Evaluation Of Clinical Significance Of ANKRD22 In Colorectal Cancer

Objective:ANKRD22 belongs to the family of Ankyrin(ANK)repeats and has 4 repeating Ankyrin motifs.Recently evidences indicate that expression of ANKRD22 is increased in various tumors such as lung cancer,breast cancer and pancreatic cancer,but its molecular mechanism in tumors needs to be further clarified.Our previously found that ANKRD22 is highly expressed in colorectal cancer and colorectal cancer initiating cells,and may be involved in the regulation of colorectal cancer progression and colorectal cancer reacquisition initiating cell characteristics.In this study,we analyzed the relationship between ANKRD22 and clinical parameters of colorectal cancer by GSEA,and prepared specific anti-ANKRD22 monoclonal antibodies to check the ANKRD22 location and its clinical values in primary colorectal cancer tissues.Our studies are of help to further understand the significance of ANKRD22 in colorectal cancer.Methods:Firstly,the relationship between ANKRD22 mRNA level and colorectal cancer related clinical parameters in 566 colorectal cancer samples from GEO database was carried out by Gene-set enrichment analysis(GSEA).Then ANKRD22/pCMV6-XL5 was used as template.The full-length sequence of ANKRD22 was amplified by PCR and subcloned into the prokaryotic expression vector pET-42a(+).After sequencing,the recombinant plasmids were transformed into Rosetta DE3 E.coli.Recombinant ANKRD22 protein was induced by Isopropylthio-β-D-galactose(IPTG)and subsequently purified by Ni2+IMAC column.Mouse monoclonal antibodies against ANKRD22 were prepared by immunized Balb/c mice with recombinant protein above.Finally,these anti-ANKRD22 antibodies were preliminarily used in colorectal cancer by Western blot and immunohistochemistry staining.Results:1.GSEA analysis showed that ANKRD22 expression was significantly increased in colorectal cancer compared with matched normal colorectal tissues;patients with higher ANKRD22 had better overall survival and prognosis.2.4 murine monoclonal antibodies specific against human ANKRD22 were obtained by prokaryotic expression,animal immunization,cell fusion and hybridoma cell screening.The characters were confirmed by Western blot and ELISA blocking experiments.These anti-ANKRD22 antibodies have displayed much higher specificity.3.Immunohistochemical staining of anti-ANKRD22 antibodies in 112 colorectal cancer tissues showed that 52 of 112 samples were positive in ANKRD22 expression(46.4%),and ANKRD22 level was positively correlated with p53 and β-catenin expression respectively(all p<0.05)Conclusions:We successfully obtained ANKRD22 recombinant protein by using prokaryotic expression system,and obtained 4 highly specific murine anti-ANKRD22 monoclonal antibodies by hybridoma technology.The results of immunohistochemical staining suggest function of ANKRD22 may be related to p53 and the Wnt/β-catenin pathway in colorectal cancer.ANKRD22 may be a potential prognostic marker for colorectal cancer.