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. 2019 Jan 12;18(1):9.
doi: 10.1186/s12943-019-0940-3.

PIWI-interacting RNA-36712 Restrains Breast Cancer Progression and Chemoresistance by Interaction With SEPW1 Pseudogene SEPW1P RNA

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Free PMC article

PIWI-interacting RNA-36712 Restrains Breast Cancer Progression and Chemoresistance by Interaction With SEPW1 Pseudogene SEPW1P RNA

Liping Tan et al. Mol Cancer. .
Free PMC article

Abstract

Background: Breast cancer is one of the most common malignancies and the major cause of cancer-related death in women. Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has been increasingly recognized, few studies have been explored the functional mechanism of piRNAs in breast cancer development and progression.

Methods: We examined the top 20 highly expressed piRNAs based on the analysis of TCGA breast cancer data in two patient cohorts to test the roles of piRNAs in breast cancer. The effects of piRNA-36,712 on the malignant phenotypes and chemosensitivity of breast cancer cells were detected in vitro and in vivo. MS2-RIP and reporter gene assays were conducted to identify the interaction and regulation among piRNA-36,712, miRNAs and SEPW1P. Kaplan-Meier estimate with log-rank test was used to compare patient survival by different piRNA-36,712 expression levels.

Results: We found piRNA-36,712 level was significantly lower in breast cancer than in normal breast tissues and low level was correlated with poor clinical outcome in patients. Functional studies demonstrated that piRNA-36,712 interacts with RNAs produced by SEPW1P, a retroprocessed pseudogene of SEPW1, and subsequently inhibits SEPW1 expression through competition of SEPW1 mRNA with SEPW1P RNA for microRNA-7 and microRNA-324. We also found that higher SEPW1 expression due to downregulation of piRNA-36,712 in breast cancer may suppress P53, leading to the upregulated Slug but decreased P21 and E-cadherin levels, thus promoting cancer cell proliferation, invasion and migration. Furthermore, we found that piRNA-36,712 had synergistic anticancer effects with the paclitaxel and doxorubicin, two chemotherapeutic agents for breast cancer.

Conclusions: These findings suggest that piRNA-36,712 is a novel tumor suppressor and may serve as a potential predictor for the prognosis of breast cancer patients.

Keywords: Breast cancer; Drug sensitivity; P53; SEPW1; piRNA.

Conflict of interest statement

Ethics approval and consent to participate

Informed consent was obtained from all participants, and this study was approved by the Sun Yat-sen University Cancer Center and Cancer Hospital, Chinese Academy of Medical Science. All experiments on the participants in this study were performed in accordance with the relevant guidelines and regulations. All animal handling and experimental procedures were also performed in accordance with the relevant institutional and national guidelines.

