Effects Of TSC2 Antisense Oligonucleotide On Proliferation And Apoptosis Of Esophageal Cancer Cell EC9706

Tuberous sclerosis complex (TSC) is an autosomal dominant hereditary disease which results from the mutations of Tsc1 and Tsc2 gene. TSC is a multi-systemic disorder characterized by a variety of organs micro structure defects and the development of numerous benign tumours (e.g.hamartomas), most commonly affecting the brain, eyes, kidney, skin, heart and lungs. As tumor suppressor factors, Tscl and Tsc2 genes play a critical adjustment effect in cell growth and proliferation process. PI3K/Akt signaling pathways stimulated by growth factor regulates downstream effects factor through phosphorylation tuberin protein, affecting growth and proliferation of cells.Research found that TSC2 can effect on the function of mTOR through Ras homologue enriched in brain (Rheb) which regulates cell growth and proliferation. TSC2 can expedite the conversion of Rheb-GTP into Rheb-GDP suppressing the downstream gene mTOR activity. The phosphorylated TSC2 lost the function to Rheb which has GTP-ase activity, resulting in Rheb-GTP gathering. Rheb-GTP can effectively activate mTOR, mTOR through phosphorylation inhibit the activity of a 4E-BP (eukaryotic translation initiation factor 4E-binding protein) and activate S6K1 (70kDa ribosomal protein S6 kinasel) and S6K2 (70kDa ribosomal protein S6 kinase2) activity, which promote cell growth, value and the differentiation that ultimately lead to tumors.In recent years, the relationship between the mTOR signaling pathways and cancer has become research hot spot, including AKT/TSC1-TSC2/mTOR pathways and related gene. Research shows that Tsc2 mutations resulting in protein dysfunction can cause AKT/TSCl-TSC2/mTOR signal transduction abnormalities, leading to non-TSC associated tumour, such as the gallbladder carcinoma, nasopharyngeal carcinoma, laryngeal cancer, colorectal cancer, lung adeno-carcinoma, lung small cell carcinoma and bladder cancer in sporadic. But the role of Tsc2 in the occurrence and development of esophageal have not been reported in the literature.In order to discuss the role of Tsc2 abnormal expression in the occurrence and development of esophageal, this study adopts TSC2 ASODN (antisense oligonucleotide) processing esophageal cancer EC9706 cells, using the MTT method detection cell growth situation; With flow cytometric technology and TUNEL method detecting apoptosis rate changes; with immunocytochemistry, Western Blot detecting TSC2 and mTOR protein expression of the EC9706 cells; employing in situ hybridization, RT-PCR technology testing cells Tsc2 mRNA and mTOR mRNA expression.Materials and methods1. Human esophageal carcinoma cell lines EC9706 were present from the Chinese Academy of Medical Sciences Cancer Institute.2. TSC2 ASODN, TSC2 SODN and N-ODN were designed and synthesized.3. The ASODN of TSC2 at 5,10, and 15μmol/L was transfected into EC9706 cells by the cationic liposome for 24,48, and 72 hours. Sense oligonucleotide and nonsense oligonucleotide at15μmol/L was transfected as sense and unrelated control, and EC9706 cells not being transfected as normal control.4. The MTT method was used to detect cell growth situation.5. With flow cytometric technology and TUNEL method detecting apoptosis rate changes. 6. Using immunocytochemistry and Western Blot detecting TSC2 and mTOR protein expression of the EC9706 cells.7. Employing in situ hybridization, RT-PCR technology testing cells Tsc2 mRNA and mTOR mRNA expression.8. Statistical analysis:The SPSS 17.0 statistical software was used.Results1 MTT test resultsCompared with the control group, the TSC2 ASODN can strongly promote the EC9706 cell proliferation (all P<0.05), and there were no obvious difference between the control groups (all P>0.05). In TSC2 ASODN transfect-ion group, the effect has concentration and time dependence, there were statistical significances differences between the various concentrations of transfection and between the different time (all P<0.05).2 The cell proliferation was detected by flow cytometryCompared with the normal control group (G0/G1:62.11±4.72%, S:17.22±1.11%),unrelated control