| Background and objectiveEsophageal cancer is a more common malignant tumor of digestive tract, and is the one of the world's most harmful to human health in all malignant tumors, China is a high incidence of esophageal cancer, a high incidence throughout more than ten provinces and cities in the north and south of China's, especially in the Linzhou city, Henan province. The occurrence and development of esophageal cancer is involved in a multi-stage and multi-gene process, and its etiology and pathogenesis remain unclear. With the improvement of medical standards; the status quo treatment of esophageal cancer has improved significantly over the past. The best treatment of early esophageal cancer is surgical excision. Up to now, the treatment of advanced esophageal cancer are not optimistic, local treatment of surgical treatment can hardly be a satisfactory outcome, the currently predominant treatment methods include radiotherapy, chemotherapy and drug treatment, but recurrence rates remain high, and five-year survival rate of patients is still is very low, it is difficult to meet the current medical development and the ultimate goal. The current rise of targeted gene therapy has been a new alternative for radiotherapy and chemotherapy in cancer patients.Therefore, a better understanding of the molecular mechanisms in carcinogenesis and progression of esophageal squamous cell carcinoma (ESCC) helps to improve therapy, prevention, the prognosis of patients with ESCCSerine/threonine protein kinase Pim is a kind of proto-oncogene; the protein kinase family has three main members, including:Pim-1, Pim-2 and Pim-3. Pim-3, as a member of the proto-oncogene Pim family that expresses serine/threonine kinase activity, was originally identified as a depolarization-induced gene, KID-1, in PC 12 cells (a rat pheochromocytoma cell line). Because KID-1 shows a high sequence similarity with the proto-oncogene Pim family that expresses serine/threonine kinase activity, it was renamed as Pim-3. More and more studies have shown that Pim-3 selectively expresses in malignant lesions of endothelial origin of the organs (including liver, pancreas), rather than the normal tissues. At present, domestic and foreign researchers have made great progress in the area of liver cancer, pancreatic cancer and colon cancer, suggesting that Pim-3 is tightly associated with the occurrence and development of tumor. However, to date, the role of Pim-3 in the occurrence and development of ESCC have not been investigated worldwide. In the present study, expressions of Pim-3 mRNA and protein were down-regulated in ESCC cell line EC9706 cells by RNA interfering technology, effects of Pim-3 siRNA on expressions of Pim-3 mRNA and protein were detected by semi-quantitative RT-PCR and Western blotting methods. Effect of down-regulation of Pim-3 expression on cell proliferation of EC9706 cells was investigated by new style CCK-8 kit, subsequently; cell cycle and cell apoptosis were detected by Flow cytometry. Finally, the expressions of cell proliferation and cell cycle related gene p21 and cell apoptosis related genes bcl-2 and bax were analyzed using semi-quantitative RT-PCR and Western blotting methods after transfection with Pim-3 siRNA. The findings may lay a foundation for making further investigations on the possible role of Pim-3 in the occurrence and development of ESCC and seeking a potential new molecular target for ESCC therapy.Methods(1) Pim-3 siRNA and control siRNA were transfected into esophageal squamous cell carcinoma cell line EC9706 cells. EC9706 cells were divided into three groups, namely, untreated group, control siRNA group and Pim-3 siRNA group, which were used in the following experiments:cell proliferation, cell cycle, cell apoptosis, semi-quantitative RT-PCR and Western blotting.(2) Cell proliferation was analyzed by new-style CCK-8 kit after transfection with Pim-3 siRNA and control siRNA.(3) Changes of cell cycle and cell apoptosis were detected using Flow cytometry after transfection with Pim-3 siRNA and control siRNA.(4) The expressions of cell proliferation and cell cycle related gene p21 and cell apoptosis related genes bcl-2 and bax were analyzed using semi-quantitative RT-PCR and Western blotting methods after transfection with Pim-3 siRNA and control siRNA.(5) Statistics analysis:The results of RT-PCR and Western blotting were analyzed by Image Pro Plus 5.0 software. All experiments results were from at least three separate experiments. The data were performed by One-way analysis of variance using SPSS version 13.0. Summary statistics were expressed as means±standard deviations, except as otherwise stated. In all statistical analysis, a P value<0.05 was considered statistically significant. Results(1) Expressions of Pim-3 gene mRNA and protein were decreased to about 1/5 of untreated group and control siRNA group after transfection with Pim-3 siRNA. There was no difference between untreated group and control siRNA group in the expression of Pim-3 mRNA and protein (P> 0.05).(2) In the untreated group and the control siRNA group EC9706 cells, cell proliferation of EC9706 cells was no significant difference (P> 0.05). However, compared to the untreated group and the control siRNA group, transfection the proliferation of EC9706 cells were obviously inhibited 48 h after transfection with Pim-3 siRNA (P<0.05).(3) The results of Cell cycle analysis showed that the cell numbers ratio of the Go/G1 phase of EC9706 cells in untreated group and control siRNA group were 39.38% and 40.25%, respectively, significantly lower than that (60.93%) in Pim siRNA group (P<0.01), while the G2/M phase cells showed the opposite result, that is, G2/M phase delay.(4) The results of cell apoptosis demonstrated that early apoptosis rate in Pim-3 siRNA group was 19.26% 48h after transfection with Pim-3 siRNA,, significantly higher than that in untreated group and control siRNA group (apoptosis rates were:5.15% and 6.05%) (P<0.05), while the numbers of live cells in untreated group and control siRNA group were 93.01% and 91.96%, respectively, significantly higher than that (78.73%) in Pim-3 siRNA group (P <0.05).(5) The results of RT-PCR and Western blotting results revealed that compared to untreated group and control siRNA group, bcl-2 mRNA and protein expression were significantly reduced in Pim-3 siRNA group (P<0.05), but expressions of p21 and bax mRNA and protein significantly increased, and the difference was statistically significant (P<0.05). In addition, there were no differences in expressions of all genes above-mentioned (P> 0.05).Conclusion(1) Pim-3 siRNA effectively down-regulates the expressions of Pim-3 mRNA and protein in esophageal squamous cell carcinoma EC9706 cells.(2) Down-regulation of Pim-3 expression obviously inhibits the proliferation of EC9706 cells, arrests cell cycle at G0/G1 phase and induces cell apoptosis..(3) Down-regulation of Pim-3 expression mediated inhibition of proliferation and cell cycle arrest may be closely associated with elevation of p21 expression. Cell apoptosis evoked by down-regulation of Pim-3 expression may be tightly related to the decrease of bcl-2 expression and increase of bax expression. |
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