miR-203 reverses chemoresistance in p53-mutated colon cancer cells through downregulation of Akt2 expression
Introduction
MicroRNAs (miRNA) are small (19–24 nucleotides), non-coding RNA molecules that function primarily to down-regulate protein expression at the post-transcriptional level by specifically binding to the 3′-untranslational region (3′-UTR) of mRNAs, and subsequently prevent their translation and/or promote their degradation [1], [2]. Notably, a single miRNA can regulate thousands of target genes simultaneously [3]. It is estimated that almost all of the protein-coding genes are subject to miRNA-mediated regulation [4]. A growing body of evidence has demonstrated that miRNAs play a fundamental role in the development, function, and maintenance of tissues and cells in various organisms [5]. Recent evidence demonstrated that miRNAs function as oncogenes or tumor suppressors to modulate multiple oncogenic cellular processes, including cell proliferation, apoptosis, invasion, and metastasis [6].
MicroRNA 203 (miR-203) is a skin-specific microRNA that is initially found to be specifically expressed in keratinocytes and promote epidermal differentiation [7]. The matured miR-203 is a 22-nt non-coding RNA. MiR-203 expression has also been found to be upregulated in psoriasis [8] and differentially expressed in some types of cancer [9]. For instance, downregulation of miR-203 expression was found in brain tumors, esophageal cancers, lymphomas, and leukemia, whereas its upregulation was also reported in breast cancer and ovarian cancer [10]. In colorectal cancer (CRC), miR-203 expression was inconsistent; some studies showed an increase in miR-203 expression [11], [12] while other studies showed a decrease [13] or no change in miR-203 expression [14]. However, the etiology of miR-203 expression in colorectal cancer has not been elucidated.
CRC is ranked third among cancers in the western world and is the fourth most common cancer in the United States [15]. The incidence of CRC in China is relatively lower, but is increasing at an alarming rate in recent years [16]. Surgery is the primary treatment for early stages of CRC. However, most cases of CRCs are detected at later stages and chemotherapy becomes the main option to slow tumor growth and reduce metastasis [17]. Even with high efficacy in tumor treatment, chemotherapeutics induced drug resistance in almost all cancers. Resistance to chemotherapeutic agents is a key barrier to the efficacy of cancer treatment and all clinically available drugs for any tumor type eventually fail [18]. The genetic basis of chemoresistance is extraordinarily complex, involving multiple processes, such as cell proliferation, DNA repair, apoptosis regulation, etc. [19]. The PI3K/Akt signaling is known to be involved in chemoresistance through regulation of cell survival, apoptosis and growth [20], [21], [22]. PI3K produces IP3, which activates Akt through phosphorylation of Akt at Ser473 and Thr308 residues [23]. Activated Akt can inhibit apoptosis through negatively regulating Bax levels [24] and evoke chemoresistance through upregulating MTDH and HSP90 expression [25], [26], [27]. Even so, total reversal of chemoresistance has not yet been established and remains elusive. Despite the significant improvements in new chemotherapeutic agents, more than 90% of cancer patients ultimately die because currently there are no chemotherapeutic options available that are completely unaffected by chemoresistance. Therefore, developing a strategy to reverse chemoresistance is of utmost importance.
Paclitaxel, previously called taxel, is a natural compound originally isolated from pacific yew tree bark and now used to treat a variety of cancers, including colon cancer [28]. However, paclitaxel is also a classic chemotherapeutic drug that induces multidrug resistance (MDR) in all of the treated cancers [29], [30]. Developing agents to reverse chemoresistance could increase the effectiveness of currently available chemotherapeutic agents, which would improve patients’ survival. In this study, the roles and mechanisms of miR-203 in reversing chemoresistance in p53-mutated and p53 wild-type colon cancer cells were examined. A significant sensitizing role of miR-203 on the cytotoxicity of paclitaxel was observed in the p53-mutated colon cancer cells and the signaling pathway that mediated the suppressive role of miR-203 was elucidated. Our study highlighted the role of miR-203 in chemoresistance and its possible application in colon cancer therapy in the clinic.
Section snippets
Cell culture
HCT-116 is a human colon carcinoma cell line, containing a wild-type p53 gene. HCT-15 and HT-29 cells are human colorectal adenocarcinoma cells, expressing mutated p53. Three cell lines were purchased from American type culture collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM, Invitrogen Inc., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), 100 μg/mL of streptomycin and 100 units/mL penicillin at 37 °C, 5% CO2.
Construction of miR-203 expression vector
The miR-203 expression vector was constructed according to
Cell proliferation assay
Fig. 1 shows the results of MTT assay. The MTT assay allows quantification of cell proliferation [31]. In the HT-29 cells, 50 nM of paclitaxel only induced about 18% decrease in cell viability. Transfection of miR-203 vector resulted in a 47% decrease in cell viability and significantly sensitized the inhibitory role of paclitaxel at all of the tested concentrations (Fig. 1A). Compared with the HT-29 cell, HCT-15 cell is a little more sensitive to paclitaxel and the 50 nM concentration induced
Discussion
Resistance to anticancer agents is one of the primary obstacles for effective cancer therapy, but the underlying mechanisms remain controversial [34]. Chemotherapy, the first-line of treatment for a large number of patients with late stage colorectal cancer, provides a modest improvement in overall survival. However, one of the major problems in treating colon cancer is chemoresistance [35]. Paclitaxel has been widely used to treat a variety of cancers, including colon cancer, but it also
Conflict of interest
None declared.
Acknowledgments
This study was supported by the National Hi-Tech Project of China (Grant Numbers: AA021810, AA021907 to YC).
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