The patients were grouped into the following categories: International Federation of Gynecology and Obstetrics (FIGO) stage IB (n = 16) and stage IIA–IIIB (n = 24). All tissues were subjected to immunohistochemical staining for IL-32 as described previously27

and clinically correlated with FIGO stage and survival, and the following results were obtained. In the serial section, immunohistochemical staining for COX-2 was also conducted to determine whether IL-32 and COX-2 are co-localized in cervical cancer cells. This study was approved by the Chungnam National University Hospital. The IL-32γ and COX-2 were amplified from the genomic DNA of human CaSki cells via PCR, using the following primers, respectively: IL-32γ: 5′-CTGGAATTCATGTGCTTCCCGAAG-3′ (forward), 5′-GAAGGTCCTCTCTGATGACA-3′ (reverse); COX-2: 5′-CCCAAGCTTGGGCTCAGACAGCAAAGC CTA-3′ (forward), 5′-CTAGTCTAGACTAGCTACAGTTCAGTCGAACGTTCTTT-3′ (reverse). Interleukin-32γ MK-2206 order check details was cloned into the EcoRI and XhoI sites of pCDNA3.1 using EcoRI and SalI, and COX-2 was ligated with pCDNA3.1 vector using the HindIII and XbaI sites. The promoters of IL-32 and COX-2 were amplified via PCR from human genomic DNA. The IL-32 promoter (−746/+25) was constructed as previously reported.21 The COX-2 promoter (−880/+9) used the following primers: 5′-CGGGATCCAAATTCTGGCCATCGCCGCTT-3′ (forward), 5′-CCAAGCTTTGACAATTGGTCGCTAA

CCGAG-3′

(reverse) cloned into the MluI and HindIII sites of the pGL3-basic vector, and the inserted sequences were confirmed via DNA sequencing. Both pTarget/E7 and pTarget/E7 antisense (E7AS) were described in a previous report.25,28 C33A/pOPI3, C33A/E7, SiHa and CaSki cells were seeded on six-well plates at a density of 3 × 105 cells per well, then grown to confluence, reaching approximately 80% at the time of transfection. For each well, plasmid DNA (1 μg) was introduced into the cells using an identical volume of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. The pTarget Cyclin-dependent kinase 3 and pTarget/E7AS plasmid were transfected into C33A/E7, SiHa and CaSki cells to confirm the E7 oncogene-specific effect on IL-32 and COX-2 expression in HPV-expressing cervical cancer cells. The pGL3 basic, pGL3b/IL-32 promoter, and pGL3b/COX-2 promoter were respectively co-transfected with pTarget, pTarget/E7 and pTarget/E7AS into C33A/pOPI3, C33A/E7, SiHa and CaSki cells to determine the specific effects of E7 on the transcriptional activities of IL-32 and COX-2. Additionally, pCDNA3.1, pCDNA3.1/COX-2, pCDNA3.1/IL-32γ, siCONTROL and siIL-32 (Dharmacon, Lafayette, CO) were respectively transfected into SiHa and CaSki cells to evaluate expression between COX-2 and IL-32 by the HPV E7 oncogene. Interleukin-32γ is the most active form of IL-32 isoforms.