Identifying And Sero-Immunoscreening Of EOC Associated Antigen And Preliminary Study On EOC Antigen Gene Function

EOC is the most lethal gynecological malignancy.Unfortunately,the majority of EOCs remain clinically undetected until patients have developed late stage disease and only a mere 25%of cancers are detected as stageⅠdisease.Once stageⅢandⅣovarian cancer,which is define by peritoneal and extra peritoneal metastatic spread,is diognosed,the survival decreases from 95%at stageⅠto approximately 20～25%.Therefor,clinical outcome and possibly survival may be significantly improved by the identification of stageⅠdisease. Nevertheless,there are not efficient methods or biomarkers that could bc used to detected ovarian cancer at early stage.SEREX provides a new way to screening caner antigens. Autoantibody against cancer antigen may be a new biomarker in detecting cancer.Currently, majority study focus on IgG autoantibody.There is rarely report about IgM autoantibody.We performed heterological sero-immunoscreening of 10 EOC associated antigen clones from ascites EOC cells cDNA library.Six clone(TM4SF1,C1D,TIZ,OV-142,FXR1,OV-189) which positive ratio in ovarian cancer is significantly higher than in cancer-free were sequenced and bioinformatics analyzed.The expression pattern of these 6 EOC associated antigens in EOC tissues were evaluated by RT-PCR.We constructed and expressed these 6 recombinant fusion antigen proteins in Escherichia coli.Antigen proteins were purified by Ni-NTA His-Bind Resins and electroeluting.We developed and optimized a novel enzyme immunoassay method(indirect ELISA)to measure IgG,IgM autoantibodies against EOC associated antigens in serum and evaluated the clinical value of autoantibody spectrum in detecting ovarian cancer.The biological function of FXR1 in EOC cell line was studied by upregulated and downregulated(RNAi)FXR1 expression.Part 1:Heterological sero-immunoscreening of EOC associated antigen and bioinformatics analysisObjective:The goal of the study was to investigate the reaction of EOC associated antigens with IgG,IgM autoantibody in serum from patients with ovarian cancer and healthy women.The antigen clones which postive ratio in serum from cancer were significantly higher than in serum from healthy women were sequenced and bioinformatics analyzed.Methods:Serological mini-arrays of recombinant tumor antigens(SMARTA)was used to investigate the reaction of 10 EOC associated antigen clones with IgG,IgM autoantibodies in serum from 62 patients with ovarian cancer and 62 healthy women.The postive clones were sequenced and bioinformatics analyzed in NCBI.Results:The positive ratio of Six clone(OV-59,84,106,142,178,189)reacted with IgG,IgM autoantibodies in patients(12.9%～27.4%)was significantly higher than control(1.6%～11.3%).After sequenced and analyzed,results showed that 4 of 6 clone were known genes.OV-159(TM4SF1):The protein encoded by this gene is a member of the transmembrane 4 superfamily,also known as the tetraspanin family characterized by the presence of four hydrophobic domains.TM4SF1 protein mediate signal transduction events that play a role in the regulation of cell development,activation,growth and motility. TM4SF1 is a cell surface antigen and is highly expressed in different carcinomas and is a target for antibody-mediated therapy.OV-84(C1D):The protein encoded by this gene is a conserved DNA binding and apoptosis-inducing protein and a major autoantigen in patients with the PM-scleroderma overlap syndrome.OV-178(FXR1):The protein encoded by this gene is an RNA binding protein.FXR1 protein shuttle between the nucleus and cytoplasm and associate with polyribosomes and have cell-growth-dependent translation activation.FXR1 protein may be a autoantigen in patient with scleroderma.FXR1 gene is most frequently overexpressed in the center of the amplified domain(3q26-27)in squamous cell carcinomas of lung.OV-106(TIZ):The novel zinc finger protein TIZ may play a role during osteoclast differentiation by modulating TRAF6 signaling activity.OV-142 has highly homology with BARD1 and is a splice variant of BARD1.OV-142 presume protein maybe has MHC-Ⅰbinding peptide and is maybe a potential immumotherapy target of ovarian cancer.OV-189 is a new gene has no homology sequence in genbank.We preliminarily presume that OV-189 is a new member of L1 family that is one class of LINE. OV-189 presume protein maybe has MHC-Ⅰbinding peptide and is maybe a potential immumotherapy target of ovarian cancer.Conclusion:Our date show EOC associated antigen not only elicit humoral immunity mediated by IgG,also induce specific IgM autoantibody in ovarian cancer patients.The high positive ratio of autoantibody against EOC associated