The construction of cDNA library and the screening of related antigen of ascitic tumor cells of ovarian cancer

Objective: To construct the cDNA library of the ascites tumor cells of ovarian cancer, which can be used to screen the related antigen for the early diagnosis of ovarian cancer and therapeutic targets of immune treatment. Materials and Methods: Four cases of ovarian serous cystadenocarcinoma, two cases of ovarian mucinous cystadenocarcinoma, and two cases of ovarian endometrial carcinoma in patients with ascitic tumor cells which were used to construct the cDNA library. To screen the ovarian cancer antigen gene, evaluate the enzyme, and analyze nucleotide sequence, serological analysis of recombinant tumor cDNA expression libraries (SEREX) and suppression subtractive hybridization technique (SSH) techniques were utilized. The detection method of recombinant expression-based serological mini-arrays (SMARTA) was used to detect the ovarian cancer antigen and the positive reaction of 105 cases of ovarian cancer patients and 105 normal women’s autoantibodies correspondingly in serum. Results: After two rounds of serologic screening and glycosides sequencing analysis, 59 candidates of ovarian cancer antigen gene fragments were finally identified, which corresponded to 50 genes. They were then divided into six categories: (1) the homologous genes which related to the known ovarian cancer genes, such as BARD 1 gene, etc; (2) the homologous genes which were associated with other tumors, such as TM4SFl gene, etc; (3) the genes which were expressed in a special organization, such as ILF3, FXR1 gene, etc; (4) the genes which were the same with some protein genes of special function, such as TIZ, C1D gene; (5) the homologous genes which possessed the same source with embryonic genes, such as PKHD1 gene, etc; (6) the remaining genes were the unknown genes without the homologous sequence in the gene pool, such as OV - 189 genes. Conclusion: SEREX technology combined with SSH method is an effective research strategy which can filter tumor antigen with high specific character; the corresponding autoantibodies of TM4SF1, ClD, TIZ, BARDl, FXRl, and OV - 189 gene’s recombinant antigen in serum can be regarded as the biomarkers which are used to diagnose ovarian cancer. The combination of multiple antigen detection can improve diagnostic efficiency.

Ovarian tumors; CA - 125 antigens; Double hybrid system technology; Gene library; Serological test.

[1] Copeland L.J.: “Epithelial ovarian cancer”. In: Disaia P.J., Creasman W. T., (eds). Clinical Gynecologic Oncology. 7th ed. St. Louis, Mo, USA: Mosby-Year Book, 2007, 313.

[2] Morgan R.J., Alvarez R.D., Armstrong D.K., Boston B., Burger R.A., Chen L.M., et al.: “Epithelial ovarian cancer”. J. Natl. Compr. Canc. Netw., 2011, 9, 82.

[3] Zhang Z., Bast R.C., Yu Y, Li J., Sokoll L.J., Rai A.J., et al.: “Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer”. Cancer Res., 2004, 64, 5882.

[4] Krackhardt A.M., Witzens M., Harig S., Hodi F.S., Zauls A.L., Ches-sia M., et al.: “Identification of tumor-associated antigens in chronic lymphocytic leukemia by SEREX”. Blood, 2002, 100, 2123.

[5] Ghorbel M.T., Murphy D.: “Suppression Subtractive Hybridization”. In: Merighi A., (ed). Neuropeptides: Methods and Protocols. New York: Humana Press, 2011, 237.

[6] Shebzukhov Y.V., Koroleva E.P., Khlgatian S.V., Belousov P.V., Kuz’mina K.E., Radko B.E., et al.: “Antibody response to a non-con-served C-terminal part of human histone deacetylase 3 in colon cancer patients”. Int. J. Cancer, 2005, 117, 800.

[7] Burger R.A., Brady M.F., Bookman M.A., Fleming G.F., Monk B.J., Huang H., et al.: “Incorporation of bevacizumab in the primary treat-ment of ovarian cancer”. N. Engl. J. Med., 2011, 365, 2473. doi: 10.1056/NEJMoa1104390.

[8] Kurman R.J., Shih I.M.: “The origin and pathogenesis of epithelial ovarian cancer-a proposed unifying theory”. Am. J. Surg. Pathol., 2010, 34, 433.

[9] Andriole G.L., Crawford E.D., Grubb R.L., Buys S.S., Chia D., Church T. R., et al.: “Prostate cancer screening in the randomized Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial: mortality results after 13 years of follow-up”. J. Natl. Cancer Inst., 2012, 104, 125. doi: 10.1093/jnci/djr500. Epub 2012 Jan 6.

[10] Van der Bruggen P., Traversari C., Chomez P., Lurquin C., Plaen E.D., Van den Eynde B., et al.: “A gene encoding an antigen recognized by cy-tolytic T lymphocytes on a human melanoma”. Science, 1991, 254, 1643.

[11] Sahin U., Türeci O., Schmitt H., Cochlovius B., Johannes T., Schmits R., et al.: “Human neoplasms elicit multiple specific immune responses in the autologous host”. Proc. Natl. Acad. Sci. U S A, 1995, 92, 11810.

[12] Clarke P.R., Sanderson H.S.: “A mitotic role for BRCA1/BARD1 in tumor suppression?”. Cell, 2006, 127, 453.

[13] Zukauskas A., Merley A., Li D., Ang L.H., Sciuto T.E., Salman S., et al.: “TM4SF1: a tetraspanin-like protein necessary for nanopodia for-mation and endothelial cell migration”. Angiogenesis, 2011, 14, 345.

[14] Shin J.N., Kim I., Lee J.S., Koh J.Y., Lee Z.H., Kim H.H.: “A novel zinc finger protein that inhibits osteoclastogenesis and the function of tumor necrosis factor receptor-associated factor 6”. J. Biol. Chem., 2002, 277, 8346. Epub 2001 Dec 20.

[15] McIntosh M.W., Drescher C., Karlan B., Scholler N., Urban N., Hellstorm K.E., et al.: “Combining CA 125 and SMR serum markers for diagnosis and early detection of ovarian carcinoma”. Gynecol. Oncol., 2004, 95, 9.