| The incidence rate of esophageal cancer is the highest in the world, especially in the Taihang area, it is the main reason of cancer deaths there. Therefore, it is the current research focus to understanding the pathogenesis of esophageal cancer in molecular level, finding the molecular target for malignant transformation of esophageal cancer and targeting therapy of esophageal cancer.Akt/mTOR signaling pathway is an evolutionarily conserved one that controls cell growth, apoptosis, angiogenesis and other biological functions. Studies have shown that the development and metastasis of tumor are closely related with this signaling pathway. Akt, also known as protein kinase B, plays a key role in the signaling pathway. Akt is a downstream component in phosphoinositide (PI) 3-kinase signaling, which is activated upon autophosphorylation of receptor tyrosine kinases induced by growth factors and other signals, stimulation of G-protein-coupled receptors, or activation of integrin signaling. PI3-kinase is the key enzyme in the generation of the second messenger Ptdlns (3,4,5) P3 from Ptdlns (4,5) P2, increase in PIP3 recruits Akt to the membrane where is activated through phosphorylation by specific kinases also dependent on PIP3. Activated Akt may regulate cell growth, proliferation, differentiation, protein synthesis, and survival, as well as angiogenesis, invasion and metastasis.The Studies found the expression of Akt in gastric cancer, breast cancer, ovarian cancer, pancreatic cancer and other malignant tumors are higher. Recently,considerable evidence shown that the expression of Akt in esophageal cancer is also higher.PHLPP, a natural newly discovered tumor suppressor gene and a leucine-rich tryptophan/threonine protein kinase, which was located on chromosome 18 and belongs to PP2C kinase. PHLPP specifically dephosphorylate the hydrophobic motif of Akt in cells, resulting in decreased activity of Akt, increased apoptosis, and inhibition of cell proliferation. It has been reported that the expression of PHLPP in colon cancer, brain glioblastoma, ovarian cancer, and so on were significantly reduced and which of Akt were significantly increased. However, there has rarely been report about hthe relationship between PHLPP and esophageal cancer research.Antisense oligonucleotides (ASODNS) technology is that synthesizing antisense complementary sequences based on the principles of nucleic acid base pairing to aim directly at certain taget gene in order to inhibit it transcription and protein synthesis, thereby to inhibit its function.Recently years, ASODN has been widely used in genetic research because of its features of specificity, simple operation, the good effects.To explore the relationship between the occurrence of esophageal cancer and PHLPP, in this study, the various concentrations of PHLPP ASODN were transfected esophageal cancer EC9706 cells. Immunocytochemistry and Western blot were used to detect the expression of PHLPP, Akt and p-Akt protein in esophageal cancer cells; and the in situ Hybridization was used to detect the the expression of PHLPP, Akt and p-Akt mRN A in each group cells; the MTT assay and the flow cytometry were used to observe the changes of the cell cycle; the TUNEL assay and the flow cytometry were used to observe the changes of apoptosis. Materials and methods1. Human esophageal carcinoma cell lines EC9706 were gift from the Chinese Academy of Medical Sciences Cancer Institute.2. EC9706 cells were recovered and then cultured in the RPMI-1640 medium containing 100g/L fetal bovine serum, at 37℃in a 5% CO2 humidified atmosphere.3. PHLPP ASODNs were transfected into EC9706 cells4. The EC9706 cells were divided into different groups. The experimental group:three different concentrations (5,10 and 15μmol/L) of the PHLPP ASODNs were transfected into EC9706 cells for 24,48 and 72h; The control group:SODN control group (15μmol/L SODN),N-ODN control group (15μmol/L N-ODN) and the normal control group (the culture medium)5. The immunocytochemistry and Western blot was used to detect the expression of PHLPP,Akt and p-Akt protein in different periods.6. The in situ Hybridization was used to detect the the expression of PHLPP, Akt and p-Akt mRN A in each group cells7. The proliferation rate of cell growth in each group and each time period were detected by MTT assay.8. The changes of cell cycle for 72h in each group were detected by flow cytometry.9. The rates of apoptosis for 48h in each group were detected by TUNEL assay and the flow cytometry.10. Statistical analysis:The SPSS 13.0 statistical software was used. The enumeration data was presented as the positive rate, Chi-square analysis was used for the comparison of positive rates, Spearman's rank correlation coefficient was used for relevant analysis; measurement data were presented as mean value±standard error