MiR-216b-5p inhibits cell proliferation in human breast cancer by down-regulating HDAC8 expression

doi:10.1016/j.lfs.2019.116945

Life Sciences

Volume 237, 15 November 2019, 116945

https://doi.org/10.1016/j.lfs.2019.116945 Get rights and content

Abstract

Aim

Over-expression of histone deacetylase 8 (HDAC8) has been demonstrated in breast cancer. But the underlying molecular mechanism of HDAC8 on the progression of breast cancer remains unknown. MicroRNAs (miRs) are proposed as important molecules in cancer progression by targeting specific oncogenes or tumor-suppressor genes. Our overall objective was to assess the miR-216b-5p role on HDAC8; and its impacts on breast cancer (BC) progression.

Main methods

We acquired cancerous and noncancerous tissues from Iran Tumor Bank (I.T.B). The MDA-MB-231, MCF-7 and MCF-10A BC cell lines were also purchased. The tissue and cell line expression levels of miR-216b-5p and HDAC8 were determined by quantitative real-time PCR (qPCR). We next measured protein levels of HDAC8 by Western blotting assay. The cell cycle, cell proliferation, and colony formation assay were determined. Finally, we investigated the role of HDAC8 using a knockout vector; and confirmed the targeting of 3′ untranslated region (3′-UTR) of HDAC8 through miR-216b-5p using a luciferase reporter assay.

Key findings

Our results demonstrated a significant decrease in miR-216b-5p, and remarkable increase in HDAC8 levels within human breast cancer tissues and cell lines. The lower levels of miR-216b-5p were negatively correlated with lymph node metastasis and advanced tumor size. The overexpression of miR-216b-5p in BC cell lines inhibited cellular proliferation and progression. HDAC8 was directly down-regulated by miR-216b-5p and knockout of HDAC8 showed the similar effects as miR-216b-5p overexpression.

Significance

Briefly, HDAC8 is an oncogene that accelerate breast cancer proliferation and progression and miR-216b-5p modulates those functions by binding to HDAC8 3′-UTR.

Introduction

Breast cancer (BC) is known as the most prevalent malignancy in women worldwide, which accounts for 26% of all cancers by 182,000 new cases, annually [1]. BC is a complicated and complex disease characterized by the growth of malignant cells in the mammary glands with a great number of aberrations at the pathologic, genomics, epigenetics, and molecular level which eventually result in the dysregulation of gene expression and signaling pathways [2].

Aberrant expression of histone deacetylases (HDACs) are related to tumor pathogenesis, progression, and prognosis [3]. Therefore, these enzymes are among the most potential biomarkers and therapeutic targets for cancer detection and treatment [3]. Recently, HDAC inhibitors have gotten more attention as potential therapeutic agents for treatment of some malignancies [3,4]. Among them, HDAC8 is the most recently identified class I HDACs which is linked to a number of diseases particularly to hematological malignancies [5]. Recent studies showed that HDAC8 is a potential Oncogene; and its high expression is directly related to several malignancies [4,[6], [7], [8]]. HDAC8 inhibition showed anti-tumor activity and increased the apoptotic cells ratio in T cell lymphomas [9]. Furthermore, it seems that HDAC8 involves in the pathogenesis of neuroblastoma [8]. It has been recently demonstrated that HDAC8 is up-regulated in BC cells and proposed this molecule as a potential oncogene in breast cancer [6].

MicroRNAs (miRs) are natural RNA molecules that play critical roles in cellular processes and regulate gene expression post-transcriptionally. MiRs are known as a type of small non-coding RNAs with 19–25 bp length that are cleaved from a 70–100 bp hairpin pre-miRNA precursors [10]. Single-stranded mature miR binds to the 3′ untranslated region (3′-UTR) of the target mRNA and regulated the block of translation or degradation of targeted mRNA molecule [10]. According to the literature, the aberrant expression of numerous miRs is associated with various human malignancies [11]. Among them, miR-216b-5p is a newly recognized miR that acts as a tumor suppressor. In nasopharyngeal carcinoma, researchers suggested that the anti-tumor effects of miR-216 applies via PKC and KRAS [12,13]; miR-216 is also inhibit proliferation of BC cells possibly by targeting syndecan binding protein (SDCBP) [14] and P2X7 receptor (P2X7R) [15].

The concurrent association between miR-216 and HDAC8 with progression of breast cancer is not understood yet. Also, to the best of our knowledge, there is no other study in evaluating the miR-216 and HDAC8 role in the carcinogenesis of breast cancer. Hence, in the this study we aimed to study the miR-216b-5p and HDAC8 levels in cancerous and adjacent none-cancerous tissues obtained from BC patients; and the specific objective was to assess the potential role of HDAC8 and miR-216 (as a potential inhibitor of the HDAC8) on tumor growth in human breast adenocarcinoma.

