miR-338在肺癌中的表达及其通过靶向作用于整合素β3抑制肺癌转移的研究

发布时间：2018-04-26 17:11

本文选题：肺癌 + miRNA　；参考：《郑州大学》2016年博士论文

【摘要】：背景及目的肺癌是严重威胁人类生命健康的恶性肿瘤,己经成为全球癌症相关死亡的主要原因。肺癌可分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)两类。手术、化疗、靶向治疗及放疗是肺癌的主要治疗手段。尽管目前肺癌的诊治水平不断进步,但肺癌患者长期生存并不乐观,总体5年生存率只有10%-20%。局部复发和远处转移是导致肺癌患者死亡的主要原因。肺癌的发生、发展是多基因改变共同作用的结果,涉及到多种癌基因的激活和抑癌基因的失活。目前基因靶向治疗己经成为肺癌研究的新热点,发现和认识新的治疗靶点并用于肺癌的诊断和治疗,具有重要的临床价值及重大的现实意义。微小RNA(miRNA,microRNA)长度为19-25个核苷酸的小非编码RNA,广泛存在于动植物体内,影响许多蛋白编码基因的表达。miRNA通过与靶m RNA特异结合可以抑制靶m RNA翻译或降解靶m RNA。miRNA参与调控细胞分化、增殖、生长、迁移、凋亡等许多细胞行为。miRNA表达异常在肿瘤发生、进展和复发中发挥重要作用,扮演着抑癌或促进癌症的作用。miR-9、miR-92b、miR-224和miR-183等为癌基因并促进肺癌转移。miR-101、miR-133a和miR-141等为抑癌基因,可以显著抑制肺癌细胞转移。此外,与肺癌相关的存在于血液,血清,血浆和唾液中的miRNA同样在肺癌中发挥着重要的作用,使miRNA成为一种新的肿瘤标志物。整合素(integrin,ITGB)属于细胞表面分子,是一类由α和β亚基以非共价键形式结合组成的跨膜蛋白受体,介导细胞之间以及细胞与细胞外基质(extracellular matrix,ECM)的粘附。整合素因其跨膜结构的特殊性而具有双向信号转导功能。目前已发现18种α亚基和8种β亚基,可形成24种异源二聚体,分布于全身不同组织和器官,发挥各自的生物学功能。同时,整合素也与疾病的发生和发展密切相关,其信号转导途径也能参与肿瘤的发生和发展。整合素的突变或异常表达通常被认为与肿瘤的发生和转移相关。整合素β3(integrin beta3,ITGB3)属于β亚基中的一员,在全身不同组织中均有表达,参与多种生物学过程,发挥不同的生理功能。ITGB3是多种蛋白质(诸如纤维粘连蛋白、层粘连蛋白、基质金属蛋白酶-2、骨调蛋白和玻连蛋白)的共同受体。已在各种恶性肿瘤中观察到ITGB3表达高度升高。ITGB3对于急性粒细胞性白血病(Acute myeloid leukemia,AML)发生发挥关键作用,使其成为潜在治疗靶点。有研究证实ITGB3是诱发结直肠癌细胞迁移和侵袭的一种重要调节因子。在乳腺癌中,m RNA表达谱排列表明,转移性肿瘤细胞的一些血管生成相关蛋白(包括ITGB3)显著上调。此外,最新研究证明,肺癌组织中表达下调的let-7c,通过靶向作用于ITGB3抑制肺癌细胞的迁移和侵袭。miR-338家族(miR-338,miR-338-3p,miR-338-5p)位于凋亡相关酪氨酸激酶7号内含子上。已经证实miR-338在肝细胞癌、口腔癌和食管鳞状细胞癌中表达下调。miR-338过度表达可以抑制胃癌细胞增殖并促进细胞凋亡。肝细胞癌中的miR-338水平恢复可使癌细胞对索拉非尼治疗敏感。研究已经证实,miR-338能够通过靶向作用于Smoothened抑制结直肠癌细胞侵袭和迁移。但仍然不清楚miR-338在肺癌转移中的作用机制。在本研究中,我们尝试使用实时荧光PCR(q RT-PCR)检测评估5个肺癌细胞株、1个人肺成纤维细胞MRC-5细胞株及115例肺癌组织、癌旁正常肺组织中miR-338的表达。构建miR-338过表达肺癌细胞株,探索在体外环境下miR-338对肺癌细胞增殖、粘附、迁移及侵袭的影响。并检测miR-338过表达肺癌细胞株中ITGB3蛋白质的表达,初步研究miR-338抑制肺癌转移的分子靶向作用机制。本课题包含以下三个部分:第一部分:miR-338在肺癌细胞株及肺癌组织中的表达及意义;第二部分:上调miR-338表达对肺癌细胞株A549、NCI-H292生物学行为的影响;第三部分:miR-338通过靶向作用于整合素β3抑制肺癌转移。第一部分miR-338在肺癌细胞株及肺癌组织中的表达及意义方法:1、培养5个肺癌细胞株(A549、NCI-H292、NCI-H460、NCI-H446、NCI-H1299)和1个人肺成纤维细胞MRC-5细胞株。2、收集手术切除的115例肺癌组织及相对应的癌旁正常肺组织标本。3、提取所有细胞株和115例肺癌组织及相对应的癌旁正常肺组织的RNA,并用q RT-PCR检测miR-338的表达。4、分析肺癌组织中的miR-338表达与患者性别、年龄、吸烟史、肿瘤大小、肿瘤癌栓、淋巴结转移、肿瘤复发及TNM分期之间的相关性。5、使用SPSS 13.0对数据进行分析,并以平均值±标准差的形式表示。使用配对样本t检验估计两组间差异。使用单因素方差分析(ANOVA)分析miR-338表达与临床病理因素之间关系,并使用对数秩检验根据Kaplan-Meier法划出生存曲线。如果p0.05,则认为组间差异具有统计学意义。结果:1、与人肺成纤维细胞MRC-5细胞相比,所有肺癌细胞株中的miR-338表达显著下调(A549 1.0vs0.15±0.01、NCI-H292 1.0vs0.35±0.12、NCI-H4601.0vs0.3±0.02、NCI-H446 1.0vs0.25±0.07、NCI-H1299 1.0vs0.275±0.01,p0.05)。2、与癌旁正常肺组织相比,115例肺癌组织中的miR-338表达显著下调(0vs-2.99±3.69,p0.001)。肺癌组织中的miR-338表达降低与患者性别、年龄、吸烟史、肿瘤大小和淋巴结转移无关(p0.05)。miR-338表达降低与肿瘤癌栓、肿瘤复发、TNM分期相关(p值分别为0.005、0.004、0.025)。低miR-338表达组的5年总生存率显著低于高miR-338表达组(中位生存时间44个月vs53个月,P=0.001)。第二部分:上调miR-338表达对肺癌细胞株A549、NCI-H292生物学行为的影响方法:1、构建、培养重组miR-338慢病毒感染的A549、NCI-H292细胞细胞株,用q RT-PCR检测细胞中miR-338的表达。2、CCK-8法检测重组miR-338慢病毒感染的A549、NCI-H292细胞及对照细胞增殖和生长能力。3、检测重组miR-338慢病毒感染的A549、NCI-H292细胞及对照细胞粘附能力。4、Transwell小室实验检测重组miR-338慢病毒感染的A549、NCI-H292细胞及对照细胞的迁移及侵袭能力。5、裸鼠移植瘤模型实验检测重组miR-338慢病毒感染的NCI-H292细胞及对照细胞对裸鼠肺移植瘤的影响。6、使用SPSS 13.0对数据进行分析,并以平均值±标准差的形式表示。使用配对样本t检验估计两组间差异。如果p0.05,则认为组间差异具有统计学意义。结果:1、重组miR-338慢病毒感染的A549、NCI-H292细胞较对照细胞miR-338的表达明显升高(A549 646.37±35.45vs23.05±3.56、NCI-H292330.86±11.05vs10.54±2.35,P0.05)。成功构建了高表达miR-338的肺癌细胞株。