regulation of VEGF. 1. using the target gene prediction database of miRNA to predict the target gene of miR-339 for the construction of pmir GLO-Wt (wild type) and pmir GLO-Mt (mutant type) luciferase double reporter gene carrier A549 cells were transfected into groups, and the double reporter gene validated the target gene.3. of miR-339 to construct pcDNA3.1-VEGF expression vector without 3'UTR VEGF, and transfected separately, respectively, and co transfected with A549 cells with miR-339mimics, Western blot method was used to detect the VEGF expression of each cell, and the Transwell chamber was used to detect the invasion ability of each cell. The regulatory mechanism of VEGF.4. uses SPSS 21 statistical software for data analysis. Results 1. it is predicted that VEGF may be the target gene of miRNA through the database, and the complementary pairing "seed region" region exists between the two and the recombinant vector of pmir GLO-Wt and pmir GLO-Mt reporter gene is successfully constructed, and the experiment of transfection of A549 cells with double Luciferase Report is shown. The luciferase activity of miR-339mimics, pmirGLO-Wt co transfected group was significantly lower than that of miR-339scramble, pmir GLO-Wt co transfection group and miR-339mimics, pmir GLO-Mt co transfection group was significantly reduced, miR-339 could act on VEGF 3 'UTR region and produce negative regulatory action to reduce the recombinant expression of.3. successfully constructed without 3' Restore experiments showed that the transfection of PC DNA3.1-VEGF recombinant vector without VEGF 3 'UTR to A549 cells could restore the inhibitory effect of miR-339 on the expression of VEGF protein and the inhibitory effect of miR-339 on A549 cells. Conclusion 1.miR-339 is expressed in lung adenocarcinoma tissue, and VEGF is highly expressed in lung cancer tissues, and between the two groups. There was a negative correlation. The expression level of miR-339 and VEGF in different lymph node metastases and different TNM stages was different between.2. and miR-339 expression in vitro. The expression of miR-339 could reduce the proliferation and invasion ability of A549 cells in lung adenocarcinoma and increase the sensitivity of cell to cisplatin. Animal experiments showed that up regulation of miR-339 expression could inhibit the proliferation of nude mice. In addition, its sensitivity to cisplatin decreased the microvessel density of tumor tissues..3.miR-339 negatively regulates the expression of VEGF through 3 'UTR region.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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