Cancer Letters

Cancer Letters

Volume 401, 10 August 2017, Pages 20-32
Cancer Letters

Original Article
MicroRNA-1301 suppresses tumor cell migration and invasion by targeting the p53/UBE4B pathway in multiple human cancer cells

https://doi.org/10.1016/j.canlet.2017.04.038Get rights and content

Highlights

  • miR1301 positively regulates p53 targeting its E3/E4 ligase UBE4B.

  • miR-1301 suppresses tumor cell migration and invasion in a p53-dependent manner.

  • It is possible to manipulate the p53/UBE4B pathway via miR-1301 to stimulate the anti-tumor activity of p53.

Abstract

The p53 protein plays a critical role in preventing tumor development. Although numerous factors have been shown to directly or indirectly regulate p53, the mechanism of how microRNAs (miRNAs) modulate p53 remains unclear. Here, we identified miR-1301, a microRNA that regulates the activity and function of p53, by directly targeting the ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase. Notably, ectopic expression of miR-1301 inhibits dissemination and metastasis of tumor cells in a p53-dependent manner. Depletion of miR-1301 downregulates p53 function. Our results reveal that there is an inverse correlation between miR-1301 and UBE4B expression and p53 status in prostate cancer. Furthermore, low miR-1301 expression is often associated with incomplete methylation of its gene in human prostate tumors. Together, our results provide the first report indicating that miR-1301 functions as a tumor suppressor that inhibits tumor cell migration and invasion in multiple human cancer cells by regulating the UBE4B-p53 pathway.

Introduction

The p53 tumor suppressor is inactivated in more than 50% of all human tumors [1], [2]. Inactivation of p53 is associated with more aggressive disease and poorer overall survival in malignant tumors, highlighting the crucial role of p53 as a tumor suppressor [3], [4]. The p53 gene regulates the cell cycle, cellular senescence, initiation of apoptotic cell death, and DNA repair [5], [6]. p53 has been called the “guardian of the genome” [7]. Mice deficient in p53 are highly susceptible to tumors; more than 75% of p53−/− mice develop tumors within six months [8]. Restoration of p53 function leads to the regression of some tumors in vivo [9], [10]. This suggests that restoration of p53 function in tumors may provide a novel effective strategy for cancer therapy.

Our previous studies have shown that UBE4B, a ubiquitin ligase with E3/E4 functions, is essential for HDM2 (Mdm2 in the mouse)-mediated p53 ubiquitination and degradation [11], [12]. The UBE4B-mediated degradation of p53 has been confirmed [13], [14]. Recently, we discovered that UBE4B targets phosphorylated p53 at serine residues 15 and 392 for degradation after DNA damage [15]. Notably, UBE4B is overexpressed in several brain tumors [11], breast tumors [16] and hepatocellular carcinoma [17], and there is an inverse correlation between the overexpression of UBE4B and a decreased p53 level in these tumors. Therefore, UBE4B is an attractive cancer therapeutic target.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are ∼22-nucleotides (nt) in length. miRNAs regulate gene expression primarily through specific interactions with mRNA targets [18], [19]. It has been demonstrated that miRNAs are involved in multiple important physiological and pathological processes, including proliferation, differentiation, apoptosis, and carcinogenesis [19], [20]. Recently, miRNAs have been reported to be an integral part of the p53 pathway [21], [22], [23], [24], [25], [26], [27]. HDM2, UBE4B, Pirh2, CHIP, and COP1 are structurally different but functionally related proteins that serve as major negative regulators of p53 [11], [28], [29], [30], [31].

It was reported that miR-1301 is dysregulated in HepG2 cells [32], [33]. Upregulation of p53 expression and downregulation of Bcl-2 and Bcl-xL expression at both mRNA and protein levels were observed in HepG2 cells transfected with an miR-1301 expression plasmid [32]. They concluded that miR-1301 induces cell apoptosis by inhibiting Bcl-2 and Bcl-xL expression in HepG2 cells [32]. However, the mechanism underlying upregulation of p53 remains unknown. Here, we report that miR-1301 stabilizes p53 and augments the function of p53 by targeting UBE4B. Our results reveal that miR-1301 inhibits tumor cell migration and invasion in a p53-dependent manner. We observed that miR-1301 is expressed at a very low level in human prostate tumors and that there was an inverse correlation between miR-1301, UBE4B expression and p53 status in these tumor samples. Furthermore, the incomplete methylation of the miR-1301 promoter region may be responsible for its low expression level in prostate cancer and some other tumor cell types. Our data provide new insight into the mechanisms of p53 inactivation in prostate tumors.

Section snippets

Cell culture and DNA transfection

MDA PCa2b cells were cultured in BRFF-HPC1 medium (Athena, USA). All other cells were maintained in α-MEM supplemented with 10% FBS. LNCaP cells were transfected using TransIT-X2™ (Mirus Bio). All other cells were transfected using the calcium phosphate method as described previously [11]. The transfection efficiency was determined by GFP expression and quantification of miRNAs.

DNA constructs

UBE4B 3′UTR was cloned into pMIR-REPORT™ Luciferase (Ambion) using primer 5′-AGCGAGCTCACCGTTCCGCCGCCCACCCTCTGCT-3′ and

miR-1301 positively regulates p53

To investigate miRNAs that regulate p53 activity, we designed a series of screening experiments. Based on the analysis of references and the miRNA prediction database, we selected forty miRNAs that potentially target p53 and several negative regulators of p53, such as UBE4B, HDM2, Pirh2, and CHIP [11], [28], [29], [30], [31] (Fig. 1A). These miRNA precursors were cloned and transfected into wild-type p53 expressing HCT116 cells. miR-192, miR-194 [25], miR-605 [26], and miR-34a [41], which have

Discussion

Recent work has revealed important roles for miRNAs and miRNA processing in tumorigenesis [18], [19], [20]. Large sets of miRNAs are under-expressed in human tumors compared to normal tissues, and interfering with miRNA processing enhances experimental tumorigenesis [20], [54]. miRNAs have also been shown to target p53 and/or components of p53 regulatory pathways, thereby directly and/or indirectly affecting its activities [21], [22], [23], [24], [25], [26], [27]. In this study, we identified

Acknowledgments

We thank the Alberta Cancer Research Biobank (ACRB) for providing tumor tissue samples. We thank Dr. Wei Wu for technical assistance, and Dr. Du and Dr. Abou Zeinab for insightful discussions. This work was supported by grants from the Women & Children's Health Research Institute (WCHRI, RES0022873), the Alberta Heritage Foundation for Medical Research (AHFMR, 201000528), the Canadian Breast Cancer Foundation (RES0009923), and the Canadian Institutes of Health Research (CIHR, MOP84216) to

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