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Investigation of the Mechanism of MYCT-1 Gene in Carcinogenesis of Gastric Cancer

Author: LiuZheng
Tutor: QiuGuangBin
School: Jilin University
Course: Clinical Laboratory Science
Keywords: siRNA MYCT-1 apoptosis cell proliferation cell cycle gene chip
CLC: R735.2
Type: Master's thesis
Year: 2010
Downloads: 26
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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.

Investigation of the Mechanism of MYCT-1 Gene in Carcinogenesis of Gastric Cancer

Abstract

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