| China is a high incidence of esophageal carcinoma area in the world andSquamous carcinoma is the most common pathological histology. Esophagealelectronic endoscopy and mucosal pathological examination are the effectivemeans for screening of high risk population and early diagnosis, but it is sohard as reconnaissance means to promote in the high risk areas of esophagealcarcinoma, so it is difficult for most of the patients with early esophagealcarcinoma diagnosis, patients are always in a middle-late period when they seethe doctors, the prognosis is poor. A large number of molecular biologyresearch data show that many factors including multi-stage, poly genes andseveral transcription factors participated in the occurring of esophageal cancer.While the exact pathogenesis is still unclear, the morbidity and mortality isstill showed an ascendant trend. Therefore, the research of abnormal activationof all kinds of signal pathways in the development of esophageal carcinoma(ERK pathway, AKT/mTOR pathway), and the new target of anticancer drugsbecome the main aim for the clinical treatment.Many reports confirmed that abnormal activations of AKT/mTOR signalingpathways existied in the process of tumor.Also, in the process of esophagealcarcinoma confirmed the existence of this situation.The main mechanism ofAKT/mTOR mediating occurrence and development of tumor is inhibiting cellapoptosis, promoting cell proliferation, controlling the cell cycle, promotingtumor vascularization and malignant invasion. mTOR is downstream signalmolecules of PI3K/AKT and participated in regulating cell growth, promotingcell migration and invasion. Its abnormal expression highly increases in tumorprogression, which show that mTOR abnormal activation relevant withtumorigenesis. So far, PTEN is the first tumor suppressor genes which hasphosphatase activity and lipid enzyme activity at the same time, while existes the function of mutation or missing in the malignant tumors. Recently,researches demeonstrate that PTEN can negative control PI3K/AKT/mTORsignaling pathways, reduce the activation of p-AKT and inhibit the abnormalexpression of downstream signaling pathways, in order to induce cellapoptosis and inhibit cell proliferation.This experiment transfected PTEN plasmid、sh-AKT plasmid (silent p-AKT) and mTOR plasmid to esophageal cancer cell lines respectively, throughdetecting the expression of PTEN、 sh-AKT and mTOR genes intra-cellularand the change of cell proliferation ability, further understood that PTENcould regulate AKT/mTOR pathway and cell proliferation in esophagealcarcinoma. All this results could provide a better and reliable theoretical basisfor the clinical treatment of esophageal carcinoma.Objective： To study the effect of AKT/mTOR pathway and theinhibition of proliferation on human esophageal carcinoma while transfectedPTEN plasmid in human esophageal carcinoma cells lines.Methods：1. To amply PTEN plasmid and then transfected the plasmid intoesophageal carcinoma cell TE1、TE13, examined the change of PTEN、AKTand m TOR in the molecular level through RT-PCR method and PTEN、AKTin the protein level through Western-blot method.2. To amply sh-AKT plasmid and then transfected the plasmid intoesophageal carcinoma cell TE1、TE13, examined the change of PTEN、AKTand m TOR in the molecular level through RT-PCR method and PTEN、AKTin the protein level through Western-blot method.3To amply PTEN plasmid and then transfected the plasmid intoesophageal carcinoma cell TE1and TE13, examined the change of cellproliferation ability on esophageal carcinoma through the Cell Count method.4.To amply mTOR plasmid and then transfected the plasmid intoesophageal cancer cell TE1、TE13, and examined the change of expression ofmTOR in the molecular level through the RT-PCR method. Results:1The effect of AKT/p-AKT after transfecting PTEN plasmid in humanesophageal carcinoma cell lines.RT-PCR results confirmed that after transfected PTEN plasmid to TE1andTE13cell lines, PTEN gene expression was obviously higher in thetransfection group than the other two groups in molecular level (TE1:0.466±0.033,0.487±0.028,1.138±0.055; TE13:0.354±0.015,0.402±0.011,0.936±0.058), the results were statistically significance difference(P＜0.05).Whiles AKT showed no obvious change (TE1:1.132±0.012,1.158±0.026,1.127±0.012; TE13:1.291±0.017,1.402±0.030,1.285±0.019, P＞0.05).After transfecting sh-AKT plasmid, PTEN expression had no significantdifference between different groups (TE1:0.466±0.005,0.530±0.005,0.409±0.009; TE13:0.451±0.026,0.492±0.024,0.466±0.060, P＞0.05), AKThad no obvious difference too (TE1:1.184±0.036,1.171±0.037,1.138±0.051; TE13:1.087±0.033,1.144±0.020,1.050±0.019, P＞0.05).Western Blot results showed that PTEN protein levels were markerly higherin the transfected group than the other two groups after transfecting PTENplasmid(TE1:0.363±0.013,0.389±0.028,0.901±0.036; TE13:0.326±0.039,0.288±0.017,0.662±0.041), the difference was statisticalsignificance(P＜0.05). However the p-AKT protein levels was lower in thetransfect group than the other two groups (TE1:0.816±0.032,0.806±0.028,0.430±0.012; TE13:0.809±0.050,0.803±0.067,0.400±0.051)(P＜0.05).After transfecting sh-AKT plasmid, the expression of PTEN demonstrated nosignificant difference among three groups (TE1:0.361±0.026,0.406±0.025,0.377±0.028; TE13:0.328±0.035,0.340±0.041,0.326±0.024, P＞0.05),and the expression of p-AKT in transfected group was lower than the othertwo groups (TE1:0.763±0.038,0.738±0.027,0.390±0.027; TE13:0.806±0.032,0.737±0.084,0.361±0.011)(P＜0.05).2The effect of cell proliferation on esophageal carcinoma whileenhancement the expression of PTEN.Cell Count method showed that the cell proliferation of human esophagealcarcinoma was inhibited significantly after transfected the PTEN plasmid. (TE1:(4.26±0.07)×10~5,(4.35±0.10)×10~5,(3.39±0.09)×10~5; TE13:(4.87±0.17)×10~5,(4.96±0.11)×10~5,(3.58±0.14)×10~5)(P＜0.05), the differencewas statistical significant difference.3The change of mTOR expression in the molecular level after differentplasmid transfected.RT-PCR results confirmed that the expression of mTOR gene showed noobvious among different groups after transfecting PTEN plasmid.(TE1:0.528±0.022,0.488±0.006,0.495±0.005; TE13:1.114±0.083,1.102±0.077，1.104±0.074, P>0.05). After transfecting sh-AKT plasmid, mTOR expressionalso had no obvious difference among different groups in both of two celllines.(TE1:0.750±0.040,0.714±0.027,0.707±0.023; TE13:0.842±0.070,0.866±0.061,0.862±0.080, P>0.05.)While after transfecting mTOR plasmid,mTOR gene expression was obviously higher in transfect group than the othertwo groups.(TE1:0.743±0.032,0.748±0.018,0.917±0.017; TE13:0.632±0.044,0.676±0.044,1.054±0.053)(P＜0.05), the results were statisticalsignificance difference.Conclusions:1. PTEN, as a tumor suppressor gene, had a negative effert on theactivation of AKT.2. PTEN may inhibit the activation of AKT, reduce the expression of p-AKT, and then suppress the proliferation of esophageal cancer cell.3. mTOR had an excessive expression in the esophageal cancer and maybe used as the new target for the treatment of esophageal cancer. |
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