2、在体外环境下上调miR-338的表达可以抑制A549、NCI-H292肺癌细胞的增殖(P0.05)、粘附(A549平均细胞计数1233±249vs310±22,p=0.035)(NCI-H292平均细胞计数180±16vs72±10,p=0.028)、迁移(A549平均细胞计数347±41vs190±8,p=0.029)(NCI-H292平均细胞计数287±33vs127±21,p=0.045)及侵袭能力(A549平均细胞计数197±12vs68±14,p=0.008)(NCI-H292平均细胞计数106±10vs41±3,p=0.006)。3、裸鼠异种移植肿瘤模型实验表明,miR-338高表达能有效地抑制裸鼠肺移植瘤的生长(平均肿瘤克隆数20±7vs4±2,p=0.004)。第三部分miR-338通过靶向作用于整合素β3抑制肺癌转移方法:1、生物信息学分析推测ITGB3为miR-338靶基因。2、用Western免疫印迹检测miR-338过度表达肺癌细胞株A549和NCI-H292及对照细胞中的ITGB3蛋白质表达。3、双荧光素酶报告检测验证miR-338靶基因。4、使用SPSS 13.0对数据进行分析,并以平均值±标准差的形式表示。使用配对样本t检验估计两组间差异。如果p0.05,则认为组间差异具有统计学意义。结果:1、miR-338过表达肺癌细胞株A549和NCI-H292中的ITGB3蛋白质表达较对照细胞均明显降低(A549 1.0vs0.35±0.04,p0.001)(NCI-H2921.0vs0.69±0.07,p=0.01)。2、双荧光素酶报告检测结果显示miR-338过表达组的荧光素酶活性较对照组降低(A549 1.0vs0.75±0.08,p=0.03)(NCI-H292 1.0vs0.62±0.06,p=0.008)。结论:1、在肺癌细胞株及肺癌组织中,miR-338表达水平较正常细胞及组织显著降低。在肺癌组织中miR-338表达水平与患者性别、年龄、吸烟史、肿瘤大小和淋巴结转移无关,而与肿瘤癌栓、肿瘤复发、TNM分期相关。低miR-338表达组的5年总生存率显著低于高miR-338表达组。2、体外上调肺癌细胞中miR-338表达水平可有效抑制肺癌细胞的增殖、粘附能力,降低肺癌细胞的迁移和侵袭能力。3、miR-338是通过作用于靶基因ITGB3 mRNA3’UTR区,从而对其表达产生负调控作用。miR-338发挥抑癌作用。ITGB3是一种新的miR-338肺癌靶基因,为肺癌基因治疗潜在的新靶点。
[Abstract]:Background and objective lung cancer is a malignant tumor that seriously threatens human life and health. It has become the main cause of global cancer related death. Lung cancer can be divided into two categories: small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC). Surgery, chemotherapy, targeted therapy and radiotherapy are the main treatment methods for lung cancer. Although the diagnosis and treatment of lung cancer is constantly at present Progress, but the long-term survival of lung cancer patients is not optimistic. The overall 5 year survival rate is only 10%-20%. local recurrence and distant metastasis is the main cause of the death of lung cancer patients. The development of lung cancer is the result of the common effect of multiple gene changes, involving the activation of a variety of oncogenes and the inactivation of tumor suppressor genes. It has become a new hot spot in the research of lung cancer. It is of important clinical value and great practical significance to discover and recognize new therapeutic targets and to be used in the diagnosis and treatment of lung cancer. Small RNA (miRNA, microRNA) is a small noncoding RNA with 19-25 nucleotides in length, which is widely used in animals and plants and affects the expression of many protein encoded genes in.MiRNA Specific binding to target m RNA can inhibit target m RNA translation or degradation of target m RNA.miRNA to participate in the regulation of cell differentiation, proliferation, growth, migration, apoptosis and many other cellular.MiRNA expressions, which play an important role in tumor occurrence, progression and recurrence, playing the role of cancer inhibiting or promoting cancer, miR-92b, miR-224 and miR-183, and so on. The oncogene and promoting the metastasis of lung cancer,.MiR-101, miR-133a and miR-141 as tumor suppressor genes, can significantly inhibit the metastasis of lung cancer cells. In addition, the miRNA associated with lung cancer in the blood, serum, plasma and saliva also plays an important role in lung cancer, making miRNA a new tumor marker. Integrin (integrin, ITGB). Cell surface molecules, a class of transmembrane protein receptors composed of alpha and beta subunits, which are composed of non covalent bonds, mediate adhesion between cells and cells and the extracellular matrix (extracellular matrix, ECM). Integrins have bi-directional signal transduction function because of their transmembrane structure. 