| Objectives:Esophageal cancer is one of the common malignant tumor in the world Ranking in the various forms of cancer mortality stand sixth on the list; new additional patients are more than300thousand each year. And most of them occured in developing countries. Our county is a place with high-rate occurrences of esophageal carcinoma, especially in cixian andshexian of hebei, Linxian of Henan,Taihang Mountain areas, Northern Jiangsu Areas, North Dabie Mountain area, northern regions of Sichuan, Chaoshan area and area inhabited by Kazakans people in Administration of Xinjiang Uygur Autonomous Region. In China, esophageal cacinoma is the second on incidence and mortality of the stomach cancer. Esophageal squamous cell carcinoma (ESCC) is the major pathologic types in China, the incidence of it accounting for about90%.The inhibitor of apoptosis proteins (IAPs),is a kind of proteins with the structure of homology cell endogenous inhibitor of apoptosis family. PED/PEA-15(phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes) is a broad-spectrum anti-apoptotic function of small molecul-eprotein, it was cloned in the organization of type II diabetes in1998Concerned with the signal transduction, apoptosis, activation of ERK and glucose transport and so on. Pay close attention toit due to its spe-cial molecular structure and function of inhibiting apoptosis, at the same time, PED/PEA-15may open new avenues for diagnosis and treatmen-t of tumors as a target protein. Caspase-3(CPP32, apopain) is the most important way of the caspase cascade. Caspase-3mediat-edapoptosis is prev-alent in a variety of cells in the body, stimulates avariety of factors to start the signal to activate Caspase-3, cytoplasm, nucleus and cytoskeleton- protein degradation inactivation. It can be said that Caspase-3is a key to enzyme in apoptosis in mammalian cells.PED/PEA-15existes widely in the cytoplasm. Its size is15kDa, lies in the1q21-22. PED/PEA-15is the substrate of the PEA-1(phosphoprotein enriched in astrocytes) on the way of the PKC (Protein kinase C) PED/PEA-15exists in Cells in the forms of two differentiated Spliceosome, which sizes are2.8kb and1.7kb respectively, and are corresponding with clone1and clone4. Studies have found that PED/PEA-15can block Caspase so that the tumor cells can evade apo-ptosis. Studies have shown that high expression of PED/PEA-15in a variety of tumor cells including lung squamous cell carcinoma, laryngeal cancer, prostate cancer. The nature of their expression levels with tumor, degree of differentiation, prognosis, the evolution of the tumor and chem-otherapy are related.This experiment examine that PED/PEA-15protein expressed in human esophageal carcinoma, analysised its relationship with esophageal carcin-oma clinical pathological features, and combined withfollow-up data to explore the significance of its expression and prognostic value. In this study,we selected the esophageal carcinoma cell lines TE1, TE2, TE10, T-Tn, Yes2, Yes4,Yes6to detect the expression of the PED/PEA-15in cell lines and selected the TE1as a research object. We established PED/PEA-15plasmid transfer cells department of PED/PEA-15gene silencing cell lines. Observed its influence on cell proliferation, apoptosis and cell cycle, and detected the expressionofcaspase-3indifferent cell lines. Explored the role of the the PED/PEA-15pathway in caspase-3apoptot-ic pathway, ADR (adriamycin) as interference agents, to induce apoptosis. We want to show the possible molecular mechanism of PED/PEA-15inhibition in esophageal in cell apoptosis, cell cycle regulation in order to reveal. We build cancer xenografts in nude mice to observe the PED/PEA-15role in promoting the growth of nude mice bearing human tumors, an-d to study the possibility of gene therapy of esophageal ca-ncinoma tar- gets.Our study discussed the possibility of PED/PEA-15inhibitors used in tumor clinical treatment to provide a theoretical basis and experimental evidence.Methods:1PED/PEA-15expression and significance in esophageal cancinoma tissuesThe histopathology of the esophageal squamous cell carcinoma were taken from the Fourth Hospital of Hebei Medical University, Thoracic Surgery2005surgical inpatients wax block85cases.54cases of male patients,31cases of female patients, male to female ratio of1.74/1, the average patient aged57.1years.The tumor located at upper11cases, tmiddle51cases, low23cases. No patients had been radistion and chemo therapy, postoperative follow-up data integrity. Control group:from January2005to December2005,90cases of benign esophageal disease tissue made gastrointestinal endoscopy and esophageal endoscopy, Immunohistochemical method for joint detection of85cases of esophageal carcinoma and90cases of normal esophageal tissue in PED/PEA-15protein expression, analysised its relationship with