| Esophageal 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, 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 ESCC helps to improve therapy, prevention, the prognosis of patients with ESCC.Pim-3, 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. Accumulating evidence indicates activated Pim family members play an important role in various types of carcinogenesis, and thus associated with occurrence and development of malignant neoplasm, further play a key role in apoptosis of tumors, certain molecular mechanism of cell apoptosis mediated by Pim-3 is as following:Pim-3 may mainly phosphorylate Bad Ser 112, further phosphorylation of Bad resulted in the liberation of Bcl-XL and Bcl-2, and thus prevent apoptosis. To date, the relationships between Pim-3 and bcl-2 in the occurrence and development of esophageal squamous cell carcinoma have not been investigated worldwide. In the present study, expressions of Pim-3 and bcl-2 mRNAs and proteins were detected in the patients with ESCC from high-incidence area of esophageal cancer in Henan province by RT-PCR, Western blotting, in situ hybrid and immunohistochemistry methods, and the relationships between expressions of Pim-3 and bcl-2 and clinicopathological features were analyzed, further correlations of Pim-3 and bcl-2 expressions were explored in ESCC. The findings may lay a foundation for making further investigations on the role of Pim-3 and bcl-2 in the occurrence and development of ESCC and seeking a potential new molecular target for ESCC therapy.Methods(1) Relative expressions of Pim-3 and bcl-2 mRNAs were detected by semi-quantitative RT-PCR method in ESCC tissue, mucosa adjacent to cancer, and normal esophageal mucosa epithelial tissue.(2) Relative expressions of Pim-3 and bcl-2 proteins were detected by Western blotting method in ESCC tissue, mucosa adjacent to cancer, and normal esophageal mucosa epithelial tissue.(3) Expressions of Pim-3 and bcl-2 mRNAs were detected by in situ hybrid method in 45 cases of ESCC tissue,22 cases of mucosa adjacent to cancer, and 45 cases of normal esophageal mucosa, and correlation between expressions of Pim-3 and bcl-2 mRNAs was analyzed.(4) Expressions of Pim-3 and bcl-2 proteins were detected by immunohisto chemistry method in 45 cases of ESCC tissue,22 cases of mucosa adjacent to cancer, and 45 cases of normal esophageal mucosa, and correlation between expressions of Pim-3 and bcl-2 proteins was investigated.(5) Statistical analysis:The data were carried out with chi square test, t test, analysis of variance and correlation using SPSS version 13.0. In all statistical analyses, a P-value<0.05 was considered statistically significant.Results(1) The results of semi-quantitative RT-PCR demonstrated that Pim-3 gene, about 500bp, was successfully amplified in ESCC tissue, but not in normal esophageal mucosa epithelial tissue. Relative expression levels of Pim-3 in ESCC tissue and mucosa adjacent to cancer were 0.809±0.015 and 0.092±0.009, respectively, while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(2) The results of semi-quantitative RT-PCR demonstrated that bcl-2 gene, about 595bp, was successfully amplified in ESCC tissue, but not in normal esophageal mucosa epithelial tissue. Relative expression levels of bcl-2 mRNA in ESCC tissue and mucosa adjacent to cancer were 0.671±0.011 and 0.102±0.007, respectively, while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(3) The results of Western blotting indicated that Pim-3 and bcl-2 proteins appeared high expressions in ESCC, but there was no expression in normal esophageal mucosa epithelial tissue. The relative expressions of Pim-3 and bcl-2 proteins (0.796±0.018 and 0.757±0.014, respectively) in ESCC tissue were significantly higher than those in mucosa adjacent to cancer (0.299±0.011 and 0.225±0.009, respectively), while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(4) The results of in situ hybrid showed that Pim-3 mRNA was mainly localized in cytoplasm, appearing ianthinus granules. There was high expression of Pim-3 mRNA in ESCC, and positive ratio was 82.22%(37/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 22.73%(5/22) and 0.00%(0/45), respectively, there were significant differences among three groups (P<0.05).(5) Pim-3 protein was mainly localized in cytoplasm, showing pale yellow to brown granules. There was high expression of Pim-3 protein in ESCC, and positive ratio was 75.56%(34/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 18.18%(4/22) and 0.00%(0/45), respectively, there were significant differences among three groups (P<0.05).(6) The results of in situ hybrid showed that bcl-2 mRNA was mainly localized in cytoplasm, appearing ianthinus granules. There was high expression of bcl-2 mRNA in ESCC, and positive ratio was 77.78%(35/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 31.82%(7/22) and 0.00%(0/45), respectively, there were significant differences among three groups (χ2=58.451, P<0.05).(7) Bcl-2 protein was mainly localized in cytoplasm, showing pale yellow to brown granules. The results of immunohistochemistry demonstrated that there was high expression of bcl-2 protein in ESCC, and positive ratio was 71.11% (32/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 22.72% (5/22) and 0.00%(0/45), respectively, there were significant differences among three groups (χ2=52.724, P<0.05).(8) Relationship between expressions of Pim-3 and bcl-2 mRNAs and proteins and clinicopathological features were analyzed using SPSS 13.0 software. The results revealed that expressions of Pim-3 and bcl-2 were obviously related to lymph node metastasis and TNM staging (P<0.05), but not related to histo grade and invasion depth (P<0.05).(9) The results of correlation analysis of Pim-3 and bcl-2 revealed that 34 cases of Pim-3 positive expression included 30 cases of bcl-2 positive expression in the 45 cases of ESCC tissues, while 11 cases of Pim-3 negative expression included 9 cases of bcl-2 negative expression. Pim-3 expression and bcl-2 expression displayed positive correlation (γp=0.664, P=0.000).Conclusion1. Pim-3 and bcl-2 appear high expressions in esophageal squamous cell carcinoma, while there are no expressions in the normal esophageal mucosal epithelial tissue.2. High expressions of Pim-3 and bcl-2 are tightly associated with lymph metastasis and TNM staging, but not related to histological grade and depth of infiltration, suggesting Pim-3 and bcl-2 play an important role in the occurrence and development of esophageal squamous cell carcinoma.3. Expression of Pim-3 is positively correlated with the expression of bcl-2, indicating cell apoptosis induced by Pim-3 may be closely associated with bcl-2 expression. |
PDF Full Text Request