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. 2020 Jun 5;18(1):84.
doi: 10.1186/s12964-020-00563-4.

Estrogen-ERα signaling and DNA hypomethylation co-regulate expression of stem cell protein PIWIL1 in ERα-positive endometrial cancer cells

Zheng Chen  1   2   3 , Hua-Jing Yang  1   2   3 , Qin Lin  1   2   3 , Min-Jiao Zhu  1   2   3 , Ying-Ying Yu  1   2   3 , Xiao-Ying He  4   5   6 , Xiao-Ping Wan  7
Affiliations

Estrogen-ERα signaling and DNA hypomethylation co-regulate expression of stem cell protein PIWIL1 in ERα-positive endometrial cancer cells

Zheng Chen et al. Cell Commun Signal. .

Abstract

Background: We previously identified PIWIL1 as an oncogene involved in endometrial carcinogenesis. However, the mechanism of Piwil1 mediated regulation of tumorigenesis remains poorly understood.

Methods: The expression levels of target genes in endometrial cancer cells were detected by quantitative reverse transcription-PCR (RT-qPCR) and western blotting. Up- or down-regulation of ERα or PIWIL1 was achieved by transient transfection with expressing plasmids or short hairpin RNA (shRNA). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to demonstrate the ERα bound to the half estrogen response element (half-ERE) located in PIWIL1 promoter. The expression of PIWIL1 and ERα in endometrial carcinoma tissues were investigated using immunohistochemistry and RT-qPCR. The proliferation ability of cancer cells were evaluated by MTT. Methylation status of the PIWIL1 promoter was detected by bisulfite sequencing PCR (BSP).

Results: In the present study, we found that PIWIL1 mediated E2-stimulated cancer cell proliferation. In ERα-positive endometrial cancer cells, we demonstrated that estrogen-ERα signaling significantly up-regulated the expression of PIWIL1, which was mediated by binding of the ERα onto the PIWIL1 promoter. Furthermore, we found that a half-ERE in the PIWIL1 promoter was essential for ERα binding. The PIWIL1 promoter was hypomethylated in ERα-positive endometrial cancer cells. Treatment with 5-aza-deoxycytidine (5-aza-dC) could up-regulate PIWIL1 expression.

Conclusions: These findings uncover a novel molecular mechanism by which estrogen-ERα signaling and DNA hypomethylation co-regulate PIWIL1 expression. These findings provide novel insights into the hormonal regulation of PIWIL1 in endometrial cancer and the PIWIL1's role in estrogen-stimulated endometrial carcinogenesis. Video Abstract. (MP4 41319 kb).

Keywords: Cell Proliferation; DNA methylation; ERα; Endometrial carcinoma; PIWIL1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effects of estrogen on PIWIL1 expression in endometrial cancer cells. a,b,c Ishikawa, RL95–2 and HEC-1B cells were treated for treated for different times(24 h, 48 h, 72 h) with different concentrations of E2 (10− 10 ~ 10− 8 mol/L). PIWIL1 mRNA and protein levels were measured by RT-qPCR and western blot. Data were represented as means ± SD for three independent experiments (RT-qPCR). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns, not significant versus control group. GAPDH was used as an internal control (western blot)
Fig. 2
Fig. 2
Involvement of the ERα in E2-induced PIWIL1 expression. a and b The ERα antagonist ICI 182,780 (10− 7 mol/L) was used to examine ERα in E2-induced PIWIL1 induction in Ishikawa and RL95–2 cells. c Ishikawa and RL95–2 cells were transfected with ERα shRNA (shERα) and HEC-1B was transfected with ERα expressing vector (exERα). The mRNA and protein levels of PIWIL1 and ERα were then assayed using RT-qPCR and western blot. Data were represented as means ± SD for three independent experiments (RT-qPCR). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns, not significant versus control group. GAPDH was used as an internal control (western blot)
Fig. 3
Fig. 3
Estrogen up-regulates the transcription of PIWIL1. a A schematic of three PIWIL1 promoter-luciferase reporter constructs (wild type, WT; Mutation of the half-ERE, MUT; Deletion of the half-ERE, DEL) was shown. b and c Luciferase activities of the WT reporter in endometrial cancer cell lines. d and e Luciferase activities of the WUT reporter in endometrial cancer cell lines. f and g Luciferase activities of the DEL reporter in endometrial cancer cell lines. The expression construct of ERα was co-transfected into ERα-negative cell lines. All experiments were performed at three times. **p < 0.01 and ns, not significant versus control group
Fig. 4
Fig. 4
E2-induced binding of the ERα onto the PIWIL1 promoter by estrogen. a A Schematic representation of the half-ERE in the PIWIL1 promoter and the three pairs of primers used for ChIP-qPCR was shown. TSS: transcription start sites. b the PIWIL1 promoter region was precipitated by the antibody against ERα in Ishikawa cells cultured in normal media. IgG served as the negative control. RT-qPCR was performed on ChIP samples. *p < 0.05, **p < 0.01 and ns, not significant versus control group. c Ishikawa cells treated with 10− 8 mol/L E2 for 0, 45, and 135 min were subjected to ChIP and the precipitated DNA was analyzed by RT-qPCR. Experimental conditions are identical to those in panel b, except that cells were cultured in hormone-free media for 72 h before E2 treatment. All experiments were performed at three times. *p < 0.05, ***p < 0.001 and ****p < 0.0001 versus control group (cells without E2 treatment). d the effects of PIWIL on E2-induced cell growth were determined by MTT assay. *p < 0.05 and **p < 0.01 versus control group
Fig. 5
Fig. 5
PIWIL1 and ERα expression in endometrial carcinoma tissues. a Example of PIWIL1 and ERα immunoreactivity in endometrial cancer tissues. Original magnification 200×, scale bar, 100 μm (left); 400×, scale bar, 50 μm (right). b PIWIL1 immunoreactivity scores of 15 ERα-positive endometrial cancer samples and 15 ERα-negative endometrial cancer samples. Values are the mean ± SD. ****p < 0.0001. c A positive correlation was detected between mRNA levels of PIWIL1 and ERα in endometrial cancer samples (r = 0.8, ****p < 0.0001)
Fig. 6
Fig. 6
Cancer-linked hypomethylation of the PIWIL1 promoter. a Results of bisulfite DNA sequencing of the PIWIL1 upstream regulatory region in Ishikawa, RL95–2 and HEC-1B cells. Black dots symbolize methylated CpGs and white dots symbolize unmethylated CpGs. b The percentage of methylated CpG dinucleotides in Ishikawa, RL95–2 and HEC-1B cells. ***p < 0.001 and ns, not significant. c RT-qPCR and western blot showed changes in PIWIL1 mRNA and protein expression in HEC-1B cells after treatment with 5-aza-dC. Data were represented as means ± SD for three independent experiments (RT-qPCR). ****p < 0.0001 versus control group. GAPDH was used as an internal control (western blot)
Fig. 7
Fig. 7
Proposed model of the cooperation between E2-ERα signaling and DNA hypomethylation for the regulation of stem cell protein PIWIL1. upper: In normal cells, hypermethylation of PIWIL1 promoter prevents the binding of E2–ERα complex to PIWIL1 promoter. Lower: In endometrial cancer cells, increased PIWIL1 expression regulated by E2–ERα signaling is due to the cancer-linked hypomethylation of the PIWIL1 promoter
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