MicroRNA-324-3p regulates nasopharyngeal carcinoma radioresistance by directly targeting WNT2B

doi:10.1016/j.ejca.2013.03.001

European Journal of Cancer

Volume 49, Issue 11, July 2013, Pages 2596-2607

https://doi.org/10.1016/j.ejca.2013.03.001 Get rights and content

Abstract

Purpose

Radioresistance severely restricts the clinical treatment of nasopharyngeal carcinoma (NPC). Accumulating evidence demonstrates that aberrant expression of microRNAs (miRNAs) contributes to cancer progression and sensitivity to radiation. Therefore, we aimed to identify miRNAs associated with radioresistance in NPC.

Methods

Aberrant miRNA-324-3p expression in NPC CNE-2 cells with radioresistance (CNE-2-Rs), compared to its parental cells, was screened by high-throughput sequencing technology and determined by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) analysis. Bioinformatic analysis was used to predict the downstream target genes of miRNA-324-3p. Then, functional and mechanical analyses of miRNA-324-3p in NPC radioresistance were performed by overexpression and down-regulation of miRNA-324-3p in CNE-2-Rs cells and its parental cells. Finally, the clinical significance of miRNA-324-3p and WNT2B was investigated in NPC tissues.

Results

Our data reveal that the expression of miRNA-324-3p is significantly decreased in CNE-2-Rs cells compared to its parental cells, and WNT2B is predicted to be the downstream target of miRNA-324-3p. Both overexpression and down-regulation of miRNA-324-3p following irradiation result in radiosensitivity alterations and protein changes of WNT2B signalling pathway in CNE-2-Rs cells and its parental cells. Importantly, down-regulation of miRNA-324-3p and up-regulation of WNT2B are significantly correlated with advanced clinical stages of NPC and this inverse expression pattern is also observed in NPC tissues before and after irradiation.

Conclusions

The present study reveals that miRNA-324-3p contributes to the radioresistance of NPC by regulating the WNT2B signalling pathway. Both miRNA-324-3p and WNT2B are potential biomarkers for radioresistance in NPC, which may serve as valuable targets for reversing radioresistance in the management of NPC.

Introduction

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated cancer, is highly prevalent in Southeast Asia, especially in the Cantonese region located around Guangzhou in China. Radiotherapy is the mainstay treatment for NPC patients.¹ Although detection of the tumour by imaging and advanced radiotherapy technique have led to an improvement in the management and treatment of NPC, patient outcomes remains unsatisfactory over the last few decades.² Radioresistance is the primary reason for the dismal prognosis in NPC treatment.³ However, the molecular mechanism of NPC radioresistance is still poorly understood. It is of great clinical value to further understand the molecular mechanism of NPC radioresistance and to identify valuable predictive markers as well as novel therapeutic strategies.

MicroRNAs (miRNAs) are a class of endogenous, small non-coding RNA molecules that completely or incompletely bind to target mRNAs, primarily at the 3′-untranslated region (3′-UTR) and/or 5′-UTR of target genes, to block translation or lead to target mRNA degradation, resulting in the inhibited expression of various target genes.4, 5 Rapidly increasing evidence reveals that differential expression of miRNAs contributes to pathogenesis and tumourigenesis in a variety of human malignancies, such as breast, lung, prostate and colorectal cancers.⁶ miRNAs can act as onco-miRNAs or anti-onco-miRNAs depending on their potential target genes.⁷ Previous studies indicate that miRNAs are involved in many important cancer-associated behaviours, including cancer-related inflammation,⁸ metastasis,⁹ angiogenesis,¹⁰ cancer stem cell property¹¹ and chemoresistant phenotype.¹² Therefore, miRNAs represent a novel therapeutic strategy for the prevention and management of cancer. Recent reports indicate that some miRNAs, such as miRNA-205,¹³ miRNA-21,¹⁴ and let7,¹⁵ are involved in the development of radioresistance in human cancers. However, the function of miRNAs associated with radioresistance in NPC remains to be investigated.

In the current study, NPC CNE-2 cells acquired radioresistance (CNE-2-Rs) by exposure to gradually increasing doses of irradiation (IR). The expression profiles of miRNAs associated with radioresistance are obtained using high-throughput sequencing technology. Furthermore, the quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) results demonstrate that miRNA-324-3p is significantly decreased in CNE-2-Rs compared with its parental CNE-2 cells, and WNT2B is predicted to be the downstream target of miRNA-324-3p. Both ectopic expression and inhibited expression of miRNA-324-3p result in radiosensitivity alterations in CNE-2-Rs and its parental CNE-2 cells, followed by inverse changes in the expression of WNT2B. Significantly, down-regulation of miRNA-324-3p and up-regulation of WNT2B are correlated with advanced clinical stages of NPC and this inverse expression pattern is also observed in NPC tissues before and after IR exposure.

