| Background and objective:Esophageal cancer is one of the common malignant tumors in China, and is the fourth cause of cancer-related death owing to its high mortality. Esophageal cancer has an obvious regional distribution in China and Shandong Province is one of the districts with high incidence of esophageal cancer. Esophageal cancer shows a highly invasive growth pattern as other digestive tract tumors, and prone to invasion and metastasis.Angiogenesis exists in many physiological and pathological processes; it can be involved not only in physiological processes such as embryonic occurrence, wound healing, female reproductive cycle of physiological process, but also in pathological processes such as inflammation, rheumatoid arthritis, diabetes, retinal disease, tumors and so on. Recently, research has found that angiogenesis plays particularly important roles in the process of growth, progression and metastasis in solid tumors. Some experimental researches have demonstrated that antiangiogenic therapy can effectively inhibit tumor growth, and decrease the ability of relapse and metastasis. Angiogenesis is an extremely complicated process involved in multiple factors, and regulated and controlled by many kinds of factors. Vascular endothelial growth factor (VEGF) is the most important promote angiogenesis factor, plays important roles in the process of angiogenesis. The roles of VEGF in liver cancer, renal carcinoma, breast tumor, lung cancer and ovarian cancer has well been confirmed, however, few studies have investigated the effects of VEGF on the biological behaviors of esophageal cancer, no data is available about this inland.RNAi refers to the phenomenon that the double-stranded RNA specific block the expression of its homologous gene after into the cell. RNAi is an important tool for the research of genetic function currently, and it has been employed to the research of gene therapy for tumor and virus infection. Lentivirus as carrier to mediate the RNAi for the investigation of gene therapy has more practical application potential, because compared to other virus carrier system, lentivirus can transduce non-dividing cells and integrate the shRNA express structure into the host cell genome, then achieve the stabile silence of purpose gene.In the present study, we detected the expression levels of VEGF-C protein in esophageal squamous cell cancer tissues and investigated the relationship of VEGF-C protein expression to various clinicopathologic factors and prognosis; RNAi was performed to destroy VEGF-C mRNA and then make the synthesis of VEGF-C protein at extremely low levels, reduce the VEGF-C protein accumulation and transcription regulation of its downstream gene, blocking the tumor cells response to a great extent and establish a kind of gene-modified treatment strategy for esophageal cancer, improving the therapeutic effects, to provide theory basis and technical countermeasures for early diagnosis and gene therapy of esophageal cancer based on the therapeutic target of VEGF-C.Part1Investigation the expression levels of VEGF-C in esophageal cancer tissuesMethods:A total of46esophageal squamous cell cancer patients undergoing surgical resection at the department of thoracic surgery, Provincial Hospital affiliated to Shandong University, between1998and2001were enrolled in this study. Among these46patients, male36cases and female10cases; ranging in age from30years to74years, median age of56years. According to the tumor-node-metastasis staging system of the International Union against Cancer (UICC)1997, T12cases, T221cases, T322cases and T41case:regional lymph node metastasis14cases and32cases without metastasis; stage Ⅰ2cases, stage Ⅱa31cases, stage Ⅱb5cases and stage Ⅲ8cases. According to the histology grading,Ⅰ8cases, Ⅱ28cases and Ⅲ10cases. None of these patients has received operative radiotherapy and/or chemotherapy. All patients were followed up and with complete follow-up data.The immunohistochemistry was performed according to the specification of SABC kit. VEGF-C polyclonal antibody was used at the dilution of1:50and F8antibody was used at the dilution of1:200. respectively. For VEGF-C protein,>30%cancer cells stained as yellow-brown and immunoreactivity higher than normal smooth muscle cells was considered as "+";<30%cancer cells immunoreactivity higher than normal smooth muscle cells or the immunoreactivity of cancer cells was close to the normal smooth muscle cells was considered as "±"; the immunoreactivity of cancer cells lower than the normal smooth muscle cells was considered as "-";"+" was considered as VEGF-C positive expression. The tumor microvessel count was performed as follows:after microscopic identifying the area of greatest vascular density (hot spot) at a low magnification of×100, then3different "hot spots’" within or around the tumor mass of each slide were counted under a high magnification of×200. Any yellow-brown stained endothelial cells or cluster clearly separated from the adjacent microvessels, cancer cells and other connective tissues was considered as a single, countable microvessle. While the vascular cavity more than the diameter sun of8red blood cells and the microvessels under the normal mucosa were excluded. The highest value of the microvessles counted in3high-power fields was regarded as the final MVD for each tumor.All statistical analyses were performed with SPSS statistical software. Chi-square test was performed to examine the association between VEGF-C protein immunoreactivity and various clinicopathologic factors. The correlation between MVD and VEGF-C protein immunoreactivity was analyzed by nonparametric test. Kaplan-Meier method was used to calculate the survival rate, and log-rank test was used to compare the survival differences. Difference was considered significant when the P value was less than0.05.Result:Immunohistochernical analysis showed that the VEGF-C protein was immunostained mainly in the cancer cell cytoplasm, and the immunoreactivity in the invasive margin was higher than that in the central area. VEGF-C protein positive expression was detected in45.65%(21/46) of