18 subunits and 8 beta subunits have been found. It can form 24 kinds of heterologous two polymers, distributed in different tissues and organs of the whole body, and play their respective biological functions. At the same time, integrin is also closely related to the occurrence and development of disease, and its signal transduction pathway can also participate in the occurrence and development of tumor. Integrin beta 3 (integrin beta3, ITGB3), a member of the beta subunit, is expressed in various tissues throughout the body, participates in a variety of biological processes and plays a different physiological function,.ITGB3 is a common receptor for a variety of proteins, such as fibronectin, laminin, matrix metalloproteinase -2, osteosin and bosin protein. A high elevation of ITGB3 expression is observed in various malignant tumors and.ITGB3 plays a key role in the occurrence of acute myelocytic leukemia (Acute myeloid leukemia, AML), making it a potential therapeutic target. Studies have shown that ITGB3 is an important regulator of the migration and invasion of colorectal cancer cells. In breast cancer, the RNA expression profile of M The list shows that some angiogenesis related proteins (including ITGB3) are significantly up-regulated in metastatic tumor cells. In addition, the latest research has shown that Let-7c, expressed in lung cancer tissue, can inhibit the migration and invasion of lung cancer cells by targeting ITGB3 (miR-338, miR-338-3p, miR-338-5p) in the.MiR-338 family (miR-338, miR-338-3p, miR-338-5p) in the apoptosis related tyrosine kinase No. On the intron. It has been confirmed that the overexpression of miR-338 in the hepatocellular carcinoma, oral and esophageal squamous cell carcinoma can inhibit the proliferation of gastric cancer cells and promote cell apoptosis. The recovery of miR-338 in hepatocellular carcinoma can make the cancer cells sensitive to the Sola Fini treatment. The research has confirmed that miR-338 can be targeted by the target. Smoothened inhibits the invasion and migration of colorectal cancer cells. But it is still unclear how miR-338 plays a role in the metastasis of lung cancer. In this study, we tried to evaluate 5 lung cancer cell lines, 1 pulmonary fibroblasts MRC-5 cell lines, 115 lung cancer tissues, and 115 lung cancer tissues, and miR-338 in normal lung tissues by using real-time fluorescence PCR (Q RT-PCR) detection. To express the expression of miR-338 over expression of lung cancer cell lines, explore the effect of miR-338 on proliferation, adhesion, migration and invasion of lung cancer cells in vitro, and detect the expression of ITGB3 protein in miR-338 overexpressed lung cancer cell lines, and preliminary study the molecular targeting mechanism of miR-338 to inhibit the metastasis of lung cancer. This topic includes the following three parts: Part one: expression and significance of miR-338 in lung cancer cell lines and lung cancer tissues; the second part: the effect of up regulation of miR-338 expression