of the mean, The differences between two groups were determined with the Student's t test, The differences among three or more groups were determined with a one-way ANOVA. Results1. The immunocytochemistry results1.1 The expressions of PHLPP in each group of cellsThree different concentrations (50,100 and 150nM) of the PHLPP ASODN were transfected into EC9706 cells for 24,48 and 72h. Compared with control groups, the expressions of PHLPP were decreased in the groups of treatment group, the differences each other were statistically significant (FRAPA=106.87,60.86,60.86; Fm.siRNA=24.14,45.78,59.19, all P<0.05), and change wih concentration-dependent and time-dependent (FRAPA=11.68,5.20,26.9; P=0.0002,0.0123,0.0001; FsiRNA=50.22,42.23,29.46, all P<0.05).1.2 The expression of Akt,p-Akt protein in each groupCompared with the control group, the expression of Akt protein in transfected groups was not significantly changed, and the difference was not statistically significant (P<0.05), while the expression of p-Akt protein expression was significantly increased, the differences were statistically significant (P<0.05), and change wih concentration-dependent and time-dependent.2. Western blot results15μmol/L PHLPP ASODN was transfected into EC9706 cells for 24,48 and 72h.Compared with the control group, the expression levels of PHLPP protein in each gtoup were lower (P<0.05), and it is the most stronggest effect to transfect for 72h. The differences between each group statistically significant (P<0.05), and change with time-dependent. The expression levels of the p-Akt protein in each groups was significantly higher (P<0.05), to after transfection 72h increased most significantly (P<0.05); However, the expression levels of Akt in each groups cells was not significantly different (P>0.05).3. The in situ hybridization results3.1 The expressions of PHLPP mRNA in each group of cellsCompared with the control group, the expression of PHLPP mRNA in each group of cells were significantly decreased, increasing with concentration and transfection time, the difference was statistically significant (P<0.05).3.2 The expression of Akt, p-Akt protein in each groupCompared with the control group, the expressions of p-Akt mRNA were significantly decreased in each experimental group, the differences were statistically significant, and change with concentration-dependent and time-dependent; the expressions of Akt mRNA in the cells of each experimental group were no significant difference.4. MTT test resultsThe relative growth rates of cells were all increased significantly with the increase of concentration and time in the three concentrations and three time periods after PHLPP ASODN was transfected into EC9706 cells. The differencs were statistically significant at the same time, different concentrations and at the same concentrations, different time (P<0.05). The difference compared with the control group were statistically significant (P<0.05).The dose of 12umol/L for 72 hours was exhibited the most stronggest effect.5. The cell proliferation was detected by flow cytometryCompared with the control groups, the number of cells in each period was significantly different:the number in G0/G1 phase remarkably decreased, the one in S phase increased, and there were statistical significances (all.P<0.05). The number of cells in G0/G1 phase remarkably decreased with the increase in the concentration of transfection, the number in S phase decreased; there were statistical significances between the three concentrations.6. The apoptosis is detected by TUNEL15μmol/L PHLPP ASODNwas transfected into EC9706 cells for 24,48 and 72h.Compared with the control groups, in the treatment groups, the number of apoptotic cells were significantly decreased, and were decreased with the time of the transfection, and the differences between them were statistically significant (all P<0.05).7. The apoptosis is detected by flow cytometryCompared with the control groups, early apoptosis, the early apoptosis rate, late apoptosis in the cell treated each individually were significantly decreased, and the difference was rapamycin (all P<0.05), and the apoptosis rate decreases with the t of transfection, There were statistical significances between the different periods (all P<0.05).Conclusion1. PHLPP ASODN can specifically inhibit the expressions of PHLPP protein and mRNA in the esophageal squamous cell carcinoma cell line EC9706, and meanwhile can down-regulate the expressions of p-Akt protein and mRNA of EC9706 cells, and thus PHLPP inhibit the expression of p-Akt cells, thereby blocking the PI3K/Akt signaling Pathway.2. PHLPP ASODN can promote the esophageal cancer EC9706 cells growth, and inhibit cells apoptosis. It tips that PHLPP can inhibit cell proliferation and induce apoptosis. |
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