patients, significantly higher than the normal esophageal mucosa (P<0.05); VEGF-C positive expression was significantly associated with invasion depth (T1-2,30.43%; T3-4,60.87%;P<0.05), differentiation (Ⅰ,0.0%;Ⅱ,50.00%; P<0.05; ⅠI,0.0%; Ⅲ,70.00%; P<0.01) and MVD (P<0.05). VEGF-C protein positive expression was not significantly associated with gender, age, tumor length, TNM stage and lymph node metastasis, respectively (P>0.05). Survival analysis showed that the overall5-year survival rate was36.96%, the5-year survival rate of patients with VEGF-C protein positive expression was14.29%. while the5-year survival rate of patients with VEGF-C protein negative expression was56.00%, the survival difference was significant (P<0.05). Kaplan-Meier survival curves showed that the accumulative survival rate of patients with VEGF-C protein negative expression was significantly higher than that of patients with VEGF-C protein positive expression (P<0.01, log-rank test).Conclusions:VEGF-C protein abnormal expression was detected in esophageal squamous cell cancer tissues; its abnormal expression involved in the process of invasion and angiogenesis, and was closely associated with poor prognosis. Our results demonstrated that VEGF-C protein can be used as an important factor to determine the malignant biological behaviors and prognosis of patients with esophageal squamous cell cancer clinically. VEGF-C might be a new target for gene therapy and antiangiogenic therapy of esophageal cancer in the near future.Part2Construction the RNAi Lentivirus carrier systems and screening the effective targetsMethods:Employ the BLOCK-iT Lentiviral RNAi Expression System of Invitrogen Company to construct the VEGF-C RNAi lentivirus carrier, construct introduction cloning and virus packaging purpose plasmid. The production of RNAi lentivirus and titer measure:four plasmids transfect293FT simultaneously to produce the virus, transduction test was used to virus function titer measure, quantitative PCR was used to molecular titer measure. Screening the cell lines with gene stabile silence: blasticidin screening was performed after lentivirus transduce EC109cells, establishing VEGF-C gene silencing cell lines and control cell lines, conduct cell instantaneous transduce experiments.Results:In this study, RNAi professional design software was employed to design3groups shRNA interference target sequence aimed at VEGF-C target gene sequence, labeled as1#/2#/3#respectively, and then built into the vshRNA restructuring carrier, shift the connected product into bacteria competent cells, PCR appraisal and sequencing comparison. Our results showed that the3groups of shRNA restructuring carrier were successfully constructed and with correct sequencing. Collection the cell liquid supernatant rich in lentivirus particles, high titer of lentivirus concentrated solution was obtained after concentration, the virus titer was determined and demarcated in293T cells. Realtime PCR was performed to detect the expression levels of target gene mRNA, and then judge the interference effects of different target. Through the fluorescence pictures, we concluded that the efficiency of virus infection was more than90%. Among these3targets,3#is the most effective RNAi target, compared with NC group, the expression level of purpose gene was decreased about30%. Compare the high and low MOI values, high MOI with a better knock effect. And further tested the synthesis of VEGF-C siRNA and inhibit expressing effects.Conclusions:Through this part of experiment, we successfully constructed3groups specific shRNA lentivirus carrier, RNAi lentivirus packaging was finished at the same time, and completed the titer demarcate after virus packaging in293T cells. Further, we screened the highest effective target through the3groups of restructuring carriers, and explicit the non-specific toxic difference of different MOI values, provided reliable basis for the next employing the highest efficiency restructuring carrier to transduce the esophageal cancer cell lines. In addition, we also primarily validated that VEGF-C siRNA performed apparent inhibition in EC109cells.Part3Assessment the RNA interference effect and the changes of cell biological behaviorsMethods:EC109cells were cultivated in good growth status; the cells were divided into the6-well cultivating board one day before virus infection, RNAi lentivirus particles were added respectively according to the experimental design groups-to conduct the infection experiment. Three days after infection, fluorescent microscope was used to detect the expression levels of GFP, collect cells5days after infection, CCK8, flow cytometry was used to investigate the influence of VEGF-C on the proliferation capacity, cell cycle and migrating ability in esophageal cancer cell lines in vitro. Cloning experiments was used to confirm the promoting proliferation role of VEGF-C. Matrigel invasive experiment was used to investigate the influence of VEGF-C on invasive capacity in esophageal cancer cell lines in vitro.Results:The cell growth curve of interference group was apparently lower than the other2groups (blank control group and meaningless sequence restructuring carrier group), demonstrating that the proliferation capacity decreased significantly and the growth capacity was restrained after the interference of VEGF-C gene. Overexpression of VEGF-C can promote proliferation, migration and progress of cell cycle of esophageal cancer cell lines in vitro, while restrain the expression of VEGF-C can perform inhibitive effects, in addition, silencing VEGF-C can significantly inhibit the invasive capacity of esophageal cancer cell lines in vitro.Conclusions:We employed lentivirus carrier system to construct shRNA restructuring carrier aimed at VEGF-C gene, and successfully achieved efficient, specific and stable silencing effects in esophageal cancer cell lines in vitro. After VEGF-C gene was silenced, the biological behaviors of esophageal cancer cell lines in vitro changed such as cell proliferation cycle extended, invasive ability restrained obviously. |
PDF Full Text Request