Elsevier

Biomedicine & Pharmacotherapy

Volume 74, August 2015, Pages 138-144
Biomedicine & Pharmacotherapy

Original article
miRNA-144 suppresses proliferation and migration of colorectal cancer cells through GSPT1

https://doi.org/10.1016/j.biopha.2015.08.006Get rights and content

Abstract

MicroRNAs play a key role in carcinogenesis or tumor progression, which negatively and posttranscriptionally regulate gene expression and function as oncogenes or tumor suppressors, as well as regulators of cell cycle, proliferation, apoptosis, migration and other processes. A number of miRNAs are reported be related to the occurrence and development of colorectal cancer (CRC). However, these studies were not involved in the effect of miRNA 144 of CRC, whose function remains unclear. In this study, we demonstrated that the expression level of miRNA 144 was markedly down-regulated in colorectal cancer HCT116 cells compared with normal control FHC cells. Meanwhile, we found that GSPT1 was over-expressed in human colorectal cancer HCT116 cells. Subsequently, GSPT1 was identified as a target of miRNA 144 through bioinformatics and luciferase reporter assays. Besides, we also confirmed that miRNA 144 can inhibit the proliferation and migration of colorectal cancer HCT116 cells . Next, we observed RNA-mediated knockdown of GSPT1 can also inhibit the proliferation and migration of colorectal cancer cells. Thus, we concluded that miRNA 144 inhibits cell proliferation and migration through GSPT1 in CRC. In addition, further mechanic investigations revealed that miRNA-144 suppressed the expression of GSPT1 to regulate the expression of c-myc, survivin and Bcl2L15 which are involved in cell proliferation, and that metastasis related factor MMP28 was also down-regulated by miRNA144. Our findings suggested that microRNA 144 might be an important element to control the status of colorectal cancer, which has provided a new insight into the mechanism of proliferation and migration and a new target in therapy against colorectal cancer.

Introduction

Colorectal cancer (CRC) is the most common cause of cancer-related mortality worldwide. The current treatment of CRC involves resection followed by radiation and chemotherapy [1]. Development of CRC is a stepwise process comprising genetic changes in tumor-suppressor genes in most of the cases. In addition, a multitude of molecular alterations of genes have been described in the literature as being associated with development and prognosis of CRC [2]. However, the mechanisms that drive the process of neoplastic transformation are not well understood. Thus, there is an urgent need to elucidate the underlying molecular mechanisms of CRC and find new molecular targets for treatment against this disease.

MicroRNAs can cause destruction of homologous mRNA or repression of cognate mRNA translation and play essential roles in maintaining the stable state of chromosome structure, thereby regulating the expression of protein-coding genes [3]. Accumulating evidence indicated that miRNAs can also function as oncogenes or tumor suppressors and contribute to cancer development [4], [5], [6]. Recent reports revealed that microRNA-144 was involved not only in many cellular biochemical reactions by regulating the expression of pivotal factors [7], [8], [9], [10], [11], but also in cell proliferation [12] and apoptosis [13]. miRNA 144 regulated the proliferation of bladder cancer cell by targeting EZH2 and regulating Wnt signaling [14], and associated with colorectal cancer progression via activation of mTOR signaling pathway [15], and promoted cell proliferation, migration and invasion in nasopharyngeal carcinoma through repression of PTEN [12]. However, the role of miRNA 144 in regulating colorectal cancer cell proliferation and migration still remains largely elusive. Polypeptide release factor, eRF3, mediates translation termination in eukaryotes through cooperating with eRF1 [16], [17]. In mammalian cell eRF3 is encoded by GSPT1 (G-to-S transition) gene [18], [19], which could be essential factor involving in regulation of the cell cycle and proliferation and apoptosis [20]. GSPT1 was considered as a potential proto-oncogene [21] involving in tumorigenesis. In this study, we showed that GSPT1 is overexpressed in HCT116 cells, and we initially aimed to identify the signals that miRNA 144 decreased GSPT1 mRNA and protein levels, reduced cell proliferation, and suppressed cell migration. Mechanic investigations were carried out to reveal the effects of down-regulation of GSPT1 by miRNA144 on the expression pattern of c-myc, survivin, Bcl2L15 and MMP 28, which were related to proliferation and migration of colorectal cancer cells. These results suggest that miRNA 144 become a tumor suppressor in colorectal cancer cells by utilizing GSPT1 as a direct and functional target in colorectal cancer.

Section snippets

Cell cultures and transfection

Cell cultures are prepared and maintained according to standard cell culture procedures. The human colorectal cancer HCT116 cells and normal colon FHC cells were cultured in Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12, Hyclone, USA) containing 10% fetal bovine serum (FBS, Sangon, China). Cell cultures were grown and cultured in a humidified 5% CO2 incubator at 37 °C. Transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, CA, U.S.) according to the manufacturer instructions.

Construction of reporter plasmids and the luciferase reporter assay

To

Expression of GSPT1 and miRNA 144 in colorectal cancer cell HCT116

To investigate the role of miRNA 144 in CRC development, we first evaluated the expression levels of miRNA 144 in colorectal cancer HCT116 cells by qRT-PCR (Fig. 1A). We found that colorectal cancer HCT116 cells had less miRNA 144 expression than normal colon FCH cells. GSPT1 has been reported to be up-regulated in breast cancer and gastric cancer. In addition, the expression levels of GSPT1 were detected. As showed in Fig 1B and C, GSPT1 expression was significantly up-regulated in HCT116

Discussion

Tumorigenesis of many cells is related to the abnormal expression of miRNAs, suggesting that the dysregulated miRNAs may play a key role in carcinogenesis or tumor progression. miRNA 144 is processed from a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1 [22]. Recent studies have shown that miRNA 144 plays an important role in many human cancers, including nasopharyngeal carcinoma [12], colorectal cancer [15], and follicular thyroid carcinoma [23].

Conclusions

In conclusion, according to the data of this work, we suggested that miRNA -144 is a suppressor factor, and contrarily, the GSPT1 is an oncogene in HCT116 cells. What's more, GSPT1 is a novel and critical miRNA 144 target. Over-expression of miRNA 144 and RNA interference of GSPT1 led to down-regulation of GSPT1, which resulted in the down-regulation of c-myc, survivin and Bcl2L15 and MMP28, and thus inhibited the proliferation and migration of HCT116 cells. Therefore, all the results indicated

Conflict of interest

The authors have not supplied their declaration of conflict of interest.

Acknowledgments

This work was supported by the National Science Foundation of China (Nos. 31172078; 30770294), the Research Fund for the Doctoral Program of Higher Education of China (20111401110008), Research Project Supported by Shanxi Scholarship Council of China and Shanxi Province Outstanding Graduate Student Innovation Project (020652901013).

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