Cinnamaldehyde induces cell apoptosis mediated by a novel circular RNA hsa_circ_0043256 in non-small cell lung cancer

doi:10.1016/j.bbrc.2017.09.136

Biochemical and Biophysical Research Communications

Volume 493, Issue 3, 25 November 2017, Pages 1260-1266

https://doi.org/10.1016/j.bbrc.2017.09.136 Get rights and content

Highlights

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We firstly explored circular RNA function involved in mechanism of cinnamaldehyde against non-small cell lung cancer.
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Circular RNA has_circ_0043256 inhibited cell proliferation and induced apoptosis in NCI-H460 and A549 cells.
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Has_circ_0043256 was for the first time reported as a cinnamaldehyde-responsive circRNA in vitro and in vivo.
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Has_circ_0043256 acted as endogenous sponge of miR-1252, relieving its target ITCH, and regulated Wnt/β-catenin pathway.

Abstract

Cinnamaldehyde (CA), the primary chemical component of the Chinese traditional herb Cinnamomum cassia, is an effective cytotoxic agent against various human cancers. Our previous study indicated that CA could trigger apoptosis in three kinds of non-small cell lung cancer (NSCLC) cells. However, CA mechanism of action in NSCLC has not been unveiled completely. Herein, we showed that a novel circular RNA hsa_circ_0043256 was upregulated in NSCLC cells in response to CA treatment, as detected by microarray and real-time PCR. Hsa_circ_0043256 could inhibit cell proliferation and induce apoptosis, while hsa_circ_0043256 knock-down could promote cell proliferation and restrain apoptosis induced by CA. Bioinformatics analysis predicted that hsa_circ_0043256 could work as a miR-1252 sponge, which could in turn directly target a vital negative regulator of Canonical Wnt signaling, Itchy E3 ubiquitin protein ligase (ITCH), as validated by dual-luciferase assay. Western blot results further confirmed that hsa_circ_0043256 could upregulate ITCH expression, whereas miR-1252 could partially abolish this effect. Interestingly, hsa_circ_0043256 knock-down could weaken Wnt/β-catenin pathway inhibition induced by CA. Finally, we discovered that CA induced apoptosis and meanwhile upregulated hsa_circ_0043256 expression in vivo. Immunohistochemical analysis revealed that ITCH expression was positively association with hsa_circ_0043256 levels. Above all, we characterized a new mechanism mediated by hsa_circ_0043256/miR-1252/ITCH axis in CA function against NSCLC, providing a novel insight into lung cancer therapy.

Introduction

Cinnamaldehyde (CA, C6H5CH=CHCHO) is the main chemical component isolated from Cinnamomumcassia [1]. In previous research, CA has shown conspicuous abilities in inducing apoptosis of multiple tumor cells either in vitro or in vivo [2]. Our previous study have preliminarily shown that CA could induce non-small cell lung cancer (NSCLC) apoptosis and the restraint of Wnt/β-catenin pathway in H1299, YTMLC-90 and A549 cells under the condition of CA [3]. Despite all these evidences, more studies are needed to provide insight into the potential antitumor mechanism involved in the regulation of CA.

Circular RNAs (circRNAs) are a brand-new kind of non-coding RNAs, becoming a hotspot research field [4]. However, circRNAs biological functions in human diseases, especially in cancer pathogenesis, are the tip of the iceberg. Recently, a handful of researches demonstrated that several circular RNAs possess microRNAs (miRNAs) binding sites, and accordingly, serve as competing endogenous RNAs (ceRNAs) to arrest the activity of miRNAs and ultimately regulate their downstream targets [5], [6]. The most well know example is CDR1as, which is highly expressed in brain tissues and can bind to several miR-7 thereby inhibit the function of miR-7, thus regulating various gene expression [7], [8], [9].

MiRNAs are known as another kind of small non-coding RNA molecules, which exert their function in post-transcriptional stages of gene expression by Argonaute 2 (Ago2) dependent manner [10]. Increasing evidence suggests that miRNAs are involved in tumorigenesis and regulate other RNA transcripts by competing for shared miRNAs [11].

Aberrant regulation of Wnt/β-catenin pathway possesses a significant role in tumor initiation, progression, and metastasis of lung cancer. Accumulation of Wnt inhibitor level is related to the reduced Wnt signaling, cell proliferation inhibition, and apoptosis [12]. Recent studies reported that non-coding RNA could also regulate Wnt/β-catenin pathway to alter cell fate. For example, miR-214 and miR-7 could promote Wnt/β-catenin pathway through degradation of Itchy E3 ubiquitin protein ligase (ITCH) in lung cancer [13], [14]. Several reports discovered that ITCH inhibits Wnt/β-catenin pathway and decrease the expression level of downstream targets, such as c-myc, CCND1, β-catenin, finally inhibiting cell proliferation and promoting cell apoptosis [15], [16].

