【作者】 刘政；

【导师】 邱广斌；

【作者基本信息】 吉林大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 胃癌是发病率最高的恶性肿瘤之一,但其发生的确切分子机制尚不清楚,临床诊治尚无特别有效的办法。因此,研究在其发生过程中不同阶段起作用的关键基因,是对胃癌进行早期诊断和有效的临床干预的基础。MYCT-1是一个新的候选抑癌基因。对胃癌中MYCT-1基因表达的研究发现,MYCT-1在76%的胃癌组织中表达下调,且表达水平在正常组织、癌组织和转移癌组织中依次降低。基因转染实验表明MYCT-1过表达可抑制胃癌BGC823细胞生长,促进细胞凋亡。说明MYCT-1表达降低在胃癌发生、发展过程中起重要作用。本研究以人胃粘膜细胞系GES-1细胞为模型,利用RNA干涉技术在mRNA水平上沉默MYCT-1基因表达,探讨其对正常胃粘膜细胞生长、凋亡及细胞周期的影响,并通过基因芯片技术分析MYCT-1调控的下游基因。结果显示：1.MYCT-1基因沉默后细胞凋亡率明显增高,细胞增殖率降低,细胞周期S期延长；2.MYCT-1基因维持一定的表达水平对正常细胞的生长是必需的,其功能主要是抑制DNA复制；3.筛选出MYCT-1基因调控的下游靶基因80个,表达上调的45个,表达下调的35个。总之,MYCT-1作为一个新的候选抑癌基因,其维持一定的表达水平对胃粘膜细胞的生长是必需的,而其表达下调可能是胃癌发生的原因之一。因此,MYCT-1基因可能成为对胃癌进行早期诊断和有效的临床干预的重要靶点,对胃癌的防治具有极其重要的作用。更多还原

【Abstract】 The incidence of gastric cancer is one of the highest in malignant tumors. Research on etiology of gastric cancer showed that gastric carcinogenesis was related to environmental factors and genetic factors. But the exact molecular mechanism of gastric cancer is not clear yet, and there is no particularly effective solution on clinical diagnosis or treatment. Therefore, the investigation of the key genes in different stages of gastric carcinogenesis is the basement to early diagnosis and effective clinical intervention, and may play an important role in treatment and prevention of gastric cancer.MYCT-1 is a novel candidate tumor suppressor gene. Data indicated that MYCT-1 was an important member in c-Myc cell signal transduction pathway. It may locate on a unique position and functions as a transcription factor in this pathway. Previous research demonstrated that MYCT-1 gene was down regulated in 76% gastric cancer tissues, and the expression level reduced in normal tissue, cancer tissues and metastasis tissues in turn. Gene transfection experiments showed that over-expression of MYCT-1 gene could inhibit the growth of BGC823 cells and promote apoptosis, which means down-expression of MYCT-1 gene plays an important role in gastric cancer occurrence and development.In this study, we aim to explore the effection of MYCT-1 gene in activation in cell growth, apoptosis and cell cycle using RNA interference in the normal gastric mucosal cell line GES-1 and analysis the MYCT-1 gene downstream regulation genes by gene chip technology to clarify the exact role of MYCT-1 gene in gastric cancer.Objective:To investigate the mechanism of MYCT-1 gene in carcinogenesis of gastric cancer.Methods:GES-1 cells were distributed into three groups:①blank group: transfecting the cells without transfection agent or siRNA;②control group: transfecting the cells with the siRNA NC;③experimental group:transfecting the cells with siRNA. The MYCT-1 siRNA was successfully transfected to the GES-1 cell by using RNA interference, The mRNA and protein expression levels were detected by RT-PCR and Western blot. The cell cycle, apoptosis and cell proliferation were detected by PI staining flow cytometry analysis, Annexin V FITC/PI double staining flow cytometry analysis and MTT assay, respectively. The gene expression was detected by the human genome-wide microarray. Results:(1) Silencing the MYCT-1 gene using RNA interferenceMYCT-1 gene specific siRNA was designed and synthesized and then transfected into GES-1 cells. RT-PCR results showed that in all groups we can find the 504bp of MYCT-1 gene and 480bp ofβ-actin DNA bands. But compared with blank group and control group, a more distinct band was detected in the experimental group, which means the MYCT-1 gene expression significantly reduced. The Western blot results was consistent with that of RT-PCR. We could detect the expression of MYCT-1 protein in the control group but a faint band in the experimental group. Compared with the control group, the expression of protein in the experimental group decreased significantly (P＜0.05). We successful reduced the MYCT-1 gene expression in cell line GES-1 by using RNA interference.(2) The effection of MYCT-1 gene silencing in the GES-1 cellsThe cell cycle detection results showed that, compared with the control group, Gl (43.04%) and G2 (2.19%) phase were reduced while S (53.76%) phase was increased in the experimental group (P＜0.05). There is no significant difference between the blank group and control group (P＞0.05). The apoptosis test showed that compared with the blank group and control group, apoptosis rate in the experimental group increased significantly (p＜0.01). The cell proliferation results showed that the siRNA effectively inhibited the growth of GES-1 cells in experimental group. The cell survival rates were 56.8±2.6% and 89.1±3.7% in the experimental group and control group respectively. The cell proliferation in experimental group was significantly lower than that in the control group (P＜0.05). We consider that the MYCT-1 gene down expression can prolong the S phase, duplicate a large number DNA, and produce a lot of polyploid cells, which can not survive and die quickly. This may lead to the apoptosis rate increased after RNA interference, DNA replication prone to a large number of genetic variation, and the majority of tumor gene is somatic cell gene mutation. Therefore, we think MYCT-1 gene is a tumor suppressor gene, which maintain a certain expression level is necessary to the growth of normal cells. MYCT-1 gene expression level and cell growth regulation is dose-response, and a further study should be performed to make sure its regulational signaling pathway.(3) Detect downstream target genes using Microarray screeningWe compared to 54000 genes expression before and after the RNA interference using the AFFYMETRIX human genome-wide chip. Total 1264 genes upregulation and 3887 genes downregulation were detected. In the upregulation genes,45 genes increase four times including 2 genes were relative with apoptosis,3 genes were relative with cell proliferation,1 gene was relative with cell cycle,2 genes were relative with transcription regulation and 15 unknown function genes. In the downregulation genes,35 genes decrease four times including 2 genes were relative with apoptosis,1 gene was relative with cell proliferation,2 genes were relative with cell cycle,1 gene was relative with transcription regulation and 11 unknown function genes.Conclusions:(1) We successfully transfected the MYCT-1 siRNA into GES-1 cells using RNA interference, and closed its expression in the mRNA level.(2) After MYCT-1 gene was silenced, GES-1 cell apoptosis significantly increased while cell proliferation reduced, and the S phase extended in cell cycle.(3) MYCT-1 gene expression maintaining a certain level is necessary to normal cell growth, and its function is to inhibit DNA replication.(4) 80 target genes in the downstream of MYCT-1 were detected, including 45 upregulated and 35 downgulated.As a novel candidate tumor suppressor gene, MYCT-1 gene maintaining a certain level of expression is essential to the gastric mucosal cell growth. Its expression downregulation may be one of the reasons of gastric carcinogenesis. Therefore, MYCT-1 gene may function as a target for early diagnosis and effective clinical interventions of gastric cancer,, which may play an important role in its prevention and treatment.更多还原