Consent for publication

We all consent for publication.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
piR-36,712 is downregulated in breast cancer and correlated with clinical outcomes in patients. a The top 20 highly expressed piRNAs in breast cancer based on the analysis of TCGA data. b The expression levels of piR-36,712 in breast cancer and paired non-tumor tissues from patients recruited at SYSUCC (Cohort 1) and CHCAMS (Cohort 2). Data are mean ± SEM, ***, P < 0.001). c The expression levels of piR-36,712 in 103 paired breast cancer and non-tumor tissues from TCGA database (mean ± SEM; ***, P < 0.001). d Northern blot of piR-36,712 in breast cancer cells. e piR-36,712 copy number per cell in MCF7 and ZR75–1 cell lines (mean ± SEM). f Distribution of piR-36,712 in cytoplasm and nucleus of breast cancer cells with U6 or GAPDH as nucleus or cytoplasm markers (% ± SEM). g Kaplan-Meier estimates of progression-free survival time in breast cancer patients at SYSUCC (Cohort 1) and CHCAMS (Cohort 2) stratified by piR-36,712 levels in tumor. HR, hazard ratio; CI, confidence interval. h The expression levels of piR-36,712 in breast cancer with (LN ≥ 1) or without (LN = 0) axillary lymph node (LN) metastasis (mean ± SEM; *, P < 0.05)
Fig. 2
Fig. 2
piR-36,712 suppresses malignant phenotypes of breast cancer cells. a, b Effect of piR-36,712 expression on MCF7 and ZR75–1 cell proliferation tested by CCK8 assay (mean ± SEM; *, P < 0.05; **, P < 0.01 and ***, P < 0.001). c, d The fluorescent thymidine analog EdU was used to identify proliferative cells by labeling their DNA (green signal). Nuclei labeled with hoechst are in blue. Representative images (left) and quantitative statistics by flow cytometry (right) (mean ± SEM, n = 3; NS, **, P < 0.01 and ***, P < 0.001). e Effect of piR-36,712 expression on colony formation ability of breast cancer cells. Results present colony formation ability relative to control (mean ± SEM; **, P < 0.01 and ***, P < 0.001). f Effect of piR-36,712 overexpression or knockdown on MCF7 and ZR75–1 cell cycle progression. g, h Effects of piR-36,712 on the abilities of MCF7 and ZR75–1 cell migration and invasion (means ± SEM; **, P < 0.01; ***, P < 0.001). i, j Effects of piR-36,712 expression on the growth of MCF7 and ZR75–1 xenografts in mice (mean ± SEM, N = 5 in each group; **, P < 0.01 and ***, P < 0.001). k, l Effects of piR-36,712 expression on MCF7 cell metastasis in mice. Luminescence imaging of metastases (k) and quantification of radiance intensity (l). Data are mean ± SEM, N = 10 in each group; ***, P < 0.001
Fig. 3
Fig. 3
piR-36,712 interacts with SEPW1P RNA. a Levels of SEPW1P RNA in MCF7 and ZR75–1 cells with stable overexpression (OE) or knockdown (KD) of piR-36,712 (mean ± SEM; **, P < 0.001; ***, P < 0.0001). b Relative reporter gene activity of psiCHECK2 vector bearing SEPW1P in MCF7 and ZR75–1 cells co-transfected with indicated amount of piR-36,712 mimic or inhibitor (mean ± SEM; **, P < 0.001; ***, P < 0.0001). c Relative reporter gene activity of psiCHECK2 vector bearing SEPW1P with mutation at putative binding site of piR-36,712 in MCF7 and ZR75–1 cells co-transfected with 100 pmol of piR-36,712 mimic. d Schematic diagram of MS2-RNA immunoprecipitation (RIP) assay. e MS2-RIP and qRT-PCR analysis shows interaction of piR-36,712 with SEPW1P RNA in MCF7 and ZR75–1 cells. f Decay of SEPW1P RNA in MCF7 and ZR75–1 cells with piR-36,712 overexpression or knockdown treated with actinomycin D determined by qRT-PCR. g, h RIP and qRT-PCR analysis shows significant increased association with PIWIL1 protein of piR-36,712 (g) and SEPW1P RNA (h) in MCF7 and ZR75–1 cells
Fig. 4
Fig. 4
Competition of SEPW1P RNA with SEPW1 mRNA for miR-7 and miR-324. a Relative luciferase activity of psiCHECK2 vector bearing SEPW1 3’UTR in MCF7 and ZR75–1 cells with overexpression or knockdown of SEPW1P by cotransfected with pcDNA3.1-SEPW1P or siSEPW1P. b Relative reporter gene activity of psiCHECK2 vector bearing SEPW1P in MCF7 and ZR75–1 cells with overexpression or knockdown of SEPW1 by cotransfected with pcDNA3.1-SEPW1 or -siSEPW1. c Levels of SEPW1 mRNA and protein in MCF7 and ZR75–1 cells with overexpression (pcDNA3.1-SEPW1P) or knockdown (siSEPW1P) of SEPW1. d Relative reporter gene activity of constructs bearing SEPW1P (d) or SEPW1 3’UTR (e) in MCF7 and ZR75–1 cells cotransfected with miR-7 or miR-324 mimic or inhibitor. f, g Relative reporter gene activity of psiCHECK2 vector bearing SEPW1P (f) or SEPW1 3’UTR (g) with mutations at putative binding site of miR-7 or miR-324 in MCF7 and ZR75–1 cells cotransfected with miR-7 or miR-324 mimic or inhibitor. Shown are mean ± SEM; **, P < 0.001; ***, P < 0.0001
Fig. 5
Fig. 5
piR-36,712 inhibits SEPW1 expression and functions by interaction with SEPW1P RNA. a Levels of SEPW1 mRNA in MCF7 and ZR75–1 cells with stable overexpression (OE) or knockdown (KD) of piR-36,712 (mean ± SEM; **, P < 0.001; ***, P < 0.0001). b MS2-RIP and qRT-PCR analysis shows interaction of piR-36,712 with SEPW1 mRNA. c, d Effects of OE or KD of SEPW1P or SEPW1 on breast cancer cell proliferation induced by OE or KD of piR-36,712 (mean ± SEM; ***, P < 0.001). e, f Effects of OE or KD of SEPW1P or SEPW1 on breast cancer cell cycle progression induced by OE or KD of piR-36,712. g, h Effects of OE or KD of SEPW1P or SEPW1 on breast cancer cell migration and invasion induced by OE or KD of piR-36,712 (mean ± SEM; *, P < 0.05; **, P < 0.01 and ***, P < 0.001)
Fig. 6
Fig. 6
piR-36,712 suppresses the malignant phenotypes of breast cancer cells via P53. a Effects of piR-36,712 on expressions of SEPW1 and its downstream P53, P21, Slug and E-cadherin in MCF7 and ZR75–1 cells. b Inhibitory effect of PFT-α on expression of P53 and its downstream in MCF7 and ZR75–1 cells overexpressing piR-36,712. c Suppression of P53 by PFT-α inhibits the inhibitory effect of piR-36,712 on proliferation of MCF7 and ZR75–1 cells. d Suppression of P53 by PFT-α suppresses the inhibitory effect of piR-36,712 on colony formation ability of MCF7 and ZR75–1 cells. e Suppression of P53 by PFT-α inhibits the effect of piR-36,712 on G1 phase cell cycle arrest in MCF7 and ZR75–1 cells. f Suppression of P53 by PFT-α inhibits the inhibitory effect of piR-36,712 on MCF7 and ZR75–1 cell migration and invasion
Fig. 7
Fig. 7
Effects of piR-36,712 on the chemosensitivity of breast cancer cells to paclitaxel (PTX) or doxorubicin (DOX). a, b Effect of overexpression (OE) or knockdown (KD) of piR-36,712 on sensitivity of MCF7 and ZR75–1 cells to PTX or DOX in vitro. c-f OE of piR-36,712 increased but KD of piR-36,712 decreased the sensitivity of breast cancer xenografts in mice to PTX or DOX. Shown are mean ± SEM (N = 5); *, P < 0.05; **, P < 0.01, ***, P < 0.001. g, h Inhibitory effect of intra-tumor administration of agopiR-36,712 on xenograft growth. Shown are pictures of tumors with or without injection of agopiR-36,712 (left panel) and the growth curves of tumors (right panel). Each point in the curves presents mean ± SEM (N = 5). *, P < 0.05; **, P < 0.01, ***, P < 0.001

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