Effects Of Rab3D On The Development Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer is one of the most common malignant tumors in China and the most common pathological type is esophageal squamous carcinom that accounts for nearly90%.According to Chen W statistics,Cancers of lung,stomach,liver,colorectum and esophagus were the most five common cancers of all new cases.Lung cancer,liver cancer,stomach cancer and esophageal cancer were the leading causes of cancer death.Although the surgery technology,radiotherapy and chemotherapy technology progress,the mortality and morbidity of esophageal cancer is still high.There was still a lack of definitive clinical therapeutic targets to esophageal squamous cell carcinoma(ESCC).Rab3D,a member of the RAS oncogene family,is widely expressed in human tissues,and those expression is significantly higher in primary tumor and cancer cell lines.However,it has not been reported in detail about the expression and mechanism of Rab3D in ESCC.ObjectivesIn this study,the expression of Rab3D protein and its correlation with clinical pathology was detected by immunohistochemical technique in 20 cases of ESCC tissues,adjacent,normal esophageal mucosa tissues and 64 cases ESCC tissues.pcDNA3-Rab3D recombinant plasmid and Rab3D-siRNA were designed and transfected respectively into the ESCC cells.The expression of Rab3D was up-regulated or down-regulated.Besides,the proliferation on ESCC cells and the expression of CyclinD1 was determined by MTT assay and Western Blot.In this study,we aimed to explore the expression of Rab3D in ESCC tissues,furthermore to provide a new method for the prognosis and treatment of ESCC.Methods1.The expression of Rab3D and its clinical pathological significance in ESCC20 cases of ESCC tissues,adjacent,normal esophageal mucosa tissues and the tissue microarray which contains 44 cases of ESCC tissue were selected.Rab3D protein expression and its clinical pathological significance in esophageal tissue was detected by immunohistochemistry technique.2.Effect of Rab3D on ESCC cell lines proliferation was detected by MTT array.The eukaryotic expression recombinant plasmid pcDNA3-Rab3D and Rab3D-siRNA were constructed and then transfected pcDNA3-Rab3D into cells with low Rab3D expression via lipofectamine.MTT assay and Western Blot were used to explore the overexpression of Rab3D on ESCC cell lines proliferation.Rab3D-siRNA was transfected into the cells with high Rab3D expression,and the low expression of Rab3D on cell proliferation was detected with the same methods.3.Effect of Rab3D on the expression of Cyclin D1 was detected by Western Blot.In order to illuminate the underlying mechanisms of Rab3D in ESCC cell lines proliferation,the pcDNA3-Rab3D recombinant plasmid and Rab3D-si RNA were transfected respectively into ESCC cells with low or high Rab3D expression.And then,MTT and Western blot were used to explore the effect of Rab3D expression on the expression of Cyclin D1.Results1.The expression of Rab3D and its clinical pathological significance in ESCCThe over-expression rate of Rab3D protein in normal tissues,tumor-adjacent tissues and ESCC tissues were 10%(2/20),30%(6/20)and 90%(18/20),respectively,with a significant difference among them(P<0.05).The expression analysis of Rab3D protein in64 ESCC tissues as followed: The over-expression rate of Rab3D protein in ESCC tissues stages Ⅰ and Ⅱ was 38.70%(12/31),that of stages Ⅲ and Ⅳ was 66.67%(22/33),there was significant difference between them(P<0.05).In addition,significant differencewas detected between the over-expression rate of Rab3D protein in ESCC tissues with no lymph node metastasis 37.93%(11/29),and lymph node metastasis 65.71%(23/35)(P<0.05).2.Effect of Rab3D on ESCC cell lines proliferation was detected by MTT array.The MTT showed that the cell proliferation rate of ESCC cell lines transfected with Rab3D plasmids has a significantly higher than that of the control group which were transfected with empty vector(P<0.05).However,the cell proliferation rate of ESCC cell lines transfected with Rab3D-siRNA was lower than that of the control group with statistically significant difference(P<0.05).3.Effect of Rab3D on the expression of Cyclin D1 was detected by Western Blot.The western blot array showed that the expression of Cyclin D1 in ESCC cells lines after transfected with Rab3D plasmids were increased compared to the control group(P<0.05).However,the expression of Cyclin D1 in ESCC cells lines declined greatly compared to the control group after transfected with Rab3D-siRNA(P<0.05).ConclusionThe expression of Rab3D is corelated with the differentiation degree of ESCC.And it was found that Rab3D expression in ESCC tissues was associated with TNM stage and lymph node metastasis.In our study we also detected that the expression of Rab3D could promote the proliferation of ESCC cell which was involved the overexpression of Cyclin D1.As a result,Rab3D expression may have potential clinical significance to predict prognosis and the progression of ESCC.Rab3D may become a target for the clinical treatment of ESCC.