Original article
Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 techniqueLa radiorésistance au cancer du sein peut être surmontée par le knock-out du gène de l’ostéopontine avec la technique CRISPR/Cas9☆

https://doi.org/10.1016/j.canrad.2020.08.048Get rights and content

Abstract

Purpose

Osteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized.

Material and methods

We used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed.

Results

A significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability.

Conclusion

Our results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.

Résumé

Objectif de l’étude

L’ostéopontine est une phosphoglycoprotéine ayant de multiples rôles physiologiques et pathologiques. L’expression élevée d’ ostéopontine favorise le comportement offensif et constitue un facteur depronostic défavorable en termes de survie des patients. L’expression élevée d’ostéopontine est un facteur de radiorésistance. L’intérêt du blocage de son gène par le système CRISPR/Cas9 en association avec une irradiation a été étudié.

Matériel et méthodes

La technique CRISPR/Cas9 a été employée pour bloquer de manière permanente le gène d’ostéopontine dans la ligne cellulaire MDA-MB-231. Après la transfection, les cellules ont été exposées à une irradiation de 2 Gy et les changements du niveau de l’expression de mRNA OPN, de l’apoptose et de la survie des cellules ont été étudiés.

Résultats

Une baisse remarquable de l’expression d’ostéopontine, seule ou accompagnée d’irradiation, a été constatée. Le blocage permanent du gène (Knock out) en association avec une irradiation a augmenté le niveau de l’apoptose. La survie de la cellule après le blocage permanent du gène d’ostéopontine a baissé. Le blocage permanent du gène (Knock out) en association avec une irradiation a abouti à une baisse plus grave de la survie des cellules.

Conclusion

Les résultats ont montré qu’après le blocage permanent du gène d’ostéopontine (Knock out), les cellules MDA-MB-231 ont retrouvé une radiosensibilité significative. Par conséquent le blocage du gène d’OPN en combinaison avec la radiothérapie (classique) pourra devenir un objectif thérapeutique efficace.

Introduction

Breast cancer is one of the most common malignancies in women all over the world. In 2018, the incidence of breast cancer is estimated to be at 2,099,600 (11.6% of the total cancer cases); meanwhile, the mortality is expected; at least, 627,000 [1], [2].

Fine needle aspiration (FNA), core needle biopsy, and excisional biopsy, as the standard diagnostic methods seem not to be able to predict the inherence of invasion accurately. It seems the assessment of some validated tumor biomarkers such as Ki-67, p53, BRCA1, BRCA2, Cyclin D1, Cyclin E, HER2, and osteopontin (OPN) may be useful in detecting tumor invasiveness in the early stages, and selection the most appropriate treatment [3], [4], [5], [6], [7], [8].

OPN gene is located on the long arm of chromosome 4 (4p22.13), encoding a member of Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) [9]. Three main human OPN variants, OPN-a (full type), OPN-b (with deletion of exon 5), and OPN-c (with deletion of exon 4) are the results of different RNA splicing. Also, OPN has two secreted (sOPN) and intracellular (iOPN) isoforms. It has been shown that both iOPN and sOPN are just two different translational products of a single full-length OPN mRNA (OPN-a) [10]. OPN, a multifunctional molecule, is produced in different normal cells and tissues such as osteocytes and osteoblasts [11]. Moreover, OPN is known to have irrefutable roles in the assessment of the prognosis, diagnosis, and treatment of a wide range of diseases, including allergy, asthma, autoimmune diseases, cancers, colitis, and inflammatory diseases [12], [13], [14]. High levels of OPN mRNA transcription or its related protein synthesis are found in breast cancer. Furthermore, overexpression of OPN protein associates with mammary calcification [15]. Using immunohistochemistry, it is showed that elevated levels of tumor OPN are an indication of the poor prognosis in breast cancer [16]. According to the various well-documented roles of OPN on the important aspects of tumor behavior, including survival, growth, and development in different tumors such as lung, prostate, pancreas, and breast cancers, it may be considered as a prognostic biomarker and a therapeutic goal in the future [17], [18], [19], [20].

Thereafter, it seems that inducing some highly selective variations in the genome of targeted tumor cells, carrying undesired overexpressed genes, including OPN, could be an efficient strategy to subside the invasive behavior of tumors. Notwithstanding the former technologies of gene-editing like designer zinc fingers (ZFs), transcription activator-like effectors (TALEs), and homing meganucleases, the necessity of other effective and precise methods which are measurable, cost-benefit, and user-friendly is still noteworthy [21]. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is introduced as a new interesting gene-editing tool, providing the opportunity to target the abnormal gene sequences directly [22]. Some former studies have revealed that the gene knockout using CRISPR/Cas9 may be a more efficient method. In fact, a couple of advantages may be counted for CRISPR/Cas9 system, including the absolute gene silencing [23].

Therefore, in this study we used CRISPR-Cas9 system to knock out the OPN gene in the cells (MDA-MB-231) in both irradiated and control groups. To our best knowledge, any study has been done before applying CRISPR/Cas9 technique and simultaneously irradiation to inhibit OPN gene.

Section snippets

Material and methods

The experimental groups used in this study were:

  • •

    non-irradiated groups including: NC: did not receive vector; pCas: receive vector without gRNA; and OPN KO: OPN gene was knockout by CRISPR/Cas9 technique;

  • •

    irradiated groups including: NC: did not receive vector; pCas: receive vector without gRNA; and OPN KO: OPN gene was knockout by CRISPR/Cas9 technique.

AJUMS's Human Ethics Committee approved all protocols (IR.AJUMS.REC.1396.1009).

gRNA cloning in GE10018 plasmid

As stated ealier, to confirm gRNA cloning the PCR products were run on the agarose gel. We observed a band with a length of 460 bp on the agarose gel (Fig. 1 B). Thereafter, to ensure the correct gRNA localization between the U6 promoter and the gRNA scaffold, a sample was examined and approved by Macrogen (South Korea) (Fig. 1C).

Transfection of MDA-MB-231 breast cancer cell line with OPN gRNA

The transfection of cancer cells was performed by TurboFect reagent. It was successfully done by the reagent's efficiency. After 24 h, we checked the efficiency of

Discussion

In this study, we used the CRISPR/Cas9 technique to knockout the OPN gene in breast cancer cell line MDA-MB-231 and then irradiated with a dose of the conventional 2 Gy. Our results showed that the synergistic interaction of gene knockout and a dose of 2 Gy of radiation had a significant suppression effect. It led to decreased cell viability and gene expression; however, the apoptosis had statistically increased in the cancer cell line. Another study has shown that the OPN expression in the

Conclusion

In this study, we knocked out the OPN gene by the CRISPR technique in the MDA-MB-231 breast cancer cell line, firstly. Then three populations of each NC, pCas, and OPN KO groups were irradiated by a conventional dose of 2 Gy. Our results illustrated that after knocking out the OPN gene, the cells showed a higher radiosensitivity. The mRNA expression levels and the cell viability decreased, and the apoptosis induction increased significantly. As we know, no similar work has been done yet. We hope

Funding

This project granted by the Ahvaz Jundishapur University of Medical Sciences (AJUMS, Ahvaz, Iran) (grant No. U-96164). The study designed for the MSc thesis of Rahil Ghanbarnasab Behbahani.

Disclosure of interest

The presented data in this study are available for all readers of this journal.

Acknowledgements

The authors express their gratitude to the help and general support of the Ahvaz Jundishapur University of Medical Sciences.

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    The presented manuscript is not under review or consideration for publication elsewhere.

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