esophageal carcinoma clinical pathological features, combined with follow-up data to explore their expression in esophageal significance and prognostic value. Combined with results of immunohistochemical detection the expression of PED/PEA-15mRNA in esophageal carcinoma cell line TE1, TE2,TE10,T-Tn,Yes2,Yes4,Yes6.2The Effect of PED/PEA-15gene silencing on apoptosis of esophageal cacinomar cellsSelected human esophageal cacinoma cell lines by RT-PCR method As The Studied Object. The full-length gene of PED/PEA-15was amplified to build a PED/PEA-15in vitro eukaryotic expression vector, and transfected into the esophagus and to cancer cell lines. Observed PED/PEA-15expression on esophageal cacinoma cell, TE1cells transfected with to PED/PEA-15specific shRNA (short hairpin RNA) knock PED/PEA-15gene, inhibition of endogenous PED/PEA-15gene expression. To observe the apoptosis and analysis of possible causes, to explore its possible mechanism of action. With ADR (doxorubicin) interfering, it induced apoptosis, and through detection of caspase-3changes of apoptosis.Human esophageal carcinoma cell lines TE1were commonly cultured in Fetal bovine serum-free RPMI-1640medium. we detected the change of Cell line cell apoptosis of the transfection PED/PEA-15of PED/PEA-15and of shRNA of the TE1; flow cytometry (FCM) and TUNEL apoptosis in experimental determination of the cell cycle and apoptosis rate; extract the different drug concentrations and time of total cellular protein by Western blot Method.3Stable expression of PED/PEA-15gene esophageal carcinoma cell line to tumor-bearing mice tumor growthCulture stably transfected with the PED/PEA-15gene esophageal cancer TE1cell lines and non-transfected TE1cell lines, from which took out of each of the4×106cells was inoculated in the left of the BALB/c-nu/nu nude mice lower abdominal subcutaneous build esophageal cacinoma cell xenografts in nude mice.10mice were randomly divided into experimental and control group (n=5). The experimental group inoculated PED/PEA-15stable transfected cells, the control group inoculated without transfected cells. Feeding mice30days, and then putting them to death, weighing tumor mass. During the experiment, every three days the tumor was measured the longest diameter (a) and shortest diameter (b) to calculate the transplanted tumor volume:V=a*b2/2, drawing the tumor growth curve, calculating the weight inhibition rate and the volume inhibition rate. observed the morphological changes of the transplanted tumors under the HE staining; FCM determinated of the cell cycle and apoptosis rate.Results:1PED/PEA-15protein expression and its significance in an esophageal carcinoma tissuesPED/PEA-15expression rate is58.8%in esophageal squamous cell carcinoma, the positive expression rate of benign esophageal disease tissue is33.3%, the esophagus distal cancerous tissues is45.9%. Statistical analysis showed that there are significant difference (P<0.05)among the three expression.Studies have shown that50cases of esophageal squamous cell carcinoma in85cases of The PED/PEA-15positive expression, the total positive expression rate was58.8%. Distribution as follows:60.0%male,40.0%female; patients over the age of60years36.0%,under the age of60patients64.0%; the tumor is located in the upper esophagus14.0%, the middle62.0%,24.0%lower segment. Statistical analysis showed that PED/PEA-15expression is no significant difference with gender, age, tumor site, these groups with no statistical significance.Organization poorly differentiated in the the positive expression of PED/PEA-15is6cases (12.0%), moderately differentiated35cases (70.0%), high-differentiated9cases (18.0%).The patients whose esophageal primary tumor invasiveness reached mucous layer and submucosal layer are2cases (4.0%), the deep muscle layer are13cases (26.0%), fiber membrane are35cases (26.0); without lymph node metastasis are5cases (10%), lymph node metastasis are45cases (90%). Statistical analysis found that the expression of the PED/PEA-15had a close relation to the degree of differentiation of the primary tumor, depth of invasion, lymph node metastasis are closely related (P <0.05). The higher positive of PED/PEA-15expression,the patient survival rate is longer (P<0.05).PED/PEA-15mRNA expression is no significant difference (P>0.05)in TE1cell lines (1.439±.605), TE2cell lines (1.1411±.497),TE10cell lines (1.338±0.588),T-Tn cell lines(1.501±0.091),Yes2cell lines(1.360±0.660), Yes4cell lines (1.275±0.437) and Yes6cell lines(1.112±0.360)2The Effect of PED/PEA-15gene silencing on apoptosis of esophageal cacinomar cellsSelected human esophageal cacinoma cell lines:TE1,TE2,TE10, T-Tn,Yes2,Yes4,Yes6, to detect mRNA by the RT-PCR method. Result showed that there was no statist ically significant difference among these groups. There are differences in the protein