P70S6K Expression And Its Effect Of Cell Proliferation In Esophageal Squamous Cell Carcinoma

Tumor is one of the major diseases to human health, and esophageal squamous cell carcinoma (ESCC) is one of the most frequently cancers in the world, it has high degree of malignancy, five-year survival rate less than5%, especially in Linxian, China. However, its exact pathogenesis is unclear. Many studies have shown that mammalian target of rapamycin (mTOR) plays an important role in cancer therapy. And mTOR/p70S6K (70kDa ribosomal protein S6kinase) signaling pathway is abnormal activated in ESCC.Since mTOR molecule is relatively large, and there are many proteins effected with it, p70S6K, which is an important downstream of mTOR/p70S6K singnaling pathway, In this study, immunohistochemistry、Western blotting、RT-PCR、MTT were used to investigate the expression of phosphorylation of p70S6K (p-p70S6K) in ESCC, and the sensitivity of EC9706cells to rapamycin after shRNA interference the expression of p70S6K. Our study may be provid a molecular targets to the therapy of esophageal squamous cell carcinoma.Methods1ImmunohistochemistryThe tissue samples of ESCC patients were collected, the expressions of p-p70S6K and mTOR were detected in the normal esophageal specimens, tumor adjacent atypical hyperplastic epithelial specimens and normal esophageal epithelial specimens by immunohistochemistry method, and statistic analysis was conducted on the relationship and significance between protein expression of mTOR and p-p70S6K in ESCC and the clinical pathological features.2Western blottingThe protein expression of p70S6K and p-p70S6K were examined in low differentiation EC1and EC9706and in high differentiation Eca109, and the effects of rapamycin to the expression of them were detected, too, by Western blotting method.3RT-PCR and MTT After p70S6K-shRNA transfected EC9706cells, the appropriate concentration of puromycin was used to screening the stable expression cells, and the effect of interferenece was detected by RT-PCR; furter, the effects of rapamycin to prolifer-ation of cells with or without p70S6K-shRNA were examined by MTT.4Statistic analysisAll experiments results were from at least three separate experiments. The data were performed by one-way analysis of variance using SPSS version13.0. A P value of<0.05was considered statistically significant, and all P values were two-side. χ2-test was used to analyze the result of immunohistochemistry, Gray scale analysis was conducted on the results of Western blotting and RT-PCR with IPP6.0.Results1The protein expression of mTOR and p-p70S6K in ESCC and the clinical pathological featuresThe expression levels of mTOR protein were closely correlated with tumor TNM grade in esophageal squamous cell carcinoma(χ2=9.121, P<0.01). The positive rates of mTOR protein expression in normal esophageal epithelium, tumor-adjacent atypical hyperplastic epithelium and carcinoma were20%(3/15),46.7%(7/15) and62.9%(22/35), respectively, which with a significant difference among the three groups (x2=7.767, P<0.05). The expression levels of p-p70S6K protein were closely correlated with lymphatic metastasis and TNM grade in esophageal squamous cell carcinoma (x2=5.846and4.523, respectively, both P<0.05). The positive rates of p-p70S6K protein expression in normal esophageal epithelium, tumor-adjacent atypical hyperplastic epithelium and carcinoma were33.3%(5/15),73.3%(11/15) and74.3%(26/35), respectively, with a significant difference among the three groups (χ2=8.350, P<0.05). There is a positive correlation between the protein expression levels of mTOR andp-p70S6K (γp=0.359, P=0.034)2The protein expression of p70S6K and the effects of rapamycin to the expression of it in ESCCThe results of Western blotting showed that p70S6K and p-p70S6K expressed in EC1, EC9706and Eca109, while the expression of p-p70S6K in Eca109is dramatically lower than that in EC1and EC9706; Rapamycin inhibited the expression of p-p70S6K with time dependence.3The sensitivity of EC9706cells to rapamycin was changed after p70S6K downregulatedp70S6levels were downregulated in EC9706by p70S6K-shRNA, which with significant difference (P<0.05), the results of MTT assay showed that cells proliferation was inhibited after interfered by p70S6K-shRNA, the sensitivity of cells proliferation to rapamycin were proved after p70S6K downregulated, which with concentration dependence.Conclusions1The expression of mTOR and p-p70S6K in esophageal squamous cell carcinoma was much higher than in the corresponding adjacent tissues and normal esophageal tissues. It suggestes that ESCC with high expression of mTOR and p-p70S6K had a stronger invasive ability. So the detection of both proteins was essentional to further understand the biological behavior of ESCC, this study provides a new molecular target for early diagnosis and treatment of ESCC.2The phosphorylation level of p70S6K is related with differentiation of the cells, the expression of p-p70S6K lower in high differentiation cells, the expression of p-p70S6K higher in low. differentiation cells; and rapamycin can inhibit the expression of p-p70S6K, which with time depedence.3p70S6K-shRNA can inhibit the expression of p70S6K, and increase the sensitivity of EC9706cells to rapamycin with dose dependence.