In the present study, we found that hsa_circ_0043256, a novel circular RNA, was upregulated in response to CA treatment in vitro and in vivo. Hsa_circ_0043256 knock-down could weaken the apoptosis triggered by CA and abolish CA ability of Wnt/β-catenin pathway inhibition, suggesting an involvement of hsa_circ_0043256 in cell apoptosis and Wnt/β-catenin pathway suppression induced by CA. We also discovered that hsa_circ_0043256 could function as miRNA sponge to block miR-1252 and promote its target, ITCH. Our results suggested a probable mechanism of hsa_circ_0043256/miR-1252/ITCH axis in promoting CA function. Thus, circular RNA hsa_circ_0043256 could represent a novel insight into CA antitumor effects, opening new doors for drugs discovery and resistance.

Section snippets

Drugs and cell lines

Cinnamaldehyde (104-55-2, sigma, purity >95%, 10 mg) was dissolved in 1 mL dimethylsulfoxide (DMSO) and the stock solution obtained was reserved at-20 °C. Human non-small cell lung cancer cell line (NCI-H460 and A549) were obtained from Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium (GIBCO Life Technologies, USA) supplemented with 10% fetal bovine serum (Tianhang Biotechnology, Hangzhou, China) and 100 U/mL Penicillin-Streptomycin Solution (Life

CA suppresses A549 and NCI-H460 cell proliferation and induces apoptosis

We assessed cell viability after CA treatment. The results showed that cell viability was suppressed approximately in a dose-dependent manner (Fig. 1A), and the IC₅₀ values of A549 and NCI-H460 were 41.27 μg/mL and 35.83 μg/mL, respectively. In addition, cell death clearly raised as the incubation time increased (Fig. 1A). Colony formation assay revealed that cell colonies number was markedly decreased when A549 and NCI-H460 cells were exposed to 40 μg/mL or 80 μg/mL CA (Fig. 1B–C). Moreover,

Discussion

Cinnamomum cassia is known to be one of the 50 fundamental herbs in traditional Chinese medicine. Cinnamaldehyde, as its primary chemical component, have anti-proliferative and anti-invasive effects for cancer cells and low toxicity for normal cells [2], [19]. Herein, we describe a novel CA mechanism mediated by the circular RNA has_circ_0043256, suggesting that this specific circular RNAs might play an important role in mechanism of action of CA.

CircRNAs, with a high degree of stability, are

Conflicts of interest

All Authors have none to declare.

Acknowledgements

This work was funded by Xiamen University, grant number 003(2). The authors are grateful to all study participants. We also thank Professor Li Qinxi at Xiamen University for his assistance with our experiments.

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Circ_0043256 could work as a miR-1252 sponge, which could in turn directly target Itchy E3 ubiquitin protein ligase (ITCH), a vital negative regulator of canonical Wnt signaling. Therefore, cinnamaldehyde suppressed NSCLC cells via modulation of hsa_circRNA_0043256/miR-1252/ITCH axis, which provides new mechanistic insights into the effects of cinnamaldehyde in lung cancer therapy [149]. Gambogic acid, a bioactive compound gotten from Garcinia hanburyi Hook.
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Some other circRNAs have been shown to indirectly regulate the Wnt/β-catenin pathway by sponging miRNAs. For instance, hsa_circ_0043256 promotes the expression of Itchy E3 ubiquitin protein ligase (a negative regulator of the Wnt/β-catenin signaling pathway) by sponging miR-1252, leading to the inactivation of the Wnt/β-catenin signaling pathway.113 Similarly, circ-ZNF124 upregulates the expression of YES proto-oncogene 1 (YES1) by sponging miR-498, resulting in the inactivation of the Wnt/β-catenin signaling pathway.111
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¹: Dr. Fang Tian and Dr. CT Yu contributed equally to this work.

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Cinnamaldehyde induces cell apoptosis mediated by a novel circular RNA hsa_circ_0043256 in non-small cell lung cancer

Highlights

Abstract

Introduction

Section snippets

Drugs and cell lines

CA suppresses A549 and NCI-H460 cell proliferation and induces apoptosis

Discussion

Conflicts of interest

Acknowledgements

Food Chem. Toxicol.

Int. J. Biochem. Cell Biol.

Cell Signal

Pharmacol. Res.

Neurobiol. Aging

Biochem. biophysical Res. Commun.

Cinnamaldehyde analogues as potential therapeutic agents

Mini Rev. Med. Chem.

Circular RNAs are a large class of animal RNAs with regulatory potency

Nature

Circular RNAs: diversity of form and function

RNA (New York, N.Y.)

Natural RNA circles function as efficient microRNA sponges

Nature

The circular RNA Cdr1as promotes myocardial infarction by mediating the regulation of miR-7a on its target genes expression

PLoS One