| Esophageal carcinoma incidence in China is still at the world's largest. Development of new drugs, molecular target therapy esophageal carcinoma become the research focus now.3-phosphoinositide-dependent protein kinase 1 (PDK1) belongs to AGC kinase family. Abnormal PI3K/Akt signalling pathway, which is mediated and regulated by PDK1 is closely related with various malignant tumor growth. PDK1 is the main upstream kinase of AGC kinases such as Akt, p70-S6K, RSK, PKC and so on. As an Akt upstream kinase, PDK1 was separated and identified by Alessi DR on 1997. It could phosphorylate Akt on Thr308. Activated Akt modulates the function of numerous substrates involved in the regulation of tarbohydrate metabolism, protien transcription, cell survival, cell cycle progression, cell migration and cellular growth. PDK1 may become the new targets of tumor treatment research.RNA interference is a kind of post-transcriptional gene silencing mechanism with advantages of high efficiency and high specificity. It has been widely used in gene function identification, post-transcriptional regulation of gene expression, as well as for a variety of diseases, especially gene therapy of malignant tumor. UCN-01 (7-hydroxy staurosporine) was the most reported PDK1 inhibitor in current research. It is the staurosporine's 7 alpha hydroxy derivatives. UCN-01 could competitive inhibit the ATP binding sites of PDK1 kinase and inhibit its kinase activity, thus down regulate the phosphorylation of Akt, drop interference the signaling pathway, then affect cell metabolism, proliferation, apoptosis and cause cytotoxic effects. In this study, siRNA and PDK1 inhibitors UCN-01 was used in esophageal cancer EC9706 cells to inhibite specifically the PDK1 gene, down regulate the phosphorylation of Akt, block PI3K/Akt signalling pathway and affect cell proliferation, apoptosis. This study could provide a theoretical basis for clinical treatment of esophageal cancer.Materials and methods1. Well-differentiated esophageal squamous carcinoma EC9706 cell line were incubated in 37℃,5%CO2 incubation box. EC9706 cells was transfected by PDK1 siRNA with the help of RNAi-Mate, or treated by UCN-01 with the concentration of 50nM, 100nM,200nM for 24h,48h,72h.2. Expression of PDK1 mRNA of EC9706 cells in each groups were detected by RT-PCR.3. Using Western Blot and immunocytochemistry to detect expression of PDK1, Akt, p-Akt protein of EC9706 cells in each groups.4. The inhibition rate of cell growth in each group and each time period were detected by MTT assay.5. Flow cytometry and TUNEL were used to count the percentage of apoptosis cells.6. Statistical analysis:The SPSS13.0 statistical software was used, a=0.05.Results1. Efficiency test of siRNA transfection:After different concertrations of negative control FAM-siRNA transfected EC9706 cells 6h later, we observed them with fluorescence microscopy. Concentration of 100nM siRNA carrying efficiency is highest, following siRNA transfection experiments all use this concentration.2. Selection of PDK1 siRNA:Compared with cell control group, Negative control siRNA or RNAi-Mate have no effect on expression of PDK1 mRNA, and no obvious difference between them(P>0.05). Each of three PDK1 siRNAs, which transfected EC9706 cells, could inhibite expression of PDK1 mRNA (P<0.05), especially PDK1 siRNA1 made the highest inhibition ratio(P<0.05). Following siRNA transfection experiments all use PDK1 siRNA1.3. Influence of PDK1 siRNA, UCN-01 on the expression of PDK1 mRNA: PDK1 siRNA1 could effectively knock down the expression of PDK1 mRNA, with the most significant inhibition after transfection 72h(P<0.05). UCN-01 could also knock down the expression of PDK1 mRNA, and change with concentration-dependent and time-dependent(P<0.05). PDK1 siRNA1 had stronger inhibitory ability than 200nM UCN-01 (P<0.05).4. Influence of PDK1 siRNA, UCN-01 on the expression of PDK1 protien and phosphorylation of Akt:PDK1 siRNAl could effectively knock down the expression of PDK1 and p-Akt protien, with the most significant inhibition after transfection 72h(P<0.05). UCN-01 could also knock down the expression of PDK1 and p-Akt protien, and change with concentration-dependent and time-dependent(P<0.05). PDK1 siRNA1 had stronger inhibitory ability than 200nM UCN-01 (P<0.05). PDK1 siRNA,UCN-01 had no influence on the expression of Akt protien(P>0.05).5.Influence of PDK1 siRNA, UCN-01 on EC9706 cell proliferation:PDK1 siRNA, UCN-01 could effectively inhibite cell proliferation, and change with time-dependent(P<0.05). The inhibition by UCN-01 was concentration-dependent (P<0.05). PDK1 siRNA1 had stronger inhibitory ability than 200nM UCN-01 (P<0.05).6. Influence of PDK1 siRNA, UCN-01 on EC9706 cell apoptosis:PDK1 siRNA, UCN-01 could effectively incresce the early apoptosis rate and the late apoptosis rate of EC9706 cells after 72h later(P<0.05). The apoptosis rate of EC9706 cells increased by UCN-01 with concentration-dependence(P<0.05). PDK1 siRNA1 had stronger increasing ability than 200nM UCN-01 (P<0.05).Conclusion1. PDK1 siRNA could effectively knock down the expression of PDK1 gene and phosphorylation of Akt in EC9706 cells, block PI3K/Akt signalling pathways, which could significantly inhibit the cell proliferation and promote the cell apoptosis. PDK1 may play an important role with esophageal cancer cell proliferation and apoptosis, and may serve as an effective target for carcinoma gene therapy. 2. PDK1 chemical inhibitor UCN-01 could also knock down the expression of PDK1 gene and phosphorylation of Aktin EC9706 cells, block PI3K/Akt signalling pathways, and inhibit the cell proliferation and promote the cell apoptosis. This may be one of the important mechanisms of UCN-01 antitumor function. UCN-01 is expected to become a new anti-tumor targeting therapy drug. |
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