Cancer Letters

Cancer Letters

Volume 300, Issue 2, 28 January 2011, Pages 197-204
Cancer Letters

Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells

https://doi.org/10.1016/j.canlet.2010.10.006Get rights and content

Abstract

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 μM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12 h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.

Introduction

The administration of 5-fluorouracil (5-FU) – the most widely used anticancer agent – is one of the standard chemotherapy regimens for colon cancers [1], [2], [3]. However, a sizable proportion of the patients show tumor recurrence after 5-FU treatment that is mainly caused by drug-resistant cancer cells. Therefore, new strategies for enhancing the sensitivity and prevention of this drug-resistance have been intensively explored.

Micro-RNAs (miRNAs) are class of small noncoding RNAs that function to control gene expression. They are closely related to the important biological processes such as embryogenesis, development, cell growth, cell differentiation and cell death. Recent research has shown the tumor suppressive and oncogenic potential of a number of miRNAs, underscoring their importance in human cancer therapeutic and diagnostic applications [4], [5], [6], [7], [8]. To obtain more insight into the drug-resistance, we have been studying miRNA expression in drug-resistant human colon cancer DLD-1 cells compared with that in the parental cells. In the current study, miRNA (miR) microarray profiling revealed differential expression of miRs between human colon cancer DLD-1 cells and their 5-FU-resistant counterpart after treatment with 5-FU. Among the miRs differently expressed between the parental and the 5-FU- resistant cells, we focused on miR-34a, the expression of which was significantly down-regulated in 5-FU-resistant cells and still remained so even after the treatment with 5-FU, whereas it was up-regulated time-dependently in the parental cells. Furthermore, Sirt1 (silent mating type information regulation 2 homolog 1) which was earlier shown to be one of the target genes of miR-34a [9], was up-regulated markedly in the 5-FU-resistant DLD-1 cells. Sirt1 is a NAD+-dependent histone deacetylase that can deacetylate histones and a number of non-histone proteins [10]. Sirt1 plays crucial roles in various cellular processes including senescence and cell survival under genotoxic and oxidative stresses. Sirt1 is up-regulated in a variety of cancers including prostate cancer and has implicated in the promotion of tumorigenesis and development of drug-resistance [11], [12], [13]. We have previously demonstrated that the expression level of Sirt1 is increased in hormone-refractory PC3 and DU145 prostate cancer cells to a greater extent than in hormone-sensitive LNCaP cells and proved that the silencing of Sirt1 attenuates cell growth and resistance to camptothecin and cisplatin in PC3 cells [14]. On the other hand, we and others reported that miR-34a regulates Sirt1 expression in a negative fashion [9], [15]. It was also reported that miR-34a inhibits Sirt1 expression directly through binding to the 3′-UTR of its mRNA in HCT116 colon cancer cells [9].

In this study, the introduction of miR-34a into 5-FU-resistant DLD-1 cells significantly attenuated their resistance to 5-FU, which was accompanied by reduced expression of Sirt1 and E2F family proteins. These findings suggest that the resistance to 5-FU in DLD-1 cells is mediated at least in part via a faulty miR-34a/Sirt1/E2F3 cascade.

Section snippets

Cell culture and cell viability

Human colon cancer DLD-1 cell line and its 5-FU-resistant cell line (DLD-1/5FU), which was obtained after the selection by drug pressure, were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS and 2 mM l-glutamine under an atmosphere of 95% air and 5% CO2 at 37 °C. The number of viable cells was determined by performing the trypan-blue dye exclusion test. 5-FU was obtained from Sigma (St. Louis, MO, USA). A PI3K inhibitor (LY294002; Cosmo Bio, Tokyo, Japan) and an Akt

Identification of miRNAs associated with resistance to 5-FU in human colon cancer DLD-1 cells

First, we examined the growth of DLD-1 cells and their 5-FU-resistant (DLD-1/5FU) counterpart after treatment with 5-FU. After the treatment with 5-FU at 30 μM for 72 h, the ratio of viable cells to non-treated control cells in each cell line was 29.0% and 72.1%, respectively, which indicated the resistance of the DLD-1/5FU cells to 5-FU (data not shown). IC50 was evaluated as 4.8 μM for the DLD-1 cells and 39.1 μM for the DLD-1/5FU ones. Thus the resistance of the DLD-1/5FU cells to 5-FU was

Discussion

In the current study, we established 5-FU-resistant cell line from human colon cancer DLD-1 cells to seek an understanding of the molecular mechanisms of 5-FU resistance with respect to miRs. At first, we approached this issue by obtaining the miR differential expression profile between the parental and its 5-FU-resistant cell line (DLD-1/5FU) by using the array-based miRNA assay. As a result, only the expression profile of miR-34a after the treatment with 5-FU was clearly different between the

Conflict of interest

None declared.

Acknowledgements

The work was supported by Grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.

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