MiR-9 promotes synovial sarcoma cell migration and invasion by directly targeting CDH1

https://doi.org/10.1016/j.biocel.2019.04.001Get rights and content

Abstract

Background

Invasion and metastasis of synovial sarcoma is the leading cause of death in patients. Epithelial mesenchymal transition (EMT) accelerates tumor cell invasion and metastasis. MiR-9 promotes tumor metastasis by inducing EMT. However, the role of miR-9 in synovial sarcoma is still not clear.

Methods

Overexpression or knockdown of miR-9 in human synovial sarcoma (HSS) cell lines was carried out by miR-9 mimics or miR-9 inhibitors transfection. Cell proliferation, apoptosis, migration and invasion were detected using MTS and colony formation assays, flow cytometry, wound healing and transwell assays, respectively. Luciferase reporter assay was applied to study the interaction between miR-9 and CDH1. Nude mice xenograft model was established, and immunohistochemistry staining assessed Ki-67 level. The related mRNA and protein expression levels were evaluated by qRT-PCR and Western blotting.

Results

The bioinformatics analyses and luciferase reporter assay showed that miR-9 can target CDH1 3′-UTR. Moreover, miR-9 could induce EMT of HSS cells via targeting CDH1. The negative regulation of miR-9 on CDH1 expression was also confirmed in a mouse xenograft model of synovial sarcoma. Furthermore, miR-9 was observed to induce HSS cell proliferation, migration and invasion and inhibit apoptosis. MAPK/ERK and Wnt/β-catenin signal pathways were activated by the miR-9 overexpression in HSS cells, and then further enhancing tumorigenesis of HSS, which was further confirmed in the mouse model.

Conclusion

MiR-9 induces EMT by targeting CDH1, and activates MAPK/ERK and Wnt/β-catenin signal pathways, thus promoting HSS tumorigenesis.

Introduction

Synovial sarcoma (SS) is a kind of soft tissue malignant sarcoma that features uncertain tissueorigins. It accounts for about 5–10% of soft tissue sarcomas, and may strike all age groups of people, yet with middle-aged and young people between 15 and 35 years old holding the largest part (Krieg et al., 2011). Although synovial sarcoma has a high malignancy and yearly increasing incidence, the comprehensive treatment dominated by surgery and supplemented by radiotherapy and chemotherapy in recent years has to a certain extent improved the survival rate of patients. However, the 5-year survival rate is still only about 50% (Stacchiotti and Van Tine, 2018). Synovial sarcoma has a high rate of metastasis with the metastasis rate 2 years after surgery being up to 40% (Krieg et al., 2011; Stacchiotti and Van Tine, 2018). Therefore, to explore the molecular biological mechanisms that affect the metastasis and recurrence of synovial sarcoma is of great significance to evaluate the prognosis of patients and molecular targeted therapy.

Epithelial mesenchymal transition (EMT) refers to the process that the epithelia with polarity in some special physiological or pathological conditions lose their polarity and are converted into the interstitial cells that have mobility and can move freely between cellular matrixes (Nistico et al., 2012). EMT is closely related to tumor invasion and metastasis, and is one of the hotspots in the research of tumor metastasis mechanism in recent years. E-cadherin (also known as CDH1), N-cadherin and β-catenin are the common markers to determine the occurrence of EMT. These proteins are involved in cell differentiation and play an important role in maintaining cell morphology and regulating intercellular adhesion (Nistico et al., 2012; Zeisberg and Neilson, 2009). E-cadherin, an EMT-related protein, is involved in epithelial adhesion and desmosomal junction, which play an important role in maintaining cell morphology, cell motility and cell adhesion (Zeisberg and Neilson, 2009; Onder et al., 2008). CDH1 is an important intercellular adhesion molecule, with the role of inhibiting tumor metastasis (Petrova et al., 2016). The adhesion function of cadherin/catenin complex depends on its integrity, so any change of its component members in quality and amount may lead to complex dysfunction, thus resulting into cell detachment, invasion and metastasis. The absence of CDH1 is an important landmark change of EMT, and a prerequisite for epithelial tumor cells possessing invasion abilities. (Saito et al. (2004) found that the down-regulation of CDH1 was related to the loss or absence of synovial sarcoma-like epithelial differentiation. The loss of CDH1 allows intercellular adhesion to loosen and thus makes cells migrate more easily to result in distant metastases. In epithelial tumors, CDH1 gene has become a new tumor suppressor gene, so that its mutation and loss are one of the key molecular events in cancer progression and metastasis.

