【作者】 李金东；

【导师】 张兴义；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 目的:探讨应用RNAi技术沉默Med19基因抑制人肺癌A549细胞生长、转移的效果。方法:①以Med19己知序列为靶点,构建siRNA-Med19重组慢病毒载体。②siRNA-Med19慢病毒表达载体与pCDNA3.1(-)- Med19过表达载体共转染293T细胞,Western blot外源筛选靶点。③将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,应用Real-time PCR和Western blot检测Med19基因的转录水平及蛋白表达;应用流式细胞术检测细胞周期及凋亡峰;应用MTT、基底膜侵袭实验、细胞克隆形成实验检测细胞增殖、侵袭能力及细胞凋亡。④将siRNA-Med19慢病毒表达载体转染人肺癌A549细胞株,复制裸鼠移植瘤模型,测量肿瘤组织体积及重量,连续病理组织切片, HE染色观察移植瘤及转移瘤结节,并计算各组转移瘤数目和转移率。结果:①成功构建siRNA-Med19重组慢病毒载体,并经基因测序证实。②成功构建目的基因过表达载体pCDNA3.1-Med19,并与siRNA-Med19重组慢病毒载体共转染293T细胞,Western blot外源验证靶点的有效性。③Real-time PCR及Western blot结果显示,转染siRNA-Med19慢病毒表达载体组中Med19的基因转录及蛋白表达水平明显降低。流式细胞术、MTT、基底膜侵袭实验、细胞克隆形成实验结果显示:siRNA-Med19慢病毒表达载体转染组抑制细胞增殖、侵袭,检测到细胞凋亡。④成功建立裸鼠移植人肺癌模型,siRNA-Med19慢病毒表达载体转染组肿瘤体积及重量均低于对照组。连续病理组织切片观察转染siRNA-Med19慢病毒表达载体组肝脏、肺脏转移率低、转移瘤数目少。结论:应用RNAi技术沉默Med19基因可以降低人肺癌A549细胞的转录、表达,导致细胞凋亡,抑制肿瘤的生长、侵袭及转移。更多还原

【Abstract】 Lung cancer as a common seen malignant tumor in the world has become major reason leading to death of cancer for most of countries, and at present it is considered the biggest malignant tumor to human health and life-threatening. In 2005, statistic data showed that there were 1,350,000 new cases of lung cancer each year in the world, and there were about 1,180,000 deaths cases of lung cancer [130], i.e., someone died of lung cancer on average less than 30 seconds in the world. Lung cancer will become the first killer in the new century. Around the world, epidemiology has shown that the incidences of lung cancer showed constant rise tendency. In America, lung cancer that has surpassed the sum of casualty of three main tumors namely prostate cancer, breast cancer and colon cancer (Greenlee RT et. al, 2001) occupies the first position in cancer casualty (Black, W.C. et. al, 2007). In 1990, there were 154,000 new cases of lung cancer in the US, 146,000 people died of lung cancer; in 2000 there were 16940,000 new cases of lung cancer and 154,900 died of lung cancer [122]. In 2002, there were 172,000 new cases of lung cancer in the United States and 157,000 people died of lung cancer, the mortality rate was 90% [131]. In recent years, with China’s social progress and the process of industrial modernization, which increases environmental pollution, as well as an increase in smokers caused china morbidity and mortality of lung cancer constant rise tendency. According to the New England Journal in 2005 reported [1] that Cancer had become the first cause of death in Chinese men (374.1/10 million), the third cause of death in Chinese women (214.1/10 million), mortality rate for men’s lung cancer was (96.9/10 million), mortality rate for women’s lung cancer was (46.7/10 million), both were the first cause of cancer casualty. Surgery has been regarded as one of the most important means in comprehensive treatment of lung cancer. Despite rapid advances in surgical technique and method, limiting in improved survival rate has been limmited. Clinical research of Lung cancer radiotherapy and chemotherapy is made by random comparison of different periods and different options. Researchers are looking for better radiotherapy and chemotherapy methods with high efficacy and lower toxicity to improve patients quality of life, alleviate a variety of chemotherapy toxicity, while attention has been paid to targeted therapy. It is gratifying that the therapy of gene level has achieved rapid development in recent years.RNA polymeraseⅡis essential for eucaryote genetic transcription, transcripts all precursors mRNA and the majority small nuclear RNA (snRNA), mRNA transcription impacts directly on the follow-up translation process from mRNA to protein, and further affects the protein function exercise. The study on RNA polymeraseⅡshowed that the latter only had transcription function on the condition adding general transcription factor compound to the purified RNA polymeraseⅡ, general transcription factor compound composed with five protein factors were required by all transcription activities, their existence was basic condition to sustain the basal transcription (basal transcription), in addition to the above-mentioned general transcription factor, RNA polymeraseⅡalso needed another protein compound. The function of the protein compound is to transmit various control signals to RNA polymeraseⅡand general transcription factor. The compound is composed with nearly 20 proteins [57]. It was named Mediator (intermediary). Mediator is an important component of the RNA polymeraseⅡgeneral transcription device, the Med compound with different kinase components plays a control function for different types of gene [8]. Conformational change of Med will produce a surface