Expression And Purification Of Protein LY6K、MAGE-A3and IGF2BP3and Application In The Early Diagnosis Of ESCC

Esophageal cancer (EC) is a common malignant tumor of esophagus withincidences of22.9/100,000and10.5/100,000in male and female Chinese, respectively.Annually, more than300,000patients are diagnosed with EC worldwide. In China, anannual increase of over150,000is reported, and most of the patients are affected byesophageal squamous cell carcinoma (ESCC). According to our knowledge, the5-yearsurvival rate of patients with early EC after surgery or radiation therapy is up to90%, butthat of patients with moderate/advanced EC is less than10%. Therefore, effectivescreening of high-risk population susceptible to EC, timely diagnosis and treatment arecrucial to reduce the mortality rate of the patients.As the invasion of EC is concealed, patients in early satge have no obvious or specific symptoms. Although endoscopy, mucosal biopsy and Positron emissiontomography-computed tomography (PET-CT) are effective for the identification of earlycarcinoma and precancerous lesions, they are not suitable for the early screening inlarge-scale population. Identification of tumor markers has been considered as animportant auxiliary method in clinical practices as it is simple, economical, rapid andhighly repeatable. Recently, extensive studies have been performed in the selection of ECspecific serum markers. For example, significant increase was noted in the expression ofp53antibody, squamous cell carcinoma-associated antigen(SCCA), carcinoembryonicantigen (CEA) and cytokeratin fragment antigen21-1(CYFRA21-1) in the serum of theEC patients compared with that of the normal control group. However, theirsensitivity/specificity is not high with values of60%/31%,26.8%/73%,17%/22%, and43%/53%, respectively. Therefore, we aim to identify the serum EC markers with highsensitivity and specificity, and verify their clinical efficiency in early diagnosis.Cancer testis antigen (CTA), a tumor-associated antigen with a specific expressionpattern, has been detected in tumor tissues, germ cells and placental trophoblast cells. Ithas reported that CTA-expressing germ cells cannot induce immune response in humanbody due to the presence of blood-testis barrier which blocked the interaction betweenleucocytes and germ cells, and the lack of human leukocyte antigenâ… (HLA-â… ). However,CTA expressed in tumor tissues could induce specific immune responses in vivo. Thus,CTA has been preferred in the screening of molecular markers in early diagnosis ofcarcinoma.Lymphocyte antigen6-complex locus K (LY6K), melanoma antigen encodinggene-A3(MAGE-A3) and insulin-like growth factor2mRNA binding protein3(IGF2BP3) are CTA subfamily members. According to the previous reports, theexpression rate of MAGE-A3in ESCC patients is62.9%, while those of the LY6K andIGF2BP3in ESCC were>90%. In normal esophageal mucosa, no expresson ofMAGE-A3, LY6K, and IGF2BP3was noted. To date, no study was performed toinvestigate the association between LY6K, MAGE-A3, IGF2BP3and esophageal cancer.Thus, expression and purification of LY6K, MAGE-A3and IGF2BP3proteins are conducted in this study. Additionally, the expression of the serum LY6K-Ab,MAGE-A3-Ab and IGF2BP3-Ab was detected in ESCC patients and the normal controlgroup, based on which to evaluate their potency in early serological diagnosis.Methods: Specific primers and enzyme digestion sites were designed to synthesizethe target gene. Then the target gene was linked with the prokaryotic expression vector,followed by transferring into E. coli. After inducing with IPTG, soluble analysis of theprotein was performed after electrophoresis through SDS-PAGE gel. Western Blottinganalysis was conducted to detect the specificity of the proteins. Subsequently, therecombinant protein was purified using affinity chromatography. Finally, indirect ELISAwas performed to evaluate the serum level of LY6K, MAGE-A3, and IGF2BP3antibodiesin EC patients (n=59) and normal subjects (n=50) using the purified recombinant proteinsas the antigens.Results:1.Recombinant proteins LY6K, MAGE-A3and IGF2BP3were successfullyexpressed and purified.2. Mann-Whitney U test and logistic regression analysis indicatedthat the level of LY6K, MAGE-A3and IGF2BP3antibodies in the ESCC patients washigher than those obtained from the normal control group. In addition, thesensitivity/specificity of LY6K-Ab, MAGE-A3-Ab and IGF2BP3-Ab in clinical diagnosisof ESCC were76.2%/78.3%(cutoff value=0.396),56.0%/45.3%(cutoff value=0.414)and55.7%/35.0%(cutoff value=0.377), respectively. The AUC of LY6K-Ab,MAGE-A3-Ab and IGF2BP3-Ab at a95%confidence level were0.835,0.548and0.502,respectively. With regards to the combined test of LY6K-Ab, MAGE-A3-Ab andIGF2BP3-Ab for clinical diagnosis of ESCC, the sensitivity/specificity was78.3%/76.0%(cutoff value=0.401) with an AUC of0.856at a95%confidence level.Conclusions: Recombinant LY6K, MAGE-A3and IGF2BP3can be used as alternative antigens for early diagnosis of ESCC. The accuracy of single MAGE-A3-Ab or IGF2BP3-Abscreen for the diagnosis of ESCC was poor, and that of the LY6K-Ab was comparatively higher. Compared with single LY6K-Ab, MAGE-A3-Ab or IGF2BP3-Ab screen,combined test showed higher sensitivity and specificity.