MicroRNA is a kind of small, non-coding RNA containing about 22 nucleotide sequences and widely spreading in animals, plants and some viruses (Ma, 2016). Its main functions include silencing RNA and conducting post-transcriptional regulation of gene expression. Recent studies showed that miRNAs played important roles in tumorigenesis and metastasis, which provided a new idea for the prevention and treatment of tumor recurrence and metastasis (Jiang et al., 2015; Colden et al., 2017). (Sarver et al. (2010) suggested that the specific expression of miR-183 family might be associated with synovial sarcoma metastasis and recurrence, and the miR-183 family might be a potential carcinogenic factor during the formation of synovial sarcoma. As a member of the miRNA family, miR-9 has been proven associated with the development, growth, and metastasis of multiple tumors. For example, in studying the bronchial squamous epithelium carcinogenesis, (Mascaux et al. (2009) found that miR-9 had varied expressions at different stages of lesion. (Luo et al. (2009) confirmed with miRNA microarray and qRT-PCR that miR-9 had a low expression in gastric cancer tissues and SGC7901 cell line. (Yanaihara et al. (2016) discovered that miR-9 was overly expressed in ovarian clear cell carcinoma, which influenced tumor metastasis by inducing epithelial-mesenchymal transition with targeting CDH1. In addition, miR-9 has been shown to be significantly up-regulated in synovial sarcomas (over 30 times) (Yu et al., 2016), but the role of miR-9 in synovial sarcoma is still unclear. This study proposed that miR-9 regulates the EMT of synovial sarcoma cells by directly binding to CDH1, thus regulating the incidence and metastasis of synovial sarcomas.

Section snippets

Cell culture

The human synovial sarcoma (HSS) cell lines SW982 and HS-SY-II (ATCC; USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum(FBS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO2and 100% humidity.

Cell transfection

MiR-9 mimics (5′-UCUUUGGUUAUCUAGCUGUAUGA-3′) and inhibitor (5′-UCAUACAGCUAGAUAACCAAAGA-3′) were purchased from GenePharma (Shanghai, China). Overexpression or knockdown of miR-9 was carried out by transfection with the miR-9 mimics or

MiR-9 inhibits the CDH1 expression by targeting the 3′ untranslated region

In order to investigate the relationship between miR-9 and CDH1, overexpression or knockdown of miR-9 was carried out by transfection with the miR-9 mimics or miR-9 inhibitor, respectively. As shown in Fig. 1A, the expression of miR-9 was significantly up-regulated when transfected with miR-9 mimics, while the expression was dramatically down-regulated by the transfection of miR-9 inhibitor in both SW982 and HS-SY-II cells (p < 0.05, versus the NC group) (Fig. 1A). In addition, the expression

Discussion

One of the most important features of malignant tumor is its ability of invasion and metastasis, which is the leading cause of death in patients with synovial sarcoma. Although the survival rate has witnessed improvement, metastasis is still an important cause of death in patients with synovial sarcoma. Therefore, looking for an effective therapeutic schedule to control tumor metastasis will be the focus and difficulty of synovial sarcoma treatment.

Currently, it has been clear that the

Conclusions

Taken together, miR-9 plays a critical role in the tumorigenesis of HSS via targeting CDH1. MiR-9 can induce EMT by targeting CDH1, thus promoting tumor invasion and metastasis. Furthermore, miR-9 was found to activate MAPK/ERK and Wnt/β-catenin signal pathways in HSS cells, thereby enhance the tumorigenesis of HSS in a mouse model. This study is conducive for us to better understanding the pathogenesis of HSS. MiR-9 may serve as a new and important molecular target for the treatment of

Funding

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

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

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