Original ArticlesMicroRNA-195 desensitizes HCT116 human colon cancer cells to 5-fluorouracil
Introduction
Chemotherapy is a useful way to treat cancer cells. However, acquisition of resistance to anti-cancer drugs is a major hindrance to effective chemotherapy. Cancer cells have multiple mechanisms to overcome toxic effects of anti-cancer drugs by altering drug transportation, inhibiting drug metabolism, increasing tolerance to genotoxic stresses, or decreasing cell death [1], [2], [3]. 5-fluorouracil (5-FU) is a widely used drug to treat a range of cancers, including colorectal, liver, and breast cancers. It interferes with DNA replication by inhibiting thymidylate synthase, thereby leading to cell cycle arrest or cell death [4]. High expression levels of thiamine synthase, Bcl-2, Bcl-XL, and Mcl-1 are related to 5-FU resistance [5], [6], [7]. Although diverse approaches have been made to manage chemo-resistance of cancer cells to 5-FU, core factors and key determinants of drug resistance are not fully elucidated yet.
MicroRNAs (miRNAs) are small noncoding RNAs that can negatively regulate gene expression by binding to the 3′ untranslated region (3′UTR) of target mRNAs [8], [9]. MiRNAs play pivotal roles in diverse processes such as differentiation, proliferation, cell death, development, and pathogenesis of different diseases, including cancer [10], [11]. Several studies have shown that miRNA expression profiles in drug-resistant cancer cells are significantly changed in comparison with those of parental drug-sensitive cancer cells [12], [13]. Aberrant expressions of certain miRNAs can lead to drug resistance by abnormally affecting expression levels of genes involved in drug transport, metabolism, stress response, cell survival, and cell death [14], [15]. For example, miR-19, -20, -21, and -204 can regulate 5-FU resistance in various types of cancer cells [16], [17], [18], [19], [20], [21]. These findings indicate that miRNAs can function as active drivers in the regulation of resistance to anti-cancer drugs.
In this study, we performed functional screening of miRNAs involved in the acquisition of resistance to 5-FU in human colorectal cancer HCT-116 cells using a lenti-viral miRNA library and identified miR-195 as a regulatory miRNA governing the acquisition of resistance to 5-FU. We demonstrated that cell growth and viability were enhanced in resistant clone stably expressing miR-195 in response to various anti-cancer drugs. Inhibition of miR-195 sensitized resistant cells to 5-FU by downregulating WEE1 and CHK1. Our results suggest that miR-195 plays an essential role in the acquisition of drug resistance. Therefore, targeting miR-195 might be able to decrease resistance of colorectal cancer cells to anti-cancer drugs.
Section snippets
Cell culture, transfection of plasmids, precursors and inhibitors of miRNA, and treatment
Human colorectal cancer HCT-116 cells were cultured in Roswell Park Memorial Institute medium (RPMI-1640, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) and antibiotics. All transient transfections including precursor and inhibitor of miR-195 (Bioneer, Daejeon, Korea) and enhanced green fluorescent protein (EGFP) reporter plasmids were performed using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). EGFP reporters were cloned by inserting 3′UTR of CHK1 or WEE1
Functional screening of cell lines resistant to 5-FU using Lenti-miRNA library
To identify miRNAs involved in the regulation of drug resistance to 5-FU, we performed functional screening using Lenti-miRNA library. Human colorectal cancer HCT-116 cells were transduced with lenti-viral particles containing 578 precursor miRNAs (multiplicity of infection = 10) and sequentially incubated with 10 μM 5-FU until control cells were dead (Fig. 1A) (2017 EMM). Each GFP-positive HCT-116 clone survived after exposure to 5-FU was isolated and established as 5-FU resistant clone (Fig. 1
Discussion
Although several studies have attempted to overcome anti-cancer drug resistance, this phenomenon remains as a major hindrance to effective cancer treatment. In this study, we performed functional screening using a lenti-miR library to identify miRNAs regulating the acquisition of resistance to 5-FU and found that four miRNAs (miR-195, miR-133a-1, miR-616, and miR-671) were involved in the survival of HCT-116 cells after 5-FU treatment. We further characterized lenti-miR-195 clone in its
Acknowledgements
This work is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (2017R1A2B2009381, 2012M3A9D1054517, and 2012R1A5A2047939).
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