Abstract
Chemotherapy resistance frequently drives tumor progression. However, the underlying molecular mechanisms remain unclear. In this study, we found that the expression level of miR-26b was down-regulated in the human colorectal cancer tissues and the resistant cells strains: HT-29/5-FU and LOVO/5-FU cells. Meanwhile, we showed that miR-26b improved sensibility of colorectal cancer cells to 5-FU in vitro and enhanced the potency of 5-FU in the inhibition of tumor growth in vivo. We further demonstrated that the tumor suppressive role of miR-26b was mediated by negatively regulating P-glycoprotein (Pgp) protein expression. Furthermore, studies of colorectal cancer specimens indicated that the expression of miR-26b and Pgp had inverse correlation. Importantly, we found that CpG islands in the miR-26b promoter region were hypermethylated in 5-FU resistant cells. Our study is the first to identify the tumor suppressive role of over-expressed miR-26b in chemo-sensitivity. Identification of a novel miRNA-mediated pathway that regulates chemo-sensitivity in colorectal cancer will facilitate the development of novel therapeutic strategies in the future.
Keywords: miR-26b, Pgp, 5-FU-resistance, colorectal cancer
Introduction
Colorectal cancer (CRC) is the most common cause of cancer-related mortality both in China and the western world. Although surgery, chemotherapy and radiotherapy for the treatment of CRC have improved in recent decades, the overall survival rate of the patients remains poor [1]. 5-Fluorouracil (5-FU) is one of the chemotherapeutic drugs, which is most widely used alone or combined with other drugs in CRC treatment. The studies have shown that 5-FU induced apoptosis and/or cell cycle arrest in cancer cells [2,3]. However, the development of chemotherapy resistance is a major obstacle against successful chemotherapy in advanced CRC treatment [4]. There is an urgent need to investigate the molecular mechanisms underlying drug resistance of CRC, which could help to develop the novel prognostic biomarkers and an efficient strategy for the treatment of CRC.
MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that bind to the 3’-untranslated region (3’-UTR) of the target mRNAs and act post-transcriptionally as negative regulators of gene expression [5,6]. Accumulating evidence has shown that miRNAs functioned as oncogenes or tumor suppressor genes in varies tumor including CRC [7,8]. Emerging evidence has revealed that abnormal expression of miRNAs also play a vital role in chemotherapeutic drug resistance [9]. Reinforced expression of miR-20a sensitized breast cancer cells to a broad spectrum of chemotherapy drugs and suppressed their proliferation both in vitro and in vivo [10]. Zhang Y et.al has shown that miR-587 is an important mediator of 5-FU-induced apoptosis in CRC [11]. In recent years, miR-26b has been a focus of interest for its role as a tumor suppressor in several cancer types, such as breast cancer, hepatocellular carcinoma, prostate cancer and colorectal cancer [12-15]. However, the potential role of miR-26b in overcoming chemotherapy resistance and the underlying mechanism in colorectal cancer remain unrevealed.
In the present study, we firstly demonstrated that miR-26b was decreased in colorectal cancer specimens and cancer cell lines, and the high degree of methylation of the miR-26b CpG islands was found in 5-FU-resistant cell. Moreover, we confirmed that miR-26b inhibited Pgp expression by directly targeting its 3’-UTR and activated the intrinsic apoptosis pathway through up-regulating cleavage of caspase-9 and caspase-3. These changes increased the sensitivity of colorectal cancer cells to 5-FU and inhibited cell proliferation in vivo and in vitro. These findings suggest that the resistance to 5-FU in colorectal cells is mediated at least in part via a miR-26b/Pgp apoptosis cascade, and provide new insights into the molecular mechanism of colorectal development and chemotherapy resistance as well as new strategy for colorectal diagnostics and treatment in the future.
Materials and methods
Tissue samples
A collection of 52 colon tissues were analyzed in the research, which included 16 human adjacent normal tissues and 36 colon tumor samples, were from Changshu No. 2 People’s Hospital. All tumor samples were collected immediately after the surgical removal and stored in liquid nitrogen. The experimental procedures were approved by the Institutional Review Board of Changshu No. 2 People’s Hospital.
Cell lines
Human colon cancer cell lines HT-29 and LOVO and the human normal colorectal epithelial cell (FHC) were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/ml of penicillin, 100 ug/ml of streptomycin and 10% fetal bovine serum. The cells were cultured at 37°C in an incubator with 5% CO2. HT-29 and LOVO cells were cultured after 22-26 passages in complete DMEM medium supplemented with 2.5 ug/ml of 5-fluorouracid (5-FU) (Sigma), replacing the drug each day. The cells became more resistant to this drug. These cells, which are named HT-29/5-FU and LOVO/5-FU cells, were subsequently cultured in complete DMEM medium containing 1.25 ug/ml of 5-FU.