Section snippets

Cell culture and the establishment of radioresistant CNE-2-Rs cells

The poorly differentiated NPC cell line CNE-2 was derived at the Central Experiment Laboratory of the Xiangya Medical School, Central South University, Changsha, China. Exponentially growing CNE-2 cells were seeded at a density of 1 × 10⁵ cells per T25 flask and initially subjected to 2 Gy of IR. The CNE-2 cells were cultured and passaged twice following the initial radiation and were then exposed to an additional 2 Gy of IR. The surviving cells were then treated as described above and subjected to

Radioresistant CNE-2-Rs cells are established and the radioresistant capacity of CNE-2-Rs is validated

To obtain miRNAs associated with radioresistance in NPC, radioresistant NPC cells were initially established by exposing CNE-2 cells to gradually increasing doses of IR, and the radioresistant capacity of CNE-2-Rs was then validated. Our data demonstrated that the survival rates of CNE-2-Rs cells assayed following different doses of IR (2, 4, 6 and 8 Gy) and at different time points (1, 2, 3, 4 and 5 days) were higher than that of its parental CNE-2 cells (Fig. 1A). A colony-forming assay

Discussion

Acquired and intrinsic radioresistance is a challenging obstacle in NPC clinical management. The acquisition of radioresistance is a complicated process, which involves the overexpression of DNA repair proteins,20, 21 aberrant activation of multiple signalling pathways,22, 23, 24 angiogenesis,25, 26 cancer stem cells²⁷ and autophagy.28, 29 Although some achievements have been made in previous studies, the exact molecular mechanisms underlying radioresistance remains to be elucidated. Functional

Conflict of interest statement

None declared.

Acknowledgements

Grants were provided by the National Natural Science Foundation of China (No. 81202128 and No. 81272974), the Research Fund for the Doctoral Program of Higher Education of China (No. 20120162120049), and the Freedom Explore Program of Central South University (No. 2012QNZT099).

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Previous studies have reported that miRNA expression is associated with several chemotherapy responses and resistance events in esophageal cancer [14], Oral squamous cell carcinoma [25], breast cancer [19], and lung cancer [26]. In another report on nasopharyngeal carcinoma, miR-324-3p and miR-519d are deregulated by inhibiting gene translation targeting WNT2B [27] and PDRG1 [28] toward radiotherapy sensitivity. In addition, mIR-29c is known to be jointly sensitive to cisplatin-based radiotherapy and chemotherapy [18,29,30].
Nasopharyngeal carcinoma (NPC) is endemic cancer in Southeast Asia with a relatively poor prognosis. Chemoradiotherapy is a primary treatment that advantages certain patients, particularly in the early stages. New predictive and prognostic biomarkers are required to guide and select the best treatment.
To evaluate the circulation expression profile of microRNAs (miRNAs) associated with responses to chemoradiotherapy in nasopharyngeal carcinoma.
Peripheral blood from 17 patients was collected before and after chemotherapy and radiotherapy. Differential expression circulating miRNAs were analyzed using microRNA Cancer Panels and were compared among patients with complete responses. Differential expression analysis using GenEx 7 Multid, statistic represented by GraphPad Prism 9. Alterations mechanism signaling pathways and biological function using IPA (Ingenuity Pathways Analysis).
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qPCR profiling in circulating blood compared before and after chemoradiotherapy in nasopharyngeal carcinoma can identify pathways involved in treatment responses. miR-483-5p, miR-584-5p, miR-122-5p, miR-7-5p, miR-150-5p, miR-421, miR-133a-3p, miR-18a-5p, miR-106b-3p, miR-339-5p are differentially regulated after chemoradiotherapy in NPC.
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¹: These authors are contributed equally to this work.

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MicroRNA-324-3p regulates nasopharyngeal carcinoma radioresistance by directly targeting WNT2B

Abstract

Purpose

Methods

Results

Conclusions

Introduction

Section snippets

Cell culture and the establishment of radioresistant CNE-2-Rs cells

Radioresistant CNE-2-Rs cells are established and the radioresistant capacity of CNE-2-Rs is validated

Discussion

Conflict of interest statement

Acknowledgements

Expression of Epstein–Barr virus-encoded proteins in nasopharyngeal carcinoma

Int J Cancer

Cancer statistics, 2010

CA Cancer J Clin

Treatment outcomes and late complications of 849 patients with nasopharyngeal carcinoma treated with radiotherapy alone

Int J Radiat Oncol Biol Phys

MicroRNAs contribute to the maintenance of cell-type-specific physiological characteristics: miR-192 targets Na+/K+-ATPase beta1

Nucleic Acids Res

MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation

Mol Cell

MicroRNA control of signal transduction

Nat Rev Mol Cell Biol

Causes and consequences of microRNA dysregulation in cancer

Nat Rev Genet

Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells

Oncogene

MicroRNA-21 suppression impedes medulloblastoma cell migration

Eur J Cancer

Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in oral cancer

Br J Cancer

Re-expression of miR-21 contributes to migration and invasion by inducing epithelial–mesenchymal transition consistent with cancer stem cell characteristics in MCF-7 cells

Mol Cell Biochem

Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model

Am J Pathol

MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN

Cell Cycle

MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN

Mol Cell Biochem

Lin28-let7 modulates radiosensitivity of human cancer cells with activation of K-Ras

Int J Radiat Oncol Biol Phys

Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics

Cancer Res

Downregulation of EphA2 expression suppresses the growth and metastasis in squamous-cell carcinoma of the head and neck in vitro and in vivo

J Cancer Res Clin Oncol

Elevated expression of HMGB1 in squamous-cell carcinoma of the head and neck and its clinical significance

Eur J Cancer

MicroRNAs contribute to the maintenance of cell-type-specific physiological characteristics: miR-192 targets Na⁺/K⁺-ATPase beta1