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. 2017 Mar 28;8(13):21861-21870.
doi: 10.18632/oncotarget.15668.

Epigenetic inactivation of HOXA11, a novel functional tumor suppressor for renal cell carcinoma, is associated with RCC TNM classification

Lu Wang  1   2 , Yun Cui  1 , Jindong Sheng  1 , Yang Yang  1 , Guanyu Kuang  1 , Yu Fan  1   3 , Jie Jin  1 , Qian Zhang  1
Affiliations

Epigenetic inactivation of HOXA11, a novel functional tumor suppressor for renal cell carcinoma, is associated with RCC TNM classification

Lu Wang et al. Oncotarget. .

Abstract

Epigenetic inactivation of HOXA11, a putative tumor suppressor, is frequently observed in a number of solid tumors, but has not been described in RCC (renal cell carcinoma). In this study, we investigated the expression, epigenetic changes and the function of HOXA11 in human renal cell carcinoma (RCC). HOXA11 was silenced or down-regulated in RCC cell lines and tissues. Methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS) revealed that the HOXA11 promoter was hypermethylated in 5/6 RCC cell lines. Demethylation treatment resulted in demethylation of the promoter and increased HOXA11 expression in these cell lines. HOXA11 methylation was also detected in 68/95 (70.5%) primary RCC tumors, but only rare adjacent non-malignant renal tissues (13%, 3/23) showed hypermethylation of promoter. We also found that the methylation of HOXA11 was associated with higher TNM classification of RCC (p<0.05). Ectopic expression of HOXA11 led to significant inhibition of proliferation, colony formation, migration and invasion abilities and induced RCC cells apoptosis. Moreover, HOXA11 was found to inhibit Wnt signaling. Thus, our study demonstrated that HOXA11 function as a tumor suppressor in RCC, while it is frequently silenced by promoter methylation in RCC.

Keywords: HOXA11; methylation; renal cell carcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Methylation and expression status of HOXA11 in RCC cell lines
A. The mRNA expression and promoter methylation of HOXA11 was detected by RT-PCR and MSP in RCC cell lines, — represents negative control; BGS analysis of HOXA11 promoter methylation in HEK-293 cell; B. Detection of HOXA11 expression by RT-PCR after demethylation treatment with Aza or Aza +TSA, A: Aza, T: TSA; C. Demethylation treatment induced demethylation in RCC cell lines by MSP, M: mehylation, U: unmethylation; D. BGS analysis of HOXA11 promoter methylation after demethylation treatment, filled circles: methylated CpG site, open circles: unmethylated CpG site.
Figure 2
Figure 2. HOXA11 expression and promoter methylation in primary RCC tissues and adjacent non-malignant renal tissues
A. MSP analysis of HOXA11 methylation in primary RCC tissues and adjacent non-malignant renal tissues. B. Quantity analysis HOXA11 mRNA expression level in paired RCC tissues, N: adjacent non-malignant renal tissues, T: primary RCC tissues; C. Representative images of HOXA11 protein expression in RCC tissues and their adjacent non-malignant tissues determined by IHC (immunohistochemistry).
Figure 3
Figure 3. Ectopic expression of HOXA11 inhibits RCC cell proliferation and colony formation abilities
A. CCK-8 assay showed an inhibition effect of HOXA11 on cell growth, *: p<0.05; B. RT-PCR showed HOXA11 expression in HOXA11- or vector-transfected cells, β-actin was used as a control. C. HOXA11 suppressed RCC cells (786-O and OSRC) colony formation, the experiment was repeated for three times as values of mean±SD, *: p<0.05.
Figure 4
Figure 4
A. HOXA11 induced apoptosis in 786-O and OSRC cells by flow cytometry analysis following Annexin V and 7-AAD staining. Quantitative analyses of apoptotic cells in 786-O and OSRC, *: p<0.05. B. Effect of HOXA11 on expression of pro-apoptosis regulators in OSRC and 786-O examined by Real-time PCR and western blot analysis.
Figure 5
Figure 5. Effects of HOXA11 on RCC cell migration and invasion
A. Ectopic expression of HOXA11 inhibit cell migration ability in 786-O and OSRC, pictures of wound-healing were captured at 0 h, 24 h and 30 h, p<0.05; B. Transwell assay shows migration results in HOXA11 unexpressed and re-expressed in RCC cells, bar graphs represent the numbers of migrating 786-O and OSRC cells, the experiment was repeated three times, *: p<0.05. C. Ectopic expression of HOXA11 inhibited migration related genes expression in OSRC and 786-O cells examined by qPCR and Western-blot.
Figure 6
Figure 6. Effect of HOXA11 on Wnt signaling down-stream target genes expression
A, B. In OSRC and 786-O cells, over-expression of HOXA11 reduced c-myc and cyclinD1 expression, values are presented as mean±SD, p<0.05.
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