Reagents
Lipofectamine 3000 Reagent (Invitrogen) was used for transfected cells, which was followed the manufacturer’s instructions. MiR-26b mimics and miR-NC were bought from ribobio. Pgp plasmid was bought from Addgene. Primary antibodies against Pgp and GAPDH were purchased from Cell Signaling Technology. Primary antibodies against Caspase-3, Caspase-9 and Cleaved-PARP were obtained from BD Biosciences, and Santa Cruz, respectively. 5-Aza-2’-deoxycytidine (5-Aza-dC) was purchased from Sigma. Cell lines were treated with 5 μM of 5-Aza-dC for 120 h.
Western blotting
Cells were lysed in RIPA lysis included protease inhibitors (Thermo scientific) and collected supernatant. The total proteins were separated by 10% gradients SDS-PAGE gels. Proteins were transferred to a polyvinylidene fluoride membrane and blocked with 5% non-fat milk before incubation with primary antibodies at 4°C overnight. Proteins were incubated with horseradish peroxidase-conjugated secondary antibodies for two hours, and detected utilizing horseradish peroxidase substrate.
RNA extraction and RT-qPCR analysis
The cells collected and lysed in Trizol (Invitrogen). The supernatant were transferred in RNA-free tube, added in 200 ul Trichloromethane. The mixture was centrifuged at 12000 g speeds for 30 minutes. Isopropanol in equal volume was added to the supernatant to precipitate RNA, centrifuged at 12000 g speeds for 30 minutes. The precipitation was washed twice by 75% ethanol and dissolved in free-RNase water. RT-qPCR assays were performed to test the expression levels of miR-26b according to the manufacturer’s instructions (TaKaRa) using an ABI Prism 7900HT instrument. The miR-26b values were normalized to U6. The 2-ΔΔCt method was used as relative quantification measure of differential expression.
Plasmids and luciferase assay
For the miR-26b-target analysis, two fragments of Pgp-WT or Pgp-Mut were cloned in the downstream of the luciferase gene in pMIR-reporter plasmid according to protocol. HT-29 cells were seeded into 24-well plate reached to 1×105 cells/ml each well and were transfected by Lipofectamine 3000 (Invitrogen) with 0.3 ug of luciferase reporter plasmid, and 0.2 ug of pre-miR-26b or control precursor. After transfection for 6 hours, medium were replaced with new DMEM. Luciferase activities were measured 48 h after transfection.
Cell viability assay and apoptosis
HT-29 and LOVO cells were transfected with anti-miR-NC or anti-miR-26b. HT-29/5-FU and LOVO/5-FU cells were transfected with miR-NC or miR-26b. Then, cells were plated in 96-well plate at concentration of 5,000 cells/well. The Cells were incubated in the presence of different concentrations of 5-FU. After 48 h, the cells viability was assayed by the CCK-8 (Cell Counting Kit-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. The study was repeated 3 times. HT-29/5-FU and LOVO/5-FU cells transfected with miR-NC or miR-26b were cultured for 48 h. The cells were analyzed using a FACSCanto-II flow cytometer (BD Biosciences).
Methylation-specific PCR
The genomic DNA was isolated and modified by the CpGenome DNA kit (Chemicon International Inc. Temecula, CA) according to the manufacturer’s protocol. The bisulfite-treated DNA was stored at -80°C until use. Methylation-specific PCR (MSP) for miR-26b promoter methylation was performed in a two-step approach as previously reported. For each PCR, methylated and unmethylated DNA was included as positive and negative controls, and water was used as a control for the PCR reaction. PCR products were separated on 3% agarose gels.
Construction of stable cell lines
The miRNA lentivirus of overexpressed miR-NC or miR-26b were purchased from Shanghai Genechem Co., LTD. HT-29/5-FU cells were cultured in 6-cm dish. When cells density reached 40%, 5 μl lentivirus and 3 μl polybrene (lentivirus was diluted to 1×108 TU/ml and polybrene was diluted to 50 μg/ml using ehhanced infection solution) were added to culture media that were replaced for every 24 hours. After three times infection, the cells were selected by adding puromycin (8 μg/ml) in medium.
Tumorigenesis in nude mice
4 weeks of age BALB/c-nude male mice were bought at the Shanghai Experimental Animal Center and were randomly divided into miR-NC, miR-26b, miR-NC + 5-FU and miR-26b + 5-FU groups. 1×106 cells in 1:1 PBS/Matrigel were planted subcutaneously into the nude mice. After 8 days, tumors were measured per two days. On the eighteenth day, tumors were harvested and weighed.
Statistical analysis
All the results were analyzed by the SPSS 11 statistical software, with P<0.05 considered statistically significant. Quantitative variables were analyzed using Student’s t-test between two groups or one-