antigen in serum from patients with ovarian cancer indicates that these autoantibodis are potent biomarker in detection ovarian cancer. Part 2:Evaluating the expression pattern of EOC associated antigen genes in ovarian cancer tissues by RT-PCRObjective:The goal of the study was to evaluate the expression pattern of EOC associated antigen genes in ovarian cancer tissues.Methods:The relatived expression level of 6 EOC associated antigen genes were assayed in 36 ovarian cancer tissues,15 ovarian benign tumor tussues,13 normal ovarian tussues by semiquantitative RT-PCR.Results:All of 6 EOC associated antigen genes almost expressed in all specimens.But the relative expression level of 6 genes in EOC tissues were higher than in benign tissues and normal ovarian tissues.The expression trends of 6 genes were grouped into three classes:1) The expression level of genes in EOC tissues was significantly higher than in normal ovarian tissues and slightly higher than in benign tissues.The expression level of genes in benign tissues was moderately higher than in normal ovarian tissues.For example:TIZ,C1D,TM4SF1,OV-142;2)The expression level of genes in EOC tissues was significantly higher than in normal ovarian tissues and benign tissues.The expression level of genes in benign tissues and normal ovarian tissues was equivalent.For example OV-189;3)The expression level of genes in EOC tissues was significantly higher than in normal ovarian tissues and benign tissues.The expression level of genes in benign tissues was significantly higher than in normal ovarian tissues.The expression level of genes in advanced EOC tissues was significantly higher than in early stage EOC tissues.The expression level of genes in high grade(2-3)EOC tissues was significantly higher than in low grade EOC tissues For example FXR1.Conclusion:Our data demonstrated that 6 EOC associated antigen genes were associated with EOC. Part 3:Expression and purification of recombination associated antigens of EOCObjective:The goal of the study was to express and purify recombination associated antigens of ECO.Methods:CDSes of ECO associated antigen genes were amplified by PCR.Products of PCR were digested with corresponding restriction endonuclease and subcloned into pET-30b(+).Recombination plasmids were identified by restriction endonuclease analysis and DNA sequencing.Positive plasmids were transformed into E.coli BL21(DE3)for expression under induction with IPTG.Recombination fusion proteins were purified by Ni-NTA His-Bind Resins and electroeluting.Products of purification were refolded and then identified by SDS-PAGE and Western blot.Results:We successfully constructed the recombination plasmids of EOC associated antigen genes and expressed the recombination fusion proteins in pET-30b(+)system.The results showed that 6 recombination antigen proteins with purity over 90%and concentration from 0.3 mg/ml to 0.8mg/ml were obtained.The recombinant antigen proteins were activity and can be used for following study. Part 4:Study on autoantibody spectrum against EOC associated antigensObjective:The objective of this study was to evaluate the clinical value of autoantibody spectrum in detecting ovarian cancer.Methods:We developed and optimized a novel enzyme immunoassay method(indirect ELISA)to measure IgG,IgM autoantibodies against EOC associated antigens in serum. Circulating autoantibodies were measured in serum from 150 patients with ovarian cancer (126 prior treatment,24 post-treatment),42 patients with benign ovarian masses,142 healthy women,20 female patients with breast cancer,20 female patients with esophageal cancer,20 female patients with lung cancer.Cut off value of IgG,IgM autoantibodies were determined by ROC curve.CA125 was measured in serum by IRMA.We evaluated the clinical value of autoantibody in detecting ovarian cancer alone,combining multiple autoantibody (autoantibody spectrum),combining multiple autoantibody with CA125.Results:Our data indicated that serum contains IgG,IgM autoantibodies against EOC associated antigens.The positive ratio of IgG autoantibodies in serum from ovarian cancer patients and cancer-free patients were 34.1%～47.6%and 13.0%～17.9%,respectively.The positive ratio of IgM autoantibodies in serum from ovarian cancer patients and cancer-free patients were 39.7%～53.2%and 12.0%～33.2%,respectively.There was not significantly clinical utility when individual autoantibodies analyzed separately.The positive ratio of IgM autoantibodies against C1D,TIZ,FXR1,OV-189 in early ovarian cancer were significantly higher than in advanced ovarian cancer.Combining five autoantibodies(TM4SF1 IgG,C1D IgG,TIZ IgG,TIZ IgM,FXR1 IgG,FXR1 IgM)showed significantly improved sensitivity (75.4%),lower specificity(78.2%)and similar accuracy(76.5%)in detecting ovarian cancer compared to those of CA125(61.1%,89.1%,77.7%).But the autoantibody spectrum showed significantly improved sensitivity in classifying early stage(76.6%),especially stageⅠ(83.3%) ovarian cancer compared to those of CA125(51.1%,44.4%).The positive ratio of the autoantibody spectrum wsa significantly lower in 24 post-treatment serum(37.5%)compared to the pairing prior treatment serum(70.8%).The positive ratio of the autoantibody spectrum in serum from breast cancer,esophageal cancer and lung cancer were 30%,20%,25%, respectively.Combining five autoantibodies(TM4SF1 IgG,TM4SF1 IgM,C1D IgG,TIZ IgM,FXR1 IgG)with CA125 showed significantly improved sensitivity(86%),specificity (91%)and accuracy(88.7%)in detecting ovarian cancer compared to those of CA125(61.1%, 89.1%,77.7%).Conclusion:Our data indicated that serum contained IgG,IgM autoantibodies against EOC associated antigens.The autoantibody spectrum(TM4SF1 IgG,C1D IgG,TIZ IgG,TIZ IgM,FXR1 IgG,FXR1 IgM)was associated with ovarian cancer with significantly improved sensitivity,lower specificity and similar accuracy in detecting ovarian cancer compared to those of CA125.This autoantibody spectrum showed significantly higher sensitivity in classifying early stage,especially stageⅠovarian cancer than CA125 alone. Combining five autoantibodies(TM4SF1 IgG,TM4SF1 IgM,C1D IgG,TIZ IgM,FXR1 IgG)with CA125 showed significantly higher sensitivity,specificity and accuracy than CA125 alone.The autoantibody spectrum was a potent biomarker in detecting EOC. Part 5:Preliminary study of the biological behaviour change of EOC cell line HO8910 by upregulated FXR1 expressionObjective:The goal of this study was to investigate the biological behaviour change of EOC cells HO8910 by upregulated FXR1 expression.Methods:FXR1 CDS was amplified by RT-PCR.The PCR product was digested with corresponding restriction enzymes and subcloned into PCDNA3.1 plasmid.HO8910 cells transfected with PCDNA3.1/FXR1 or PCDNA3.1 were selected in G418 to generate HO8910/ PCDNA3.1/FXR1 and HO8910/PCDNA3.1.Cells growth curve,clone formation assay,adhesion assay,vitro migration and ivasion assay,flow cytometric assay for cells cycle and apoptotic were examined.Results:We successfully constructed recombinant PCDNA3.1/FXR1 plasmid.The stable transfection strains of HO8910 transfected with PCDNA3.1/FXR1 or PCDNA3.1 were obtained in G418 and identified by RT-PCR and Western blot.Cell growth curve showed upregulated FXR1 expression in HO8910 significantly increased their energy for growth. Clone formation assay showed upregulated FXR1 expression in HO8910 significantly increased cells proliferation.Upregulated FXR1 expression in HO8910 significantly increased cells adhesion and did not affect vitro migration,invasion.Flow cytometric assay showed that upregulated FXR1 expression in HO8910 decreased percentage(67.1%)of G1 phase cells compared with negtive control(80%)and did not affect cells apoptotic.Conclusion:Upregulated FXR1 expression in HO8910 significantly increased cells energy for growth,proliferation and adhesion. Part 6:Preliminary study of the biological behaviour change of EOC cell line A2780 by RNAi FXR1 expressionObjective:The goal of this study was to investigate the biological behaviour change of EOC cell line by RNAi FXR1 expression.Methods:Three paires siRNAs targeting FXR1 were designed and Synthesised.The highest inhibition efficient siRNA was selected by transient transfection and subcloned into siRNA expression vector PGPU6/GFP/Neo.A2780 cells transfected with positive siRNA expression vector and negtive control were selected in G418 for stable transfection strains. Cell growth curve,clone formation assay,adhesion assay,vitro migration and ivasion assay, flow cytometric assay for cells cycle and apoptotic were examined.Results:The inhibition ratio of siRNA subcloned into PGPU6/GFP/Neo is 80%.The stable transfection strains of A2780 transfected with siRNA expression vector PGPU6/GFP/Neo were obtained in G418.The highest inhibition ratio of the stable transfection strains is 60%.Downregulated FXR1 expression in A2780 mild decreased their energy for growth and significantly decreased their energy for clone formation. Downregulated FXR1 expression in A2780 did not affect the migration,invasion and adhesion.Flow cytometric assay showed that downregulated FXR1 expression on A2780 increased percentage(49.2%)of G1 phase cells compared with negtive control(40%)and did not affect cells apoptotic.Conclusion:Downregulated FXR1 expression in A2780 mild decreased cells energy for growth and significantly decreased cells proliferation and did not affact adhesion.