on the biological behavior of lung cancer cell line A549 and NCI-H292; the third part: miR-338 inhibits metastasis of lung cancer by targeting integrin beta 3. The expression of first division miR-338 in lung cancer cell lines and lung cancer tissues Significance methods: 1, 5 lung cancer cell lines (A549, NCI-H292, NCI-H460, NCI-H446, NCI-H1299) and 1 individual pulmonary fibroblast MRC-5 cell lines.2 were used to collect 115 cases of surgically removed lung cancer tissues and corresponding normal lung tissue specimens adjacent to the cancerous lung tissue, and extract all the cell lines and 115 cases of lung cancer tissue and the corresponding normal lung tissue adjacent to the cancerous lung tissues. The expression of.4 in miR-338 was detected by Q RT-PCR, and the correlation between miR-338 expression in lung cancer tissues and the sex, age, smoking history, tumor size, tumor tumor thrombus, lymph node metastasis, tumor recurrence and TNM staging were analyzed, and the data were analyzed with SPSS 13 and expressed in the form of mean mean standard deviation. The paired sample t test was used. A single factor variance analysis (ANOVA) was used to analyze the relationship between the miR-338 expression and the clinicopathological factors, and the logarithmic rank test was used to draw a survival curve according to the Kaplan-Meier method. If P0.05, the difference between the groups was statistically significant. Results: 1, all lung cancer cells were thinner than the human lung fibroblast MRC-5 cells. The expression of miR-338 in the cell lines was significantly down (A549 1.0vs0.15 + 0.01, NCI-H292 1.0vs0.35 + 0.12, NCI-H4601.0vs0.3 + 0.02, NCI-H446 1.0vs0.25 + 0.07, NCI-H1299 1.0vs0.275 + 0.01, P0.05).2. Compared with normal lung tissue adjacent to the carcinoma, 115 cases of lung cancer tissues were significantly down regulated. The decrease of expression was not related to the sex, age, smoking history, tumor size and lymph node metastasis (P0.05).MiR-338 expression decreased with tumor thrombus, tumor recurrence, and TNM staging (P value was 0.005,0.004,0.025). The total 5 year survival rate of low miR-338 expression group was significantly lower than that of high miR-338 expression group (median survival time was 44 months vs53 months, P=0.001 The second part: the second part: up regulation of the effects of miR-338 expression on the biological behavior of lung cancer cell line A549, NCI-H292, 1, construction, culture of A549, NCI-H292 cell lines, Q RT-PCR detection of miR-338 expression.2, CCK-8 method for detecting recombinant lentivirus infection Colonization and growth capacity.3, the adhesion ability of recombinant miR-338 lentivirus infection A549, NCI-H292 cells and control cells.4, Transwell chamber test to detect the A549 of recombinant miR-338 lentivirus infection, the migration and invasion ability of NCI-H292 cells and control cells,.5, nude mice transplantation tumor model test for the detection of recombinant miR-338 lentivirus infection NCI-H292 The effect of cell and control cells on lung xenografts in nude mice.6, SPSS 13 was used to analyze the data and expressed in the form of mean standard deviation. The difference between the two groups was estimated by paired sample t