br Luciferase reporter assay br The

2.14. Luciferase reporter assay
The promoter of p27kip1 cDNA cDNA (700–1200 bp) was amplified by using PCR and cloned into luciferase reporter plasmids (pGL3-basic). The luciferase reporter plasmids were co-transfected with pcDNA3.1-ZNF692 or si-ZNF692 in HEK293T cells. After 48 h, Liproxstatin-1 were harvested and assessed for luciferase activity using the Dual Lucif-erase Reporter Assay System (Promega, Madison, WI, USA). Relative lu-ciferase activity was corrected for Renilla luciferase activity of pGL3-basic, and normalized to the activity of the control (pcDNA3.1 or si-NC).
The experimental protocols were evaluated and approved by the Nanjing Medical Animal Care Committee. Eight female nude mice (4–6 weeks old) were purchased from Nanjing Medical University School of Medicine's accredited animal facility. HeLa cells were transfected with sh-ZNF692 or sh-NC as previously described, and 5.0
× 106 logarithmic growth cells were implanted subcutaneously in the armpit of the mice. Tumour volume (length × width2 × 0.5) was mea-sured weekly using callipers. Four weeks after injection, the animals were sacrificed, and the tumours were measured. Meanwhile, the tu-mour tissues were immunohistochemically stained with ZNF692 (Abcam, 1:200), Ki67 (Spring Bioscience, 1:100), and p27kip1 (Cell Sig-nalling, 1:200).
2.16. Statistical analysis
Data are shown as the means ± S.D., and Student's t-test, chi-square test, one-way ANOVA were used to analyse the data using SPSS statistics software (version 20.0, Chicago, III). P b 0.05 was considered statistically significant.
3. Results
3.1. ZNF692 was identified as a candidate oncogene in CC
3.2. Knockdown of ZNF692 suppressed the aggressiveness of CC in vitro
3.3. Overexpression of ZNF692 promoted the malignant phenotype in HeLa cells
3.4. ZNF692 promoted cell proliferation and invasion through suppressing p27kip1 expression by directly binding its promoter region
GO analysis was used to elucidate how ZNF692 exerts its carcino-genic effect. As shown in Fig. 4A, most of the genes were enriched in reg-ulation of transcription and many genes were enriched related to cell cycle regulation. Our present results showed that si-ZNF692 impaired
CC cell proliferation and generated G1 phase arrest, which is consistent with the GO analysis results. Therefore, we evaluated expression of G1 phase-related cell cycle regulation genes, and the finding suggested that p27kip1 was increased or decreased at the mRNA level after knock-down or ectopic expression of ZNF692 in CC cell lines, respectively (Fig. 4B-D). When ZNF692 was knocked down, p27kip1 was obviously increased and PThr160-CDK2 expression was moderately decreased at the protein level; however, CDK2 and CCNE1 was not obviously affected at the protein level (Fig. 4E). Meanwhile, overexpression of ZNF692
showed the opposite results at the protein level (Fig. 4F). Moreover, the expression of p15ink4b, p16ink4a, p21cip1, CCNB1, CCND1, CDK4, and CDK6 was not influenced either at the mRNA or the protein level (Fig. 4B-F).
ZNF proteins function as transcription factor by binding the specific nucleotide sequences of DNA [22]. To further verify whether ZNF692 binds to the promoter region of p27kip1 to suppress its transcription, we performed ChIP assay. According to the sequence of promoter re-gion, 7 primers are designed for different sections. After immunoprecip-itation by Flag-ZNF692, the 7 primers were used for PCR. Results showed that only primer 5 (815–1022 bp) was enriched in the immu-noprecipitation complex both in Siha and Hela cells. The PCR amplifica-tion product was then used for agarose gel electrophoresis, and compared with IgG group, Flag-ZNF692 enriched more DNA fragments amplified by primer 5 (Fig. 4G). In addition to this, ectopic ZNF692 or si-ZNF692 significantly reduced or increase luciferase activity when co-transfected PGL3-p27kip1 promoter cDNA (700–1200 bp) (Fig. 4H). These results suggest that ZNF692 directly binds the promoter region of p27kip1 to suppress its transcription.