| Objective:1,To study the antigen(CD44) which immuno-conjugated with the monoclonal antibody KMP1and the biological effects of KMP1.2,To investigate the effect of CD44targeted RNA interference (RNAi) on the growth and migration of human bladder carcinoma EJ cells, and research the change of EJ cells immuno-bounding to the monoclonal antibody KMP1.3,To investigate the change of KMP1immuno-bounding to the antigen and EJ cells which were degraded carbohydrate moiety, identify the glycosyltransferase expression levels on bladder tumor tissues of patients and normal bladder tissues, investigate the effect of GALNT1targeted RNA interference (RNAi) on the growth and migration of human bladder carcinoma EJ cells in vitro and in vivo, and research the change of shGALNT1-EJ cells immuno-bounding to the monoclonal antibody KMP1.Methods:1,Preparation of monoclonal antibody KMP1against bladder carcinoma, antigen identification, and its biological effects:We developed an antibody KMP1against bladder cancer that recognized the CD44of EJ cells. Antibody was screened by ELISA, Flow cytometry and immunohistochemistry. Antigen purification, staining with silver, and the amino acid sequence was studied. Soft agar colony formation assay and wound healing assay were performed to investigate the cell proliferation and cell migration in vitro. In vivo,3days after tumor inoculation in20mices, each mice in group B is injected intraperitoneally200ug KMP1while mice in group A is injected intraperitoneally200ug mIgG instead. Tumor cell growth and distribution were monitored by tumor volume measured by vernier calipers and dynamic in vivo fluorescence imaging.2,CD44knockdown decreases immuno-bounding to the monoclonal antibody KMP1and inhibits the growth and migration of EJ cells:We transfected bladder cancer cell line EJ with well-designed CD44siRNA. Western blot, EL1SA and flow cytometry analysis were used to assess the CD44immuno-bounding to the monoclonal antibody KMP1. Wound healing assay and Boyden chamber were used to investigate the change of shCD44-EJ cell's migration. Crystal violet colorimetric analysis were used to assess the change of adhesion of shCD44-EJ cells. Proliferation of shCD44-EJ cells was measured using a [3H]-thymidine incorporation assay.3,GALNT1knockdown decreases EJ cells immuno-bounding to the monoclonal antibody KMP1and changes the biological effects of EJ cells:Inhibition of glycosyltransferase by Benzyl-N-acetyl-D-galactosaminide (BG),mild periodate oxidation and immuno-attachment were used to analyze the change of EJ cells and the antigen. DNA microarray assay the glycosyltransferase expression levels on bladder tumor tissues of patients and normal bladder tissues. We transfected bladder cancer cell line EJ with well-designed GALNT1siRNA. Immunohistochemistry,flow cytometry analysis and Western blot were used to assess the antigen and EJ cells immuno-bounding to the monoclonal antibody KMP1. Proliferation of shGALNT1-EJ cells in vitro were measured using Soft agar colony formation assay and [3H]-thymidine incorporation assay. Wound healing assay and Boyden chamber were used to investigate the change of shGALNT1-EJ cell's migration. Subcutaneous bladder tumors in BALB/c nude mices were induced by inoculation of shCD44-EJ cells and shGALNT1-EJ cells, growth of the mices were observed after inoculation, measured the diameters of tumors very5days and worked out the gross tumor volumes. Results:1,Preparation of monoclonal antibody KMP1against bladder carcinoma, antigen identification, and its biological effects:KMP1is an IgGl antibody which can bound to EJ, BIU-87, T24cell lines and bladder carcinoma tissue, not to Lovo, HeLa, K562, HepG2, Jurkat,293, HCV29cell lines, human red blood cell, human lymphocytes and normal bladder tissue. KMP1recognizes the CD44of EJ cells, KMP1inhibits cancerous proliferation and migration of EJ cells. In EJ-GFP nude mouse tumor model, the addition of KMP1significantly inhibited tumor growth and extended the average life span of nude mice. These indicate that KMP1has a promising antitumor effect in vivo.2,CD44knockdown decreases immuno-bounding to the monoclonal antibody KMP1and inhibits the growth and migration of EJ cells:CD44knockdown extremely decreased KMP1staining signal compared with the EJ cells transfected by pSuper-puro empty vector control, Western blot, ELISA and flow cytometry analysis found that CD44knockdown decreases EJ cells immuno-bounding to the KMP1. Western blot analysis revealed a remarkable decrease of CD44expression in shCD44-EJ cells, but not in pSuper-puro vector transfected. CD44silencing also significantly inhibited cell migration through wound healing assay. The modified Boyden chamber assay showed similar results also. Crystal violet colorimetric analysis showed that the adhesion of shCD44-EJ cell's was63%to vector group.[3H]-thymidine incorporation assay revealed that CD44silencing can extremely decrease the proliferation of EJ cells.3, GALNT1knockdown decreases EJ cells immuno-bounding to the monoclonal antibody KMP1and changes the biological effects of EJ cells:Mild periodate oxidation was performed to degrade carbohydrate moiety of the antigen,KMP1did not react to the antigen. Benzyl-N-acetyl-D-galactosaminide (BG) treatment markedly reduced KMP1recognized EJ cells signal. Microarray analysis revealed that GALNT1mRNA in bladder tumor tissues was11.2-fold higher than in normal bladder tissues, while other N-acetylgalactosaminyltransferases (GALNT2-9) and Fucosyltransferases(FUT1-10)had no significant changes in bladder tumor tissues and normal bladder tissues. GALNT1expression in EJ cells was knocked down through transfection by pSUPER-shGALNT1vector. GALNT1knockdown extremely decreased KMP1staining signal compared with the EJ cells transfected by pSUPER empty vector control. GALNT1knockdown decreased24.5%colony formations of EJ cells compared with the empty vector transfected control cells. GALNT1knockdown markedly reduced incorporation of [jH]-thymidine into DNA of EJ cells at all time points compared with the empty vector transfected control cells. However, GALNT1knockdown inhibited cell migration only7.2%compared with the Wide type EJ cells through Wound healing assay. GALNT1knockdown also had no remarkable changes(only12.3%) compared with the the empty vector transfected control cells through a modified Boyden chamber assay. Silenced CD44or GALNT1expression significantly inhibited subcutaneous tumor growth compared with the control groups by injection of the empty vector transfected control cells. At the end of observation course (40days), the inhibitory rate of cancerous growth for CD44or GALNT1knockdown was38.0%and52.5%, respectively.Conclusions:1,Preparation of monoclonal antibody KMP1against bladder carcinoma and its biological effects:The CD44may contribute to recur and planting migration of bladder cancer. KMP1can recognize CD44of EJ cells specially and inhibits its proliferation and migration in vitro, it also may well inhibit tumor growth and metastasis in vivo. Thus, KMP1may have a considerable potential to prevent the recurrence of bladder transitional cell carcinoma.2,CD44knockdown decreases immuno-bounding to the monoclonal antibody KMP1and inhibits the growth and migration of EJ cells:CD44silencing can effectively decrease EJ cells immuno-bounding to the monoclonal antibody KMP1and inhibit the migration and the proliferation of EJ cells in vitro.3,GALNT1knockdown decreases EJ cells immuno-bounding to the monoclonal antibody KMP1and changes the biological effects of EJ cells:These results indicate that the GALNT1might cause aberrant glycosylation of CD44in bladder cancer.GALNT1knockdown had no remarkable changes in cell migration, Silenced CD44or GALNTl expression significantly inhibits proliferation and tumor growth of bladder cancer. |
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