miR-544a Stimulates endometrial carcinoma growth via targeted inhibition of reversion-inducing cysteine-rich protein with Kazal motifs

Highlights

• MiR-544a can function as an oncogene in Endometrial carcinoma (EC) through targeted inhibition of RECK.

• MiR-544a antagonist can inhibit tumor growth in EC mice and prolong the mice᾽s survival time.

• MiR-544a mimics can reverse the inhibition of RECK on EC cell᾽s function.

Abstract

Endometrial carcinoma (EC) is a female-specific malignant tumor. Although current treatments can achieve good outcomes and improve patient survival, there remains a high incidence of treatment-induced infertility, a serious side effect that is unacceptable to those of childbearing age. Studies have demonstrated that micro ribonucleic acids (microRNAs or miRNAs) such as miR-544a regulate tumor-related gene expression. However, whether miR-544a is involved in the progression of EC is unknown. This study aimed to investigate the biological functions and underlying mechanisms of miR-544a in EC in vivo and in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed miR-544a overexpression in EC tissue and cell lines, which was associated with a decreased in overall survival as revealed by Kaplan–Meier analysis. Functionally, the miR-544a inhibitor restricted the proliferation [detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay], invasion, and migration (detected by transwell assay) of human endometrial adenocarcinoma cells (HEC-1B and Ishikawa) and facilitated cell apoptosis (detected by flow cytometry assay). Western blotting analysis revealed that the miR-544a inhibitor decreased the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 and elevated the levels of cleaved caspase3 and cleaved poly (ADP-ribose) polymerase. Furthermore, animal experiments indicated that the miR-544a antagonist (antagomir-544a) suppressed tumor growth significantly in a mouse xenograft model. The mechanistic, qRT-PCR, and immunohistochemical indications were that a reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and miR-544a had inverse expression changes in EC. Bioinformatics analysis revealed RECK as a potential target for miR-544a, and this was verified by the dual-luciferase reporter assay. Subsequently, in vitro experiments, including transwell assay, MTT assay, flow cytometry assay, and Western blotting analysis, demonstrated that RECK exerted antitumor effects on EC, which were negatively regulated by miR-544a. Taken together, our study findings suggested miR-544a as a valuable target in EC therapy.

Introduction

Endometrial carcinoma (EC) is a malignant tumor that develops in the female reproductive system and has a serious impact on the quality of life and health of patients with EC [1]. Although postmenopausal women constitute the major high-risk group for EC, 3%–14% of patients with EC are of childbearing age, i.e., under 40 years, and this proportion is increasing every year [2,3]. Currently, the main treatment for EC is surgery combined with radiotherapy and chemotherapy, which has proved to be effective in saving lives [4]. However, all or part of the uterus is surgically removed and some patients, especially women of childbearing age, find this difficult to accept. In addition, the recurrence rate of EC is approximately 15%, with recurrence occurring mostly within the first 3 years of treatment [5]. Furthermore, chemotherapy drugs have strong side effects owing to their high toxicity and some patients may even be drug resistant, both of which can seriously affect the physical and mental health of patients [6,7]. Therefore, an in-depth study of the molecular mechanisms of EC and the development of novel treatments are essential.

Micro ribonucleic acids (microRNAs or miRNAs), derived from hairpin or double-stranded RNA precursors, are types of noncoding highly conserved RNA with a length of 20–25 nucleotides [8]. MiRNAs repress protein synthesis by interacting with a target messenger RNA (mRNA). Incomplete complementation of miRNAs with the target mRNA inhibits its translation, whereas complete complementation degrades the mRNA; this interaction inhibits protein synthesis [9,10]. In addition, substantial evidence suggests that miRNAs regulate tumor progression via cell growth suppression [11] and autophagy [12], accelerate apoptosis [13], and reduce cell differentiation [14], thus making it a novel target for cancer diagnosis and treatment [15].

As an important member of the miRNA group of genes, the role of miR-544a has been widely studied in multiple cancers [[16], [17], [18], [19], [20]]. For instance, miR-544a promotes epithelial–mesenchymal transition and accelerates tumor development in gastric cancer cells [17]. It also improves the invasion and migration capacities of breast cancer cells [18]. In addition, several studies have shown that miR-544a inhibits tumor cell apoptosis and promotes tumor cell proliferation, which are diagnostic markers for colorectal cancer [20]. However, to the best of our knowledge, the biological function and target genes of miR-544a in EC have not been reported to date.

The recently discovered novel matrix metalloproteinase (MMP) inhibitor―reversion˗inducing cysteine˗rich protein with Kazal motifs (RECK)― inhibits the various MMP expressions at the posttranscriptional level and suppresses tumor angiogenesis, thereby inhibiting tumor invasion and metastasis [21]. Zhang et al. verified that RECK knockdown promotes MMP-9 expression in stromal and endometrial epithelial cells [22]. Wang et al. revealed that miR-21, which was targeted to inhibit RECK expression, facilitates cell proliferation, migration, and invasion in esophageal cancer [23]. Moreover, Xia et al. suggested that miR-96 inhibition enhances RECK expression and improves chemotherapy and radiotherapy sensitivity toward esophageal cancer [24]. These studies suggest RECK as an active factor in EC tumorigenesis and development. Bioinformatics analysis revealed the presence of a miR-544a recognition binding site in the RECK mRNA sequence.

The present study demonstrated that miR-544a was highly expressed in EC cell lines and tissues. Both in vitro and in vivo experiments confirmed that miR-544a inhibition enhances RECK expression, thereby suppressing EC progression, and this may provide a novel insight in developing therapeutic targets for EC.