MiR-124 suppresses cell proliferation in hepatocellular carcinoma by targeting PIK3CA

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Abstract

MicroRNAs (miRNAs) have crucial roles in the development and progression of human cancers, including hepatocellular carcinoma (HCC). Recent studies have shown that microRNA-124 (miR-124) was downregulated in HCC; however, the underlying mechanisms by which miR-124 suppresses tumorigenesis in HCC are largely unknown. In this study, we report that phosphoinositide 3-kinase catalytic subunit alpha (PIK3CA) is a novel target of miR-124 in HepG2 cells. Overexpression of miR-124 resulted in decreased expression of PIK3CA at both mRNA and protein levels. We found that miR-124 overexpression markedly suppressed cell proliferation by inducing G1-phase cell-cycle arrest in vitro. Consistent with the restoring miR-124 expression, PIK3CA knockdown suppressed cell proliferation, whereas overexpression of PIK3CA abolished the suppressive effect of miR-124. Mechanistic studies showed that miR-124-mediated reduction of PIK3CA resulted in suppression of PI3K/Akt pathway. The expressions of Akt and mTOR, key components of the PI3K/Akt pathway, were all downregulated. Moreover, we found overexpressed miR-124 effectively repressed tumor growth in xenograft animal experiments. Taken together, our results demonstrate that miR-124 functions as a growth-suppressive miRNA and plays an important role in inhibiting the tumorigenesis through targeting PIK3CA.

Highlights

► PIK3CA is a novel target of miR-124 in HepG2 cells. ► MiR-124 suppresses cell proliferation by downregulating PIK3CA expression. ► MiR-124 regulates the PI3K/Akt pathway in HepG2 cells. ► MiR-124 overexpression inhibits the tumorigenesis in nude mice.

Introduction

MicroRNAs (miRNAs) are a class of endogenous, short (19–22 nucleotides), noncoding RNA molecules that function as critical gene regulators [1]. It is currently estimated that miRNAs could potentially regulate close to one third of the coding genes in human genome [2], indicating that miRNAs play substantial roles in physiological and pathological processes. Accumulating evidence has suggested that the deregulation of miRNAs is implicated in many human diseases, including cancers [3]. In human cancers, miRNAs are frequently located in genomic breakpoint regions and can function as tumor suppressor genes or oncogenes during tumor development and progression [4].

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide [5], [6]. Despite the clinical implementation of numerous therapeutic strategies, HCC remains a major public health concern. The molecular pathogenesis of HCC is complicated and poorly understood. Recently, an increasing number of reports have showed that miRNAs play important roles in HCC progression, providing new avenues for HCC diagnostic and therapeutic application [7]. To date, multiple miRNAs have been shown to be dysregulated in HCC, such as miR-122, miR-7, miR-29, miR-221, and miR-151 [8], [9], [10], [11], [12], which contribute to the development and progression of HCC. Among them, miR-124 is found to be downregulated in HCC tissues [13], [14]. MiR-124 has been previously shown to suppress cell proliferation in several cancers, including squamous cell carcinoma and gastric cancer [15], [16]. These data suggest a potential tumour suppressive function of miR-124. However, the role of miR-124 in hepatocarcinogenesis and the molecular mechanisms by which miR-124 exerts its functions remain to be largely known.

In this study, we identified PIK3CA as a novel target of miR-124 in HepG2 cells. PIK3CA functions as an oncogene which plays important roles in many cancers, including HCC [17], [18], [19]. Furthermore, we show that overexpression of miR-124 in HepG2 cells suppressed cell proliferation and xenograft tumor growth through the repression of PIK3CA. Finally, we show that upregulation of miR-124 led to constitutive suppression of PI3K/Akt pathway. Our data suggest that miR-124 may be a new therapeutic target for HCC.

Section snippets

Cell lines and cell culture

HepG2 and 293T cell lines were provided by Institute of Biochemistry and Cell Biology of Chinese Academy of Science (China) and originated from ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin sulfate. Cells were incubated at 37 °C under a 5% CO2 condition.

Vector constructs

The human pre-miR-124 sequence, the coding sequences of PIK3CA were amplified by PCR and cloned into pCDH-CMV-MCS-EF1-copGFP

PIK3CA is a novel target of miR-124 in HepG2 cells

Using TargetScan, we identified PIK3CA as being a potential target of miR-124. The 3′-UTR of PIK3CA mRNA contains a complementary site for the seed region of miR-124 (Fig. 1A). To test whether PIK3CA could be directly targeted by miR-124, we cloned the PIK3CA 3′UTR (wt 3′UTR) or the mutant sequence (mt 3′UTR) into a luciferase reporter vector and performed luciferase reporter assays. The result showed that miR-124 overexpression decreased the PIK3CA 3′UTR luciferase reporter activity, and this

Discussion

Growing evidence has suggested that dysregulation of miRNAs contributes to tumorigenesis [20]. Changes in miRNA profiling are implicated in almost all aspects of cancer biology, including cell proliferation [21]. Thus, miRNAs are increasingly viewed as a potential diagnostic and therapeutic tool [22], [23]. In this study, we focused on miR-124, which has been suggested to inhibit tumor growth in several human cancers, such as Glioblastoma multiforme [24] and cervical cancer [25]. Xie and

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