expression among the cell strains by Western blot examination.In order to facilitate the research, we selected two better transfection esophageal carcinoma cell lines TE1and T-Tn to detect the influence on PED/PEA15-15gene of esophageal cancer cell proliferation, the result showed that cell proliferate more quickly with the PED/the PEA-15gene expression increased. MTT result showed that TE1cells and T-Tn cells of the transfection PED/PEA-15can promote cell proliferation, and the time-dose-dependent cell proliferation rate IR(χ±s)%:TE1transfection group concentration of2μg when compared with the control group,, there was no significant difference (P>0.05) for12h,24h;,there are significant differences with the control group (P<0.05) for48h,60h; transfection group concentration for4μg with the control group compared to12h no significant difference (P>0.05); and there is significant difference (P<0.05) for24h,48h,60h. The T-Tn transfer dye group concentration is2μg when the control group compared to12h,24h,48h there was no explicit forward difference (P>0.05), there is significant difference (P<0.05) when it is for60h; transfected group concentrationcompared with the control group for4μg12h no significant difference (P>0.05); and the control group was significantly different (P<0.05) for24h,48h,60h.Joined the ADR (adriamycin), the Western-blot results showed that PED/PEA-15protein levels in TE1cell line decreased expression of the difference among these groups was significant (P<0.05), at the same time detecting apoptotic cells by Caspase apoptosis kit, found that there is no significant in Pro caspase-3protein expression joined the ADR participation, and caspase-3activation cleavage, indicating that the increased activity of ADR under the action of apoptosis, PED/the role of the PEA-15is inhibited.Transfected with empty vector plasmid and exogenous TE1esophageal cancer cells were transfected with the PED/PEA-15gene in the same ADR processing conditions, Western-blot empty vector transfected group Caspase-3is activated to indicate apoptosis The PED/PEA-15transfection group, while no apoptotic cells between the two groups was statistically significant difference (P<0.05). Shows that the PEA15protein inhibition of tumor cell apoptosis.The Effect of PED/PEA-15gene silencing on apoptosis of esophageal cacinomar cells:TUNEL apoptosis experiment results showed that PED/PEA-15was knocked out,TE1cell apoptosis was significantly increased. FCM detection apoptosis, results showed that:the apoptosis rate of the control group (9.83±1.34)%(P<0.01) was significant lower than the shRNA apoptosis rate (19.53±0.80)%. PED/PEA-15gene silencing can increase TE1cells apoptotic activity. Western-blot results showed that:ADR processing after two cell lines transfected with transfected the PED/PEA-15specific shRNA cell lines increased apoptosis, caspase-3activity, significant difference between the groups.3Stable expression of PED/PEA-15gene esophageal carcinoma cell line to tumor-bearing mice tumor growthHuman esophageal carcinoma xenografts in nude mice was successfully constructed, tumor formation rate is100%. The end of the experiment, the size and weight of the transplanted tumors in experimental group were significantly greater than the control group (P<0.05), volume inhibition rate was-175%, weight inhibition rate was-78.6%; compared with the control group and experimental group The transplanted tumor cell apoptosis rate was significantly lower (7.03±1.63)%, in G0/G1phase cells was significantly lower, and S-phase cells was significantly increased (P<0.05).Conclusions:1PED/PEA-15high expression in human esophageal squamous cell carcinoma, and have a close relation with the depth of tumor invasion, tumor grade and lymph node metastasis are closely related. PED/PEA-15and the prognosis of esophageal cancer is directly relation, can be as the judgment of esophageal cancer prognosis indicators.2PED/PEA-15expression in different cell lines, and the expression was no significant difference. PED/PEA-15gene in human esophageal cancer cell proliferation influential,with increased expression levels of PED/PEA-15gene, cell proliferation accelerated.3High expression of PED/PEA-15inhibited the apoptosis of esophageal carcinoma cell line. PED/PEA-15gene silencing, and esophageal canc-er cells can inhibit proliferation and induce apoptosis. The PED/PEA-15expected as an effective target for gene therapy of esophageal cancer.4In vivo test proved that PED/PEA-15promoted the gro-wth of human esophageal cancer cell lines TE1nude mice transplanted tumor. PED/PEA-15promote tumor effect by promoting tumor cell proliferation, inhibit apoptos-is andcause the cells in G0/G1phase to accelerate its mechanism may be implementedto prevent caspase activation. |
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