Role and mechanism of miR-4778-3p and its targets NR2C2 and Med19 in cervical cancer radioresistance

https://doi.org/10.1016/j.bbrc.2018.11.110Get rights and content

Abstract

The aim of this study was to investigate the effect of miR-4778-3p on the radiosensitivity of cervical cancer cells and to elucidate the underlying mechanism. Tissue samples were collected from eight patients with cervical cancer prior to chemoradiotherapy. MicroRNA chip analyses, RT-PCR, gene transfection, CCK8, wound healing and Transwell assays, colony-forming assay, western blot, and the Dual-Luciferase Reporter Assay System were used to evaluate the role of miR-4778-3p in cervical cancer radiosensitivity and its relationships with target molecules NR2C2 and Med19. Thirty-two differentially expressed miRNA molecules (fold-change > 2; p < 0.05) associated with cervical cancer radioresistance were identified. The expression of miR-4778-3p was significantly lower in recurrent or metastatic patients than in control subjects. In vitro studies using radioresistant HeLa and SiHa cervical cancer cell lines showed that miR-4778-3p upregulation significantly inhibited cell proliferation, invasiveness, and migration after irradiation. There was also a significant increase in apoptosis and a significant decrease in the proportion of cells at the G2/M phase. Further, miR-4778-3p upregulation led to increased expression of apoptosis-related molecules, such as Bax, Caspase-3, Caspase-8, and Caspase-9. Reporter gene assays showed that miR-4778-3p bound specifically to NR2C2 and Med19 and negatively regulated their expression. Thus, miR-4778-3p reduces the vitality, proliferation, and migration of radioresistant cervical cancer cells and may regulate the radiosensitivity of cervical cancer by targeting and regulating NR2C2 and Med19 expression.

Introduction

Radiotherapy is an important treatment modality for locally advanced cervical cancer. However, cancer recurrence or metastasis still occurs in approximately 30% of radiotherapy-treated patients with locally advanced cervical cancer [1,2]. A reason for radiotherapy failure may be tumor radioresistance. The mechanisms underlying tumor radioresistance are complex. Currently known factors contributing to resistance include aberrant DNA damage and repair, hypoxia or formation of hypoxia-inducing factors, the presence of cancer stem cells, and production of relevant microRNAs. Understanding the underlying mechanisms and molecular networks involved in tumor radioresistance is important to improve radiotherapy treatment efficacy.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs found in eukaryotes that play a major role in gene silencing. Studies have shown that abnormal miRNA expression is closely related to chemoresistance, recurrence and metastases, and poor prognoses in patients with lung adenocarcinoma, breast cancer, and pancreatic cancer [[3], [4], [5]]. Lui et al. [6] compared differences in miRNA expression of 166 samples from normal and cancerous cervical tissues, as well as miR-143 and miR-21 expression in 29 matched samples of cancerous/normal cervical tissues by northern blot. They found that miR-21 was expressed in all cancerous tissues, but rarely expressed in normal tissues. Conversely, miR-143 was mostly expressed in normal tissue; however, its expression was low or absent in cancerous tissues. These results suggest that miR-143 exerts an inhibitory effect against cervical cancer, whereas miR-21 promotes cervical cancer growth. Wang et al. [7] applied deep sequencing technology to study the miRNA expression profile of Caski cervical cancer cells. Of 46 miRNAs, the expression levels of miR-21, miR-24, miR-27a, and miR-205 were found to be comparatively high. Furthermore, the growth-inhibitory effects of miR-143 and miR-145 and the growth-promoting effects of miR-146a against HeLa cervical cancer cells were validated miR-143, miR-145, and miR-146a expression in HeLa cells. Other studies using miRNA chip analyses or deep sequencing of cervical cancer tissue or cells lines have shown reduced expression of miR-203, miR-23a/b, and miR-34a, and increased expression of miR-15 and miR-16-1, in cervical cancer [[8], [9], [10]]. However, few reports discuss the relationships between aberrant miRNA expression and the radiosensitivity or radioresistance of cervical cancer.

This study screened for differentially expressed miRNAs before and after cervical cancer tissue irradiation. A radioresistant cervical cancer cell line was also successfully established. The functions and mechanisms of miRNAs were evaluated in the parent and radioresistant cervical cancer cell lines. Our study provides a new direction and basis for predicting cervical cancer prognosis following radiotherapy, reversing cervical cancer radioresistance, and increasing the cure rate of cervical cancer in clinical settings.

Section snippets

Patient selection and human tissues

Eight tissue samples were acquired from patients with cervical cancer before radical radiotherapy in the Department of Radiation Oncology, Xijing Hospital, Fourth Military Medical University (FMMU). All patients consented to a 3-years follow-up investigation. Three patients had good local control and no evidence of recurrence or metastasis; their tissue samples were grouped into control (radiation-sensitive). The remaining five tissue samples were allocated to the experimental group

Decreased miR-4778-3p expression in tumor tissue of recurrent or metastatic patients and in radioresistant cell lines

miRNA chip results showed that 32 miRNA molecules had differential expression (fold-change > 2). Compared with expression in radiosensitive tissue from control patients, 10 miRNA molecules were upregulated and 9 were downregulated in tissue from patients with recurrence. Further, 9 miRNA molecules were upregulated and 4 were downregulated in tissue from patients with metastasis. Specifically, miR-4778-3p expression was significantly downregulated in both recurrent and metastatic patients.

Discussion

One of the most prevalent treatment modalities for locally advanced cervical cancer is radiotherapy combined with concurrent chemotherapy, which has greatly improved tumor control and cure rate [[11], [12], [13]]. However, many patients still experience recurrence or metastasis. Radioresistance may be a key reason underlying treatment failure. Therefore, we aimed to improve cervical cancer radiosensitivity, thereby increasing the clinical efficacy of radiotherapy [14]. Present data on miRNAs

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

None.

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