Restored expression of KLF4 by 5-Aza inhibits the proliferation of cervical cancer cells.

<p>(A and B) KLF4 protein expression gradually increased in response to different doses of 5-Aza in SiHa and C33A cells, as detected by ICC. (C and D) The proliferation of SiHa (C) and C33A (D) cells treated with different doses of 5-Aza was determined by counting cells longitudinally. (E and F) The viability of SiHa and C33A cells treated with 10 mM 5-Aza was determined by the MTT assay. (G and H) The cell survival rate of cervical cancer cell lines SiHa and C33A treated by chemistry agent cisplatin was detected by the MTT assay. Bars indicate SE. *, P<0.05.</p

Promoter Hypermethylation of KLF4 Inactivates Its Tumor Suppressor Function in Cervical Carcinogenesis

<div><p>Objective</p><p>The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However, the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression.</p><p>Methods</p><p>The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR, immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay.</p><p>Results</p><p>The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (P<0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (P<0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = −0.486, P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza), the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased, the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased.</p><p>Conclusion</p><p>KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene’s function as a tumor suppressor in cervical carcinogenesis.</p></div

Correlation of methylation status of KLF4 gene and the protein expression in SCC and NC.

<p>Correlation of methylation status of KLF4 gene and the protein expression in SCC and NC.</p

KLF4 promoter methylation is negatively correlated with the gene expression.

<p>(A) The relative mRNA levels of KLF4 in cervical cancer and normal cervix tissues. (B) The methylation ratio of the KLF4 promoter BSQ3 region. (C) An inverse correlation is observed between KLF4 promoter methylation and mRNA levels (r = −0.486, P = 0.003).</p

KLF4 expressions at both the transcriptional and the translational levels is drastically enhanced after treatment with the demethylating agent 5-Azacytidine.

<p>(A) Bisulfite sequencing of the KLF4 promoter in SiHa cells after treatment with different doses of 5-Aza. (B) KLF4 mRNA levels were quantified by PCR for three independent RNA samples from SiHa cells after treatment with different doses of 5-Aza, *, P<0.05. (C) KLF4 protein expression in SiHa cells was gradually enhanced in response to increasing doses of 5-Aza. (D) The relative expression of KLF4 protein in SiHa cells treated with different doses of 5-Aza. (E) KLF4 protein expression in SiHa cells was gradually enhanced during the time-course of treatment with 5 mM 5-Aza; it was reduced upon 5-Aza withdrawal following a 72-hour treatment. (F) Bisulfite sequencing of the KLF4 promoter in C33A cells after treatment with different doses of 5-Aza. (G and H) KLF4 expression was detected by PCR and western blot in C33A cells treated with different doses of 5-Aza in three independent repeats, *, P<0.05. (I) The relative expression of KLF4 protein in C33A cells treated with different doses of 5-Aza. (J) KLF4 protein expression was monitored during the time-course of treatment with 5 mM 5-Aza and during agent withdrawal following a 72-hour treatment. The relative levels of KLF4 protein normalized to β-actin are shown. Bars indicate SE. *, P<0.05.</p

The KLF4 promoter region is hypermethylated in cervical cancer.

<p>(A) A schematic representation of the CpG islands found in the promoter region of the KLF4 genomic locus. Numbers indicate positions in bp relative to the transcription start site. The two CpG island regions marked in red were bisulfite sequenced. (B) Bisulfite-converted DNAs from cervical cancer tissues (n = 24) and normal tissues (n = 11) were amplified at the KLF4 promoter, and the fragments were sequenced. CpG sites are represented as boxes, with shaded regions indicating methylation, and unshaded regions indicating no methylation. (C) The methylation level ratio of the CpG sites in cervical cancer and normal cervix tissues. Bars, SE. *, P<0.05.</p

Methylation of the KLF4 promoter in cervical cancer cell lines.

<p>(A and B) KLF4 expression in the 4 cervical cancer cell-lines HeLa, CaSki, SiHa and C33A was detected by IHC (A) and by PCR and western blot (B). We applied the human embryonic stem cell line H7 as a positive control and the rabbit IgG polyclonal antibody as the isotype control in immunocytochemistry. (C) Bisulfite sequencing of the KLF4 promoter in cervical cancer cell-lines. (D) Statistical analysis of KLF4 promoter methylation in cervical cancer cell-lines. (E) MS-PCR for a region of the KLF4 promoter in the 4 cervical cancer cell lines. A methylated band was amplified in SiHa and C33A cells. Globally methylated DNA from normal fetal cord blood samples was included as a positive control for the methylated (M) and unmethylated (U) primers.</p

Tafazzin (TAZ) promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis

<div><p>Tafazzin (TAZ) is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27), high-grade squamous intraepithelial lesions (HSIL, n = 26) and squamous cervical carcinoma (SCC, n = 41) by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. <i>In vitro</i>, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. <i>In vivo</i>, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis.</p></div

TAZ increases the growth of cervical cancer cells <i>in vitro</i>.

<p><b>(A)</b> Immunocytochemical staining showing TAZ expression in HeLa, SiHa, C33A, Caski and HT3 cells (magnification, ×100). (<b>B)</b> Western blot analysis of TAZ expression in HeLa, SiHa, C33A, Caski and HT3 cells. Stably transfected cell lines were identified by western blotting: <b>(C)</b> control (SiHa-GFP) and TAZ-overexpressing SiHa (SiHa-TAZ) cells; (F) control(SiHa-shControl) and TAZ-knockdown SiHa (SiHa-shTAZ) cells; (I) control(HeLa-shControl) and TAZ-knockdown HeLa (HeLa -shTAZ) cells. The growth and viability of cells were detected using growth curves and 3-(4,5-dimethylthiazole-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in SiHa-GFP and SiHa -TAZ cells <b>(D, E)</b>, SiHa-shControl and SiHa-shTAZ cells <b>(G, H)</b>, and HeLa-shControl and HeLa-shTAZ cells <b>(J, K)</b>. Values are shown as the mean±SD obtained from 3 separated experiments, * <i>P</i><0.05 <i>vs</i>. control; ** <i>P</i><0.01 <i>vs</i>. control.</p

TAZ inhibits apoptosis in cervical cancer cells both in vitro and in vivo.

<p>Apoptosis of TAZ-mediated cervical cancer cells was monitored with fluorescence-activated cell sorting (FACS) analysis. The representative cell apoptosis histograms and the percentage of apoptosis cells of TAZ-mediated cervical cancer cells are shown: (A, B) SiHa-GFP and SiHa-TAZ cells; (E, F) SiHa-shControl and SiHa-shTAZ cells; (I, J) HeLa-shControl and HeLa-shTAZ cells. Values are expressed as the mean±SD of three experiments in duplicate. Apoptotic cell death in tumor xenografts formed by TAZ-mediated cervical cancer cells was measured by TUNEL assay, and representative micrographs are shown (magnification, ×40):(C) SiHa-GFP and SiHa-TAZ cells; (G) SiHa-shControl and SiHa-shTAZ cells; (K) HeLa-shControl and HeLa-shTAZ cells. Quantitative analysis of apoptosis cells in xenograft samples formed by TAZ-mediated cervical cancer cells: (D) SiHa-GFP and SiHa-TAZ cells; (H) SiHa-shControl and SiHa-shTAZ cells; (L) HeLa-shControl and HeLa-shTAZ cells. Six tumor samples were measured and analysed every group. Values are shown as the mean±SD. *<i>P</i><0.05 <i>vs</i>. control; ** <i>P</i><0.01 <i>vs</i>. control.</p