MicroRNA-370-3p inhibits human glioma cell proliferation and induces cell cycle arrest by directly targeting β-catenin

Highlights

• miR-370-3p is down-regulated in glioma tissues and cell lines.

• miR-370-3p inhibits glioma cell growth by targeting β-catenin.

• miR-370-3p is inversely correlated with β-catenin in glioma tissues.

Abstract

Objective

The aim of this study was to explore the expression and biological role of miR-370-3p in human gliomas.

Methods

Clinical specimens from the brains of 20 glioma patients and 10 healthy controls were obtained to quantify the expression level of miR-370-3p using quantitative real-time PCR. Oligonucleotide mimics of miR-370-3p were transfected into U251 and U87-MG cells for a gain of function assay. The CCK-8 assay, colony formation assay, EdU assay and flow cytometry were used to evaluate the roles of miR-370-3p in cell proliferation and the cell cycle regulation. Western blot and luciferase activity assays were used to investigate the reciprocal relationship between miR-370-3p and its predicted target, β-catenin.

Results

miR-370-3p expression was frequently found to be decreased in glioma tissues, and its expression level was negatively correlated with the malignant degree of the glioma. Overexpression of miR-370-3p showed a significant inhibitory effect on cell proliferation and accompanied cell cycle G0/G1 arrest in U251 and U87-MG cells. Furthermore, miR-370-3p inhibited the expression of the canonical Wnt pathway downstream targets cyclin D1 and c-myc via direct binding interaction with the 3′-untranslated region of β-catenin mRNA. Reintroduction of β-catenin could partially reverse the anti-proliferation effect of miR-370-3p. Finally, in 20 glioma tissues the expression of miR-370-3p was negatively correlated with both protein and mRNA levels of β-catenin.

Conclusion

miR-370-3p suppresses glioma cell growth by directly targeting β-catenin, suggesting that the miR-370-3p/β-catenin axis may be a target for glioma therapy.

Introduction

Malignant glioma, which is characterized by high morbidity and mortality rates, is the most common type of adult brain tumors (Taylor, 2010). The median survival of patients with glioblastoma multiforme (GBM), the most common histological subtype of high-grade gliomas, ranges from 9 to 12 months (Surawicz et al., 1998). Despite advancements in both diagnostic modalities and therapeutic strategies over the past several decades, the prognosis of malignant gliomas still remains poor (Van Meir et al., 2010). To develop more effective treatments, current research is focused on understanding the biology and molecular mechanisms of glioma development and progression.

MicroRNAs (miRNAs) are recognized as a class of endogenous, small non-coding RNA molecules composed of approximately 20 nucleotides, which negatively regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs (Bartel, 2004, Bartel, 2009). About one-third of human genes might be regulated by miRNAs and each miRNA could target hundreds of mRNAs (Lewis et al., 2005). For those reasons, miRNAs are extensively involved in human physiological and pathological processes. Increasing evidence suggests that the abnormal expression of some specific miRNAs are closely related to cancer progression, and some miRNAs also serve as tumor suppressors or promoters by directly regulating known oncogenes or tumor suppressor genes (Lu et al., 2005, Iorio and Croce, 2009). Thus, a full understanding of the roles of miRNAs in glioma may provide a new mechanism for cancer treatment.

MiR-370-3p is located within the DLK1/DIO3 domain, which has been identified as a cancer-associated genomic region on human chromosome 14 (Kircher et al., 2008, Benetatos et al., 2013). Recently, several studies reported that the aberrant expression of miR-370-3p was involved in various human cancer processes; however, its role in tumorigenesis remains controversial. Substantial evidence demonstrated that miR-370-3p plays a role as a tumor suppressor in cholangiocarcinoma (Meng et al., 2008), acute myeloid leukemia (Zhang et al., 2012), oral squamous carcinoma (Chang et al., 2013), hepatocellular carcinoma (Xu et al., 2013) and ovarian cancer (Chen et al., 2014). In contrast, some studies found that upregulation of miR-370-3p contributes to the progression of gastric carcinoma (Lo et al., 2012), Wilms tumor (Cao et al., 2013) and prostate cancer (Wu et al., 2012). For human malignant glioma, the biological role and functional mechanisms of this miRNA are still largely unknown.

β-catenin is a key mediator of canonical Wnt signaling and is closely related to the initiation and progression of glioma. It has been demonstrated that overexpression of β-catenin consistently appears in astrocytic tumors and correlates with poor prognosis and reduced survival in GBM patients (Rossi et al., 2011). Knockdown of β-catenin using siRNA significantly inhibited U251 cell proliferation (Pu et al., 2009). In the present study, we found that miR-370-3p had low expression levels in glioma tissues and that it served as a tumor suppressor by inhibiting glioma cell growth. Cell growth was inhibited by directly targeting β-catenin and negatively regulating the expression of two genes downstream of the Wnt/β-catenin pathway, cyclin D1 and c-myc.