group (G0/G1:64.80±1.25%, S:17.28±1.33%) and sense control group (G0/G1:66.75±2.78%,S:18.65±1.34%), the number of cells in each period was significantly different:the number in GO/G1 phase remarkably decreased, the one in S phase increased, and there were statistical significances (all P<0.05). In TSC2 ASODN transfection group, the effect has concentration and time dependence, there were statistical significances between the three concentrations (all P<0.05).3 The apoptotic was detected by TUNEL and flow cytometryTwo methods both results show that cell apoptosis rates of experimental groups concentration at 5,10,and 15μmol/L respectively are:early apoptosis:4.27±0.28 3.04±0.26 and 1.96±0.08%, late apoptosis:4.66±0.30,3.25±0.3 and 1.99±0.18%, AI:13.11±0.13,9.31±0.29 and 4.38±0.43%. Compared with the normal control group (early apoptosis:4.91±0.14%, late apoptosis:10.35±0.47%, AI:16.23±0.45%), unrelated control group (early apoptosis:5.12±0.12%, late apoptosis:9.68±0.55%, AI: 16.63±0.34%) and sense control group (early apoptosis:5.13±0.23%, late apoptosis: 9.77±0.54%, AI:16.46±0.43%), The apoptosis rates were significantly reduce, and the difference was statistically significant (all P<0.05). The apoptosis rate reduced with the concentration of TSC2 ASODN, there were statistical significances between the different concentrations (all P<0.05).4 The immunocytochemistry and the Western Blotting results4.1 The expressions of TSC2 protein in each group of cellsCompared with the control group, the expressions of TSC2 protein in TSC2 ASODN transfection group was decreased significant, and the differences each other were statistically significant(F immunocytochemistry=574.30,732.06,2293.70, F western blotting=50.6,330.69,1221.28,all P<0.001), and change with concentration-dependent and time-dependent(F immunocytochemistry=253.54,270.00,269.64, F Western blotting=123.47,83.59,13.29, all P<0.001). Namely, with the increase of TSC2 ASODN concentration and the extension of time, restrain TSC2 protein expression ability enhancement.4.2 The expressions of mTOR protein in each group of cellsCompared with the control group, the expressions of mTOR protein in TSC2 ASODN transfection group was increased significant, and the differences each other were statistically significant(F immunocytochemistry=87.15.41.89,36.34, F western blotting=72.55,114.27.302.39 all P<0.001), and change with concentration-dependent and time-dependent(F immunocytochemist,y=72.55,114.27,302.39, F Western blotting=87.15. 41.89.36.34,all P<0.001).Namely, with the increase of TSC2 ASODN concentration and the extension of time, promote mTOR protein expression ability enhancement.5 The in situ hybridization and RT-PCR results5.1 The expressions of TSC2 mRNA in each group of cellsCompared with the control group, the expressions of TSC2 mRNA in TSC2 ASODN transfection group was decreased significant, and the differences each other were statistically significant(F in-situ hybridization=67.81.139.61.392.57, F RT-PCR=1472.41,2733.14.656.62,all P<0.001), and change with concentration-dependent and time-dependent (F in-situ hybridization=260.23.572.22,1004.35, F RT-PCR=53.56.260.40,333.24,all P<0.001.Namely, with the increase of TSC2 ASODN concentration and the extension of time, restrain TSC2 mRNA expression ability enhancement. 5.2 The expressions of mTOR mRNA in each group of cellsCompared with the control group, the expressions of mTOR mRNA in TSC2 ASODN transfection group was increased significant, and the differences each other were statistically significant(F in.situ hybridization=381.78,772.79,1303.82, F RT-PCR=140.66,153.57,160.65,all P<0.001), and change with concentration-dependent and time-dependent (F in-situ hybridization=1064.87,1334.50,2793.36, F RT-PCR= 167.59,247.39,541.54, P<0.001).Namely, with the increase of TSC2 ASODN concentration and the extension of time, promote mTOR mRNA expression ability enhancement.Conclusion1. TSC2 ASODN inhibits cell apoptosis, make S period cells increased, G0/G1 phase cells reduce, strengthen the cell growth.2. TSC2 ASODN can specific inhibit TSC2 expression of esophageal cancer cells EC9706, and raised its mTOR expression.