| Background:Esophageal cancer is one of the most common malignancies of the upper aerodigestive tract.Stage 1-3 esophageal cancer is usually treated with surgery to remove the affected section of esophagus.However,approximately half of the patients were diagnosed at a late stage with an unresectable disease.Treatment for these patients aims to improve quality of life and prolong survival.Although a number of cancer types have experienced double-digit improvements in survival statistics in the last few decades,the 5-year survival rate of esophageal cancer remains still poor and esophageal cancer is now considered as an extremely difficult cancer to beat.Having analyzed the clinical data,it is obvious that survival rates are unlikely to improve significantly until new anticancer drugs are available.To develop novel molecularly targeted therapeutic agents,it is absolutely needed to identify the molecular events and main targets associated with the development and progression of this disease.Extracellular matrix proteins represent a class of attractive targets to be explored in esophageal cancer treatment.Cancers develop and progress in the context of the extracellular matrix(ECM).The ECM is essential for normal development and maintenance of the microenvironment of embryonic and adult tissues.The ECM is composed of structural proteins,glycosaminoglycans,growth factors and cytokines,secreted(pro)enzymes,and glycoproteins called matricellular proteins.The ECM not only provides structural stability to tissues and organs,but also regulates activities of growth factors and cytokines by directly tethering inactive pro-forms to structural components of the ECM.The composition of the ECM has profound effects on cell behavior,including adhesion,proliferation,and migration,as well as on the maintenance of the stem cell niche.In addition,matricellular proteins participate in dynamic matrix-cell interactions and fine tune cellular functions.Fibulin-5(FBLN-5)is one of seven members of the fibulin family of extracellular matrix proteins,also known as the EVEC or DANCE,in one chromosome in 14 q32.1,amember of Class II short fibulins,founded in 1999 by two attempted a new blood vessel growth regulator's team identified for the first time.It is a typical rod-shaped morphology characterized small secreted glycoprotein,molecular weight of 66 k Da,containing 448 amino acids,consists of two kinds of structure module,namely EGF_like domain repeats and a unique C_terminal fibulin_type module.In the first calcium-binding epidermal growth factor-like duplication there is a proline-rich insertion sequence that interacts with a large number of ECM and secretory proteins through the structural domain.FBLN-5contains an integrin_binding arginine-glycine-aspartic acid(RGD)motif motif.Functionally,FBLN-5 binds ?v?3??v?5 and ?9?1 integrins,which regulates the adhesion of endothelial cells through its RGD motif.Previous studies have shown that Fibulin-5 has various biological potentials in different types of cancers,displaying both tumor-promoting and tumor-protective functions by mechanisms that are not totally defined.Prior to this study,the role of Fibulin-5 in esophageal cancers has not been reported yet.Therefore,we first investigated the expression of Fibulin-5 in human esophageal cancer.Then,in vitro and in vivo studies,we verify the carcinogenic properties of fibulin-5 in esophageal epithelial cells.Finally,the molecular mechanism of Fibulin-5 in esophageal epithelial cells with carcinogenic activity was preliminarily investigated.Part ? Investigation on the expression of Fibulin-5 protein in patients with esophageal cancerObjective: To investigate the expression of Fibulin-5 in esophageal cancer and its relationship with clinical indicators.Methods: We collected the pathology diagnosed esophageal cancer tissue microarray,including 100 esophageal cancer and 80 tissue adjacent to carcinoma tissue.The expression of Fibulin-5 in esophageal carcinoma and adjacent tissues was analyzed by immunohistochemistry.And We analyzed the relationship between the expression of Fibulin-5 in the cytoplasm and nucleus and the expression of tumor cell proliferation marker ki67,and clinical indicators including age,sex,tumor size,histological grade,T stage,lymph node metastasis and clinical stage.The relationship between Fibulin-5expression and clinical adverse prognostic factors and survival time was also analyzed.Results: Compared with adjacent tissues,Fibulin-5 in cancer tissues has higherpositive staining signals in cytoplasm and nucleus,and Fibulin-5 cytoplasmic expression and the expression of tumor cell proliferation markers ki67 were positively correlated.The correlation analysis of clinical indicators revealed that the expression level of Fibulin-5 in cytoplasm was positively correlated with histological classification,lymph node staging and clinical staging,while nuclear grading was only related to lymph node staging.Survival analysis showed that patients with high expression of Fibulin-5 cytoplasm had worse prognosis than those with low expression of Fibulin-5 cytoplasm.Conclusion: Fibulin-5 is highly expressed in human esophageal carcinoma and may play a promoting role in esophageal cancer.Part ? The study on Fibulin-5 transformed esophageal epithelial cellsObjective: To investigate the carcinogenic properties of Fibulin-5 in esophageal epithelial cells.Methods: We first constructed the expression plasmid of Fibulin-5 mammalian cells,and established the stable transfected esophageal epithelial HET-1A cell line of p MSCV-Fibulin-5-puro.Then we used soft agar colony formation assays to examine its ability to transform normal esophageal epithelial HET-1A cells.After 4 weeks,cell colonies in the negative control HET-1A cells transfected with pc DNA3.1 and without any plasmid transfected was compared;Finally we used a mouse tumor xenograft model to investigate its potential in the tumorigenicity.We subcutaneously injected 10 mice with fibulin-5 overexpression HET-1A cells.After 4 weeks,We observed the tumor growth in mice and compared with the mice injected with normal HET-1A cells.Results: The normal esophageal epithelial cells were transfected with p MSCV-Fibulin-5-puro and p MSCV-puro plasmid.After screening with puromycin,stable transfected cell lines were constructed around 4 weeks later.By Western Blotting test,We found that there was no Fibulin-5 expression signal in the cells without transfection and transfected with blank vector p MSCV-puro,while the stable Fibulin-5 transfected cell line had obvious immune signal,Which confirmed the overexpression of Fbulin-5;Many cell colonies in HET-1A cells with Fibulin-5 overexpression after 4 weeks were observed in soft agar colony formation experiments,but we didn't find any cell colonies in HET-1A cells transfected with pc DNA3.1 negative control and without plasmid transfection;Nine out of 10 mice subcutaneously injected with Fibulin-5 overexpression HET-1A cells developed tumors of average size after 4 weeks,while no tumors were found in mice injected with normal HET-1A cells.Conclusion: Fibulin-5 has a translational effect on esophageal epithelial cells and may function as a driver gene in the development of esophageal cancers.Part ? The study on the active molecular mechanism of Fibulin-5 proto-carcinomaObjective: To preliminarily investigate the molecular mechanism of fibulin-5 in esophageal cancer cells with carcinogenic activity.Methods: We first constructed fibulin-5 gene knockout vector,the stable transfected PX330-FBLN5 and PX330 esophageal cancer EC109 cell lines were established,and the phosphorylation water of AKT Ser473 was detected by Western Blotting.Results: A stable fibulin-5 gene knockout cell line was successfully constructed around 4 weeks.Western Blotting test found that compared with the blank not transfection and transfection carrier PX330 cells,fibulin-5 gene knockout cell expression signal significantly reduced,which confirmed fibulin-5 gene knockout success;Compared with the normal cultured cells,there was no obvious difference on AKT protein expression levels in the Fibulin-5 overexpression and reduced expression cells.On the contrary,AKT Ser473 phosphorylation levels were significantly different in the three cells.Conclusion: Fibulib-5 may enhance its tumor promotion function in esophageal cancer by activating AKT signal transduction. |
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