miR-598 inhibits metastasis in colorectal cancer by suppressing JAG1/Notch2 pathway stimulating EMT

https://doi.org/10.1016/j.yexcr.2017.01.022Get rights and content

Highlights

  • miR-598 expression in CRC tissues was significantly lower than corresponding adjacent noncancerous tissues.

  • miR-598 suppresses CRC cells metastasis and EMT.

  • JAG1 is direct target of miR-598.

  • miR-598 inhibits metastasis in colorectal cancer by suppressing JAG1/Notch2 pathway stimulating EMT.

Abstract

MicroRNAs (miRNAs) are a class of endogenous, evolutionarily conserved small non-coding RNA molecules that mediate the posttranscriptional process of target gene, leading to translational repression or degradation of target mRNAs. A series of studies have indicated that miRNAs play an important role in tumor initiation, development and progression. In this study, we found that down regulation of miR-598 was a frequent event in CRC tissues compared to the paracarcinoma tissues. And the study demonstrated that miR-598 was implicated in CRC metastasis. Transwell migration assay revealed that elevated miR-598 expression reduces CRC cell migration. Moreover, our study showed that suppression of miR-598 expression induces CRC cell epithelialmesenchymal transition(EMT) and overexpression of miR-598 inhibits CRC cell EMT. In addition, bioinformatics target prediction identified JAG1 as a putative target of miR-598. Knockdown of miR-598 was shown to upregulate JAG1 expression. Furthermore, overexpression of miR-598 suppressed the expression of JAG1. Consistent results were also obtained when the regulation of JAG1 expression by miR-598 was further specified in CRC tissues. Moreover, overexpression of JAG1 induces epithelialmesenchymal transition(EMT) and promotes the metastasis of CRC cells. Decreased Notch2 expression suppresses CRC cells metastasis and EMT. Together, these results indicate that miR-598 is a novel regulator of colorectal cancer metastasis. Our data suggest miR-598 is implicated in regulating Epithelial-mesenchymal transitions by directly suppressing its downstream target gene JAG1 to inactivate Notch signaling pathway.

Introduction

Colorectal cancer (CRC) is one of the most common, incident and deadly malignancies in the world [1], with almost 1.23 million new cases and 0.6 million deaths annually [2]. Invasion and metastasis are the major reasons of high mortality in patients with CRC worldwide, yet the underlying molecular mechanisms that regulate the invasion-metastasis cascade are not well studied [3]. Further understanding of the metastatic mechanisms of CRC may greatly reduce the mortality and improve the poor survival of CRC patients.

MicroRNAs (miRNAs) are endogenous, evolutionarily conserved small non-coding RNA molecules that mediate the post-transcriptional process of target gene, leading to translational repression or degradation of target mRNAs [4]. It is well known that miRNAs are involved in various physiological and pathological process [5], [6], [7], [8], [9], [10], [11], [12], [13]. A series of studies have indicated that dysregulated expression of miRNAs has been potentially associated with cancers and miRNAs play an important role in tumor initiation, development and progression [14].

Metastasis is a complicated process influenced by many cell-intrinsic and extrinsic factors. In recent years, studies have found that miRNAs are related to the carcinogenesis and metastasis of CRC. Evidences support that miRNAs play an important role in metastasis of CRC [15]. Furthermore, studies have indicated miRNAs participate in the metastasis of CRC by influencing cancer stem-cell biology, angiogenesis, epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) or drug resistance [16]. Abnormal expression of specific miRNAs has been thought as a new insight into molecular mechanism underlying metastatic procession of CRC, they also may provide new therapeutic targets for CRC patients [17], [18], [19].

In this study, microarray-based strategy was applied to identify differentially expressed miRNAs in CRC by comparing miRNAs profile between CRC tissues and para-carcinoma tissues from four CRC patients. miR-598 was highly downregulated in colorectal cancer tissues. And we found that the low expression of miR-598 was related to colon cancer metastasis and poor prognosis. Next, we will try to characterize its function in CRC metastasis and elucidate the mechanism.

Section snippets

Cell lines and cell culture

Human CRC cell lines HT-29, HCT116, SW620 and normal colon cell line NCM-460 were purchased from American Type Culture Collection and were maintained in Dulbecco's Modified Eagle medium (DMEM; GibcoBRL, Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum, 100 U/ml penicillin sodium and 100 mg/ml streptomycin sulfate in humidified 5% CO2 at 37 °C.

Cell transfection and infection

Transfection of miR-598 mimics, miR-598 inhibitor, negative control, Notch2-siRNA and JAG1 plasmid was performed using the Lipofectamine

miRNA-598 is downregulated in colorectal cancer tissues compared to the corresponding normal

To investigate the role of miRNAs in CRC, miRNAs expression was profiled in CRC tissues and paired para-carcinoma normal tissues. Then cluster analysis was performed of the miRNAs on four CRC tissues and corresponding adjacent noncancerous tissues (Fig. 1A) and we found miR-598 was downregulated in the CRC tissues. In order to validate the microarray platform, we used quantitative reverse transcription PCR (qRT-PCR) to analyze the same RNA samples that were used for the microarray. And we

Discussion

miRNAs are a class of critical gene regulators at post-transcriptional level which play an important role in carcinogenesis and metastasis [21]. In this study, miR-598 was found highly decreased in four CRC patients by miRNAs profile and qRT-PCR. It was also found that expression of miR-598 in three CRC cell lines (HT-29, HCT116, SW620) was lower than that in normal colon cell line NCM-460.

Though recently some researchers have discovered that miR-598 participate in carcinogenesis, development

Funding

“The National Natural Science Foundation of China (No. 81201913 and No. 81672335)”, “The Natural Science Foundation of Shanghai (No. 13ZR1430800)”, “The Research Foundation of Shanghai Jiao Tong University (No. YG2016ZD10)” and “The project of Cancer Research Center, Shanghai Xuhui Central Hospital, Shanghai Clinical Center (No. CCR2011003)”.

Conflicts of interest

There are no conflicts of interest in this work.

Acknowledgments

This project was supported in part by grants from “The National Natural Science Foundation of China (No. 81201913 and No. 81672335)”, “The Natural Science Foundation of Shanghai (No. 13ZR1430800)”, “The Research Foundation of Shanghai Jiao Tong University (No. YG2016ZD10)” and “The project of Cancer Research Center, Shanghai Xuhui Central Hospital, Shanghai Clinical Center (No. CCR2011003)”.

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    Both these authors contributed equally to this work.

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