to collect and assemble to PIC components in RNA polymeraseⅡand general transcription factor, this is an important aspect of the Med at the role of transcription mediation[9], and plays a key role in activation and inhibition of eukaryotic mRNA synthesis. Genetic name of Med19 in human encode is associated protein of metastasis of lung cancer, plays an important role in the cell growth. It can directly or indirectly participate and mediate a variety of signaling pathways which are highly expressed in lung cancer cell line 95D with high metastasis capacity, but lowly expressed in lung cancer cell lines 95C with low metastasis capacity. To adjust and control transcription and expression of Med19Gene’s can prevent invasion and metastasis of lung cancer. Therefore, the application of siRNA interfering Med19 gene may be one of the most effective option prospects of gene therapy for lung cancer.Med19 gene is closely related with cell proliferation, differentiation and apoptosis. Difference expression study with DNA chip through closed antisense digonucleotides LCMR1 is adopted to investigate many signals transduction genes capable of inhibiting tumor cell proliferation, invasion, and promote tumor cell apoptosis such as tumor necrosis factor receptor (TNF RSF10), tumor necrosis factor super family (TNF SF10), serine (cysteine) protease inhibitors (SERPINB8), bone morphogenetic protein receptorⅡ(BMPR2), serine/threonine kinase 2 (STK2), serine/threonine kinase 3 (STK3), G proteinβ5 (GNB4), G protein 4 (GNG4), IL-3 byα(L3RA) and so on. Signal transduction gene which plays a very important positive role in cell proliferation, differentiation and the construction of the cytoskeleton is lowly expressed, mainly includes ras homology gene family member C (ARHC), ras homology gene family member A (ARHA). Signal transduction and gene in regulation of apoptosis and cell cycle occupies a large proportion in the differential expression gene, but three parts correlate and influence each other, and constitute an extremely complex and sophisticated regulatory network.Initial speculation is that LCMRI gene can probably play a role in signal transduction pathway by regulating cell proliferation, differentiation, cell cycle regulation and suppression of apoptosis. We believe that the construction of siRNA-lentivirus expression vector interfere with Med19 can inhibit cell growth and proliferation, and promote apoptosis.It has been confirmed that blocking Med19 gene can induce tumor apoptosis and growth inhibition. Therefore, cancer gene therapy regarding Med19 as target can block upstream and downstream cancer-causing factors and cancer-causing gene only by blocking a Med19 protein which is more stronger specificity and higher application value to therapy of some or suppression of cancer comparing to therapy for a simple oncogene or tumor suppressor gene and provides an ideal candidate target goal for gene therapy for malignant tumorChoice of target genes should be the first consideration for gene therapy. As tumor recurrence and development relates to oncogene change, blocking a bridge of general transcription device–Mediator can probably be one of the most effective methods for the cancer therapy. Studies have shown that blocking Med19 can block various downstream anomalistic tumor signals leading to transcription and translation stop, consequently Med19 may have potential as the ideal molecular target for treatment of malignancies. Theoretically, blocking tumor cell Med19 gene signal transduction pathway can probably play a role in the treatment for cancers.RNAi refers to one phenomenon which the normal biology inhibits gene expression. Once double-stranded RNA(double stranded RNA, dsRNA) homology with endogenous mRNA encoding region is introduced into cells, it can trigger a post-transcriptional control procedures to specifically identify mRNA with homologous sequences give rise to block of translation function, a phenomenon, that is known as posttranscriptional gene silencing (posttranscriptional gene silencing, PTGS). Due to RNAi with specificity and efficiency, this technology has become an important tool for the study of gene function, and can probably play a role in viral, genetic diseases and the treatment of tumor diseases. The siRNA lentivirus expression vector by application of encoding shorthairpin RNA (short hairpin RNA, shRNA) can inhibit gene expression in the human cells for steady-long time.Med19 gene has not been fully understood about its molecular mechanism in the pathogenesis of lung cancer. This study constructs siRNA lentivirus eukaryotic expression vector of Med19 specificity and transfected into human lung cancer cell line A549, understand