Section snippets

Patients and tissue collection

We obtained 32 tissue specimens from Iran Tumor Bank (I.T.B) during the period January 2016-January 2018. We then obtained control samples from normal adjacent tissue; and subsequently, we confirmed both the malignant and normal tissues, pathologically. We then obtained the written informed consent from all studied subjects. In this case, we got the approval from ethics committee of Kurdistan University of Medical Sciences. We then excluded those patients with a history of other organ cancers

Expression of HDAC8 and miR-216b-5p in breast cancer cell lines and clinical specimens

In analyzing the clinicopathologic status, we detected the expression of HDAC8 and miR-216b-5p in 32 primary breast cancer tissues and 32 normal adjacent tissues using qRT-PCR. The expression of miR-216b-5p in cancerous tissues were significantly decreased in compared with normal adjacent tissues (0.0024 ± 0.00025 (r.u.) vs. 0.004 ± 0.00044 (r.u.), respectively) (Fig. 1A and B) (p value = 0.002). Our results also revealed that miR-216b-5p expression is decreased in metastatic breast cancer cell

Discussion

In the present study, we evaluated the impact of HDAC8 and miR-216b-5p in breast cancer progression. Here, we showed that miR-216b-5p is significantly down-regulated in human breast cancer tissue and BC cell lines. We also observed that miR-216b-5p has a negative correlation with the HDAC8 level. Our results demonstrated that miR-216b-5p down regulates the expression of HDAC8 by directly targeting of the HDAC8 3ʹ-UTR. We proved that upon miR-216b-5p overexpression, HDAC8 is significantly

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the ethics committee of Kurdistan University of Medical Sciences and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Funding source

This work was supported by a research grant from Kurdistan University of Medical Sciences, Sanandaj, Iran (Grant/Award Number: ‘IR.MUK.REC.1395/279’).

Financial disclosure

The author has no financial relationships relevant to this article to disclose.

Author contributions

M-N.M. and N.D. carried out the experiment. K.R. helped supervise the project. A.A., S.M-Y., and V.H. conceived and planned the experiments. A.E. provided the BC samples. M.A. took the lead in project, conceived the original idea, supervised the project, analyzed the results and wrote the manuscript with support from K.R. and S.M-Y.

Declaration of Competing interest

Dr MN Menbari declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Dr K Rahimi declares that he has no conflict of interest. Dr A Ahmadi declares that he has no conflict of interest. Dr A Elyasi declares that he has no conflict of interest. Dr. N Darvishi declares that she has no conflict of interest. Dr V hosseini declares that she has no conflict of interest. Dr S Mohammadi-Yeganeh declares that she has no conflict of

Acknowledgments

The author wish to thank all patients and health stuffs who participated in this study. Financial support from Kurdistan University of medical sciences is highly appreciated.

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MiR-216b-5p inhibits cell proliferation in human breast cancer by down-regulating HDAC8 expression

Abstract

Aim

Main methods

Key findings

Significance

Introduction

Section snippets

Patients and tissue collection

Expression of HDAC8 and miR-216b-5p in breast cancer cell lines and clinical specimens

Discussion

Ethical approval

Informed consent

Funding source

Financial disclosure

Author contributions

Declaration of Competing interest

Acknowledgments

Trends Pharmacol. Sci.

Eur. J. Pharmacol.

Adv Biol Regul

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Gene

Life Sci.

Biochem. Biophys. Res. Commun.

Cell

Cancer statistics

2018. CA Cancer J Clin

Low uptake of tamoxifen to prevent breast cancer

Lancet Oncol.

Histone deacetylases function as novel potential therapeutic targets for cancer

Hepatol. Res.

Exploiting histone deacetylases for cancer therapy: from hematological malignancies to solid tumors

Sci. China Life Sci.

miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8 oncogene

J. Cell. Physiol.

Histone deacetylase 8 in neuroblastoma tumorigenesis

Clin. Cancer Res.

Transcription and beyond: the role of mammalian class I lysine deacetylases

Chromosoma

Overview of microRNA biology

Semin. Liver Dis.

miR-216b suppresses tumor growth and invasion by targeting KRAS in nasopharyngeal carcinoma

J. Cell Sci.

MiR-216b is involved in pathogenesis and progression of hepatocellular carcinoma through HBx-miR-216b-IGF2BP2 signaling pathway

Cell Death Dis.