| Esophageal squamous cell carcinoma is one of the most common gastrointestinal malignancy. LinZhou city of Henan province is also a high incidence area. The progressions of Surgery, chemotherapy, radiotherapy and biotherapy, have promoted development of esophageal squamous cell carcinoma treatment, But the exact mechanism in tumorigenesis remains to be elucidated.Therefore, early diagnosisrate and discovery of new anti-cancer drugs are still huge challenge.The 70 kDa ribosomal S6 kinase 1 (p70S6Kl), a downstream target of mammalian target of rapamycin (mTOR), is an important regulator of cell cycle progression,cell proliferation, and cell survival, and plays an important role in cell signal networks. Now there is a widespread concern over the mTOR inhibitors, and also various mTOR inhibitors have been used into clinical trials, and achieved a preliminary results. mTOR inhibitors can also improve the chemoradiation sensitivity of tumors. However, blocking strategy of p70S6K1 is still tiny, and relationship of p70S6K1 activation level and the sensitivity to mTOR inhibitors in cancer cells remains to be elucidated. In this study, we try to construct a vector,which is expected to specically inhibit expression of p70S6K1 in mRNA level and p-p7OS6K1 protein level in Esophageal squamous carcinoma cell line EC9706 cells. Methods 1 construction of shRNA expression vectorsAccording to siRNA designing principles, designed three p70S6K1 specific target sequence in Ambion company's website and synthesised sense and antisense DNA fragments, which added five T in 3'terminal as terminator of RNA polymeraseⅢ, and also added Restriction Enzyme cutting sites. Sense and antisense 65 nt oligonucleotides annealed into double strand of DNA(dsDNA), and ligated to the linear RetroQ-pSIREN-DsRed-Express vectors, then, we got recombinatant plasmids and named them respectively p1shRNA-p70S6K1, p2shRNA-p70S6K1 and p3shRNA-p70S6K1. Transfected these vectors into competent E.coli, and cutured in ampicillin resistant solid board overnight at 37℃. Picked to positive cloning next day, and sent bacterium fluid for sequencing identification.2 Transfectionp1shRNA-p70S6K1 were transfected into EC9706 cells by LipofectamineTM 2000 according to manufacturer's instructions. Experiment was divided into two groups (1) the control group(transfected liposome no plasmid); (2) the experimental group (transfected liposome and plasmid); We watch the expression of red fluorescent after 24 h of transfection.3 RT-PCRRT-PCR was used to detect the expression of p70S6Kl gene after diferent timepoints of transfection, which were respectively on 24,48,72,96 and 120 h;All experiments results were from three separate experiments.4 Western blottingWestern blotting were used to detect the expression of phosphorylated p70S6Kl protein after diferent timepoints of transfection, which were respectively on 24,48, 72,96 and 120 h;All experiments results were from three separate experiments.5 Image processing and statistical analysisAnalysis the images of RT-PCR and Western blotting by image processing software IPP5.0.2. The datum were performed by one-way ANOVA using SPSS version 10.0. A P value<0.01 was considered statistically significant, and pairwise comparison by LSD-t test, A P value<0.01 was considered statistically significant. Results1 Identification of the recombinant vectorsSequencing reports showed that the sequences ligated to pSIREN-RetroQ-DsRed-Express consistent with the designed sequences.2 Fluorescence expressionThere were red fluorescence expression after 24 h of transfection.3 RT-PCRExpression of p70S6K1 mRNA were downregulated at 24 h,48 h and 72 h after transfection (P<0.05). p1shRNA-p70S6K1 had the highest inhibitory effect at 48 h, with the highest interfering rate of 55.3%.3 Western blottingExpression of p70S6K1 mRNA were downregulated at 24 h,48 h,72 h and 96 h after transfection (P<0.05). p1 shRNA-p70S6K1 had the highest inhibitory effect at 48 h, with the highest interfering rate of 58.0%.ConclusionIn this study, we constructed a shRNA expression vector p1 shRNA-p70S6K1 and transfected it to EC9706 cells. p1shRNA-p70S6K1 could inhibit expression of p70S6K1 in mRNA level and protein level in EC9706 cells. We found that the recommanted vector significantly downregulated p-p70S6K1 protein expression and p70S6K1 transcriptional activation.So the recommanted vector can be used to further research of p70S6K1 in ESCC.Expression of p70S6K1 were downregulated from 24 h to 48 h, and the expression was the lowest at 48 h. Later increased to the level of control group. The possible reasons are that,with the time going after transfecting some vectors lost from cells, so expression of shRNA decrease. Therefore,we have to improve the technology of transfection, in order to prolong the interference duration. |
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