test. If P0.05, the difference between the groups was statistically significant. Results: 1, the A549 of recombinant miR-338 lentivirus infection, NCI-H292 cells were compared. The expression of miR-338 in the control cells increased significantly (A549 646.37 + 35.45vs23.05 + 3.56, NCI-H292330.86 + 11.05vs10.54 + 2.35, P0.05). The lung cancer cell line with high expression of miR-338 was successfully constructed. The expression of miR-338 under the environment in vitro could inhibit the proliferation of A549, NCI-H292 lung cancer cells (P0.05), and the average cell count was 1233 + 249. Vs310 + 22, p=0.035) (average cell count of NCI-H292 180 + 16vs72 + 10, p=0.028), migration (average cell count of A549 347 + 41vs190 + 8, p=0.029) (NCI-H292 mean cell count 287 + 33vs127 + 21, p=0.045) and invasion (average cell count 197 + 14 + 14.) The model experiment showed that high expression of miR-338 could effectively inhibit the growth of lung xenografts in nude mice (average number of tumor clones 20 + 7vs4 + 2, p=0.004). Third part of miR-338 could inhibit the metastasis of lung cancer by targeting integrin beta 3: 1, bioinformatics analysis of ITGB3 as a miR-338 target gene.2, using Western immunoblotting MiR-338 overexpressed the expression of ITGB3 protein in the lung cancer cell lines A549 and NCI-H292 and the ITGB3 protein in the control cells, and the dual luciferase reporter detection and verification of the miR-338 target gene.4. The data were analyzed using SPSS 13 and expressed in the form of mean standard deviation. The paired sample t test was used to estimate the difference between the two groups. If P0.05, it was considered between the groups. The difference was statistically significant. Results: 1, the expression of ITGB3 protein in the miR-338 overexpressed lung cancer cell lines A549 and NCI-H292 was significantly lower than that of the control cells (A549 1.0vs0.35 + 0.04, p0.001) (NCI-H2921.0vs0.69 + 0.07, p=0.01).2. The results of the double Luciferase Report showed that the luciferase activity of the miR-338 overexpression group was lower than that of the control group. Low (A549 1.0vs0.75 + 0.08, p=0.03) (NCI-H292 1.0vs0.62 + 0.06, p=0.008). Conclusion: 1, in lung cancer cell lines and lung cancer tissues, the expression level of miR-338 is significantly lower than that of normal cells and tissues. The level of miR-338 expression in lung cancer tissues is not related to sex, age, smoking history, tumor size and lymph node metastasis, but with tumor thrombus and swelling. Tumor recurrence and TNM staging. The total 5 year survival rate of low miR-338 expression group was significantly lower than that of high miR-338 expression group.2. The expression level of miR-338 in lung cancer cells in vitro could effectively inhibit the proliferation of lung cancer cells, adhesion, and reduce the migration and invasion of lung cancer cells.3, miR-338 is through the target gene ITGB3 mRNA3 'UTR area, from the target gene. .miR-338 plays a negative role in regulating its expression..ITGB3 is a new target gene for miR-338 lung cancer. It is a potential new target for gene therapy of lung cancer.

【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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