PAX2 Expression in Ovarian Cancer

Abstract

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty.

Figure 1

Expression of PAX2 in various ovarian cancer cell lines. (a) Real-time RT-PCR analysis of PAX2 mRNA expression in twenty-six ovarian cancer cell lines with different histology origins; (b) Western blot analysis of PAX2 protein expression level in seven selected ovarian cancer cell lines.

Figure 2

(a) Western blot analysis of TOV21G clear cell line and RMUGL mucinous cell line stably transfected with PAX2-targeted shRNAs 15839, 15840, and 15841. PLKO and non-target are vector controls; (b) Cell proliferation of PAX2 shRNAs stably transfected cells measured by WST-1 assay.

Figure 3

(a) Images of wound closure assay of TOV21G cell line stably transfected with PAX2 specific shRNAs during a 24 h period. PLKO and non-target shRNA were the negative controls, and shRNAs 15839, 15840 and 15841 were PAX2-targeted shRNAs; (b) the images were analyzed by the TScratch program [26] to estimate the percentage of wound closure. PAX2 stable knockdown TOV21G cells had slower rate of wound closure.

Figure 4

Up-regulation of Annexin A1 and increase in apoptotic cells in PAX2 knockdown cell lines. (a) Reverse phase protein array (RPPA) analysis showed PAX2 knockdown ovarian cancer cell lines had a higher expression of Annexin A1 than control cells; (b) Western blot analysis was used to measure Annexin A1 expression in RMUGL and TOV21G ovarian cancer cell lines with or without PAX2 knockdown; (c) Flow cytometric analysis of percentage of apoptotic cells using APC-Annexin V staining.

Figure 5

(a) The most differentially expressed genes in the PAX2 stable knockdown TOV21G clear cell ovarian cancer cell lines identified by microarray analysis; (b) Validation of down-regulation of PAX2 and the up-regulation of G0S2, WFDC1, and GREM1 in PAX2 knockdown TOV21G cell lines by RT-PCR.

Figure 6

Cell viability of pCMV6-Myc-PAX2 transfected ovarian cancer cells. 2774 and TOV21G are PAX2-positive cells, while OVCA432 and OVCAR3 are PAX2-negative cells. The reduction in cell viability of OVCAR3 and OVCA432 cells after transfected with pCMV6-Myc-PAX2 plasmid in comparison to pCMV6-Neo vector control was significant (p < 0.05).

Figure 7

Examples of nuclear immunostains for PAX2 in a clear cell, mucinous and endometrioid ovarian carcinomas.