the reorganization lentivirus vector interference result on Med19 gene of A549 cells, as well as influence to lung cancer cell growth and proliferation, invasion and metastasis, cell cycle and apoptosis, further explores Med19 gene role in the occurrence and development of lung cancer, provides a new means and theoretical foundation for RNA interference (RNAi) molecular target gene therapy for lung cancer.Objective: to construct siRNA-Med19 lentivirus expression vector and study its inhibitory effect on human lung cancer A549 cells in vitro and in vivo.Method:(1) Construction of lentivirus expression vector: according to known sequence of Med19 gene mRNA in the genebank to determine the appropriate target site, synthesize DNA templates of encoding siRNA, connect Med19siRNA annealed template oligonucleotide to linearized pGCSIL-GFP expression vector, and to construct siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion;(2) Construction of target gene over-expression vector: primer design, the use of PCR to amplify Med19 functional gene, to connect with eukaryotic over-expression vector after digestion or linearization, to construct pCDNA3.1(-)-MED19 over-expression vector by sequencing and enzyme digestion;(3) Selection of exogenous target by Western blot: cotransfection of 293T cells with siRNA-Med19 lentivirus expression vector and pCDNA3.1(-)-MED19 over-expression vector, exogenous target selection with Western blot.(4) Packaging and titer determination of siRNA-Med19 lentivirus vector: to pack three plasmid vector with the best target siRNA-Med19 lentivirus expression vector and pHelper 1.0 vector and vector pHelper 2.0 in 293T cells, and to demarcate virus titer.(5) In vitro study: transfection of human lung cancer A549 cell line with siRNA-Med19 lentivirus expression vector; application of Real-time PCR and Western blot to determine transcription levels of Med19 gene and protein expression; applying flow cytometry to test cell cycle and apoptosis peak; to applying MTT, the basement membrane invasion assay and cell colony to form assay cell proliferation, invasion ability and apoptosis. (6) In vivo study: Nude mice transplanted human lung cancer model was establish by s.c. injecting transfected A549 cells with siRNA-Med19 lentivirus expression vector followed by; measurement of tumor size and weight, and observation of the liver and lung metastasis, and tissue section and HE staining histology to apply to study transplanted tumor and metastatic tumor nodules, and calculate the number of metastatic tumor and metastatic rate in each group.Results:(1) successful construction of siRNA-Med19 lentivirus expression vector by sequencing and enzyme digestion(2) successful construction of target gene over-expression vector pCDNA3.1 (-)- MED19. by sequencing and enzyme digestion.(3) co-transfection of 293T cells was performed with greater than 90% fluorescence expression of the rate. Exogenous selection by Western blot showed that target 4 # is the best target.(4) High-titer lentivirus liquid was prepared after packaging and enrichment of siRNA-Med19 lentivirus vector. The virus titer was 2E+9 TU/ml, which was measured and calibrated in 293T cells.(5) In vitro data revealed that siRNA-Med19 lentivirus expression vector can inhibit proliferation, invasion, and promote apoptosis of human lung cancer A549 cells. Real-time PCR and Western blot results showed that expression level of Med19 mRNA and protein of Med19 was significantly reduced after transfection of A549 cells with siRNA-Med19 lentivirus expression vector. Flow cytometry, MTT, basement membrane invasion assay, and cell colony-forming assay showed that siRNA-Med19 can significantly inhibit cell proliferation, invasion, induce apoptosis of A549 cells.(6) We have successfully established human lung cancer A549 xenograft model in nude mice. Tumor volume and weight in animals treated with Med19 mRNA was lower than the control groups. Continuous observation of the pathological tissue revealed that liver and lung metastases were significantly lowered in animals treated with Med19 mRNAe.Conclusion: (1) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can specifically and efficiently interfere with human lung cancer A549 cells, and inhibit gene expression of Med19 leading to inhibition of proliferation and invasion and apoptosis induction of A549 cells. Med19 prompt signal pathway is in an ideal molecular target for gene therapy of human lung cancer which is expected to be one of the reasonable strategies.(2) This study represents the first study to demonstrate that siRNA-Med19 lentivirus can effectively inhibit growth and metastasis of transplant A549 tumor mice model in vivo.更多还原