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Effect of Down-Regulation of Pim-3 Expression on Cell Proliferation, Cell Cycle and Cell Apoptosis of Esophageal Squamous Cell Carcinoma Cell Line EC9706 Cells

Author: WangZuo
Tutor: LiHuiXiang；LvXinQuan
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: Pim-3 EC9706 cells Cell proliferation Cell cycle Apoptosis
CLC: R735.1
Type: Master's thesis
Year: 2010
Downloads: 23
Quote: 0
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Abstract


Background and purpose of esophageal cancer is one of the more common malignancies, is one of the most harmful to human health in the world today malignant, China is a high incidence of esophageal cancer, the high incidence of more than 10 provinces and cities throughout the country, especially in Henan Province Linzhou. The development of esophageal cancer is a multi-stage and multi-gene evolving process, but its etiology and pathogenesis is not yet very clear. With the improvement of medical standards Current treatment of esophageal cancer than in the past there has been significant improvement, but the recurrence rate is still high, the patient five-year survival rate is still very low, it is difficult to meet the ultimate goal of the current medical development. The current rise of targeted gene therapy has become a put, not satisfied with the effect of chemotherapy treatment of cancer patients a new alternative means. Thus, from the molecular level to explore the molecular mechanism of the development of esophageal squamous cell carcinoma, and to find effective therapeutic targets for the development of for to inhibit esophageal squamous cell carcinoma occurs, the clinical treatment of esophageal squamous cell carcinoma, prevention, and patient survival the improvement of quality of life are extremely important. Serine / threonine protein kinase Pim is a proto-oncogene, protein kinase family has three members, including: Pim-1, Pim-2 and Pim-Pim-3 as a table Dasi / threonine kinase one, for the first time in the activity of the proto-oncogene pim family pheochromocytoma PC12 cells as depolarization-induced genes KID-1 was confirmed in rats addicted. KID-1 gene and the proto-oncogene Pim-1 and Pim-2 sequence highly consistent, so named Pim-3. The study showed that Pim-3 selectively in endothelial origin of organs (including the liver, pancreas) malignant lesions, but is not expressed in normal tissue. At present, domestic and foreign researchers in the field of liver cancer, pancreatic cancer, colon cancer, Pim-3 gene has been great progress, suggesting that it is closely related with the development of tumor. However, to date, both at home and abroad were no Pim-3 in esophageal squamous cell carcinoma in the study reported in this study, the authors used RNA interference technology, lowered esophageal squamous cell carcinoma cell line EC9706 cells in Pim- The 3 expression transferred by RT-PCR and Western blotting technology research Pim-3siRNA the Pim-3 mRNA and protein expression in EC9706 cells. Pim-3 expression EC9706 cell proliferation, CCK-8 cell proliferation kit cut down Pim-3 expression on EC9706 cell cycle and apoptosis by flow cytometry analysis, Real-time final adoption PCR and Western blotting studies with cell proliferation and cell cycle-related genes p21 and apoptosis inhibitory factor bcl-2 and apoptosis-promoting factor bax mRNAs and protein expression changes, the study was designed to explore Pim-3 gene in esophageal scales shaped cell carcinoma, the possible role in the development and lay the foundation for further search of new molecular markers targeted therapy of esophageal squamous cell carcinoma. Method 1 Pim-3 siRNA and control siRNA using Lipofectamine 2000 transfection esophageal squamous cell carcinoma EC9706 cells, the cells were divided into three groups, namely the untreated group, the control siRNA group and Pim-3 siRNA group, respectively, for The following cell proliferation, cell cycle, apoptosis, and semi-quantitative RT-PCR and Western blotting analysis. 2 new CCK-8 kit Pim-3 transfected and control siRNAs esophageal squamous cell carcinoma EC9706 cell proliferative capacity. 3 the use of flow cytometry cut Pim-3 expression in esophageal squamous cell carcinoma EC9706 cell cycle and apoptosis changes. 4 by semi-quantitative RT-PCR and Western blotting was used to detect changes in the three groups of cells in the proliferation and cell cycle-related factors p21 and apoptosis-related factor bcl-2 and bax mRNA and protein expression levels. 5 RT-PCR and Western blotting applications Image Pro Plus 5.0 software gray value analysis, all of the above experiments were repeated at least three times. Application SPSS13.0 statistical software for statistical analysis, statistical data are expressed as mean ± standard deviation (x ± s) more than the number of samples were single factor analysis of variance (One-way ANOVA), P lt; 0.05 has significant. Results 1 Pim-3 siRNA transfection, Pim-3 mRNA and protein expression were decreased to about 1/5 of the untreated group and control siRNA group. Pim-3 expression was no difference between the untreated and control siRNA group (P gt; 0.05). EC9706 cells in the untreated group and control siRNA group, no significant difference (P gt; 0.05) EC9706 cell proliferation. However, compared with the untreated group and control siRNA group, Pim-3 siRNA group 24h after transfection EC9706 cell proliferation was significantly inhibited (P lt; 0.05). The cell cycle analysis showed that the untreated group and control siRNA group EC9706 cells in the G0/G1 phase of the cell count ratio were 39.38% and 40.25%, significantly lower than the Pim siRNA group Go/G1 cell number (60.93 %) (P lt; 0.01), and G2 / M phase cells showed the opposite result, that the G2 / M phase blocked. 4 cells results show that 48h after transfection, Annexin Ⅴ detection of Pim-3 siRNA cells early apoptosis rate was 19.26%, significantly higher than the untreated group and control siRNA group early apoptosis rate (apoptosis rates were : 5.15% and 6.05%) (P lt; 0.05), the number of living cells without treatment group and control siRNA group EC9706 were 93.01% and 91.96%, significantly higher than Pim-3 siRNA group, the number of living cells (78.73 %) (P lt; 0.05). 5 RT-PCR and Western blotting results show that, compared with the untreated group and control siRNA group, Pim-3 siRNA group bcl-2 mRNA and protein expression were significantly down-regulated (P lt; 0.05), p21 and bax mRNA and protein expression levels but significantly increased, and the difference was statistically significant (P lt; 0.05) In addition, the expression levels of these genes between the untreated group and control siRNA group average no differences (P gt; 0.05). Conclusion 1 using RNA interference technology effectively lowered esophageal squamous cell carcinoma EC9706 cells of Pim-3mRNA in protein expression. 2 Pim-3 genes were down 3 Pim-3 gene expression mediated reduction in proliferation inhibition and cycle still could significantly inhibit the proliferation of esophageal squamous cell carcinoma EC9706 cells in Go/G1 and cell apoptosis induced cell cycle still may significantly associated with p21 upregulation is closely related to their mediated apoptosis and down antiapoptotic gene bcl-2 expression and the rise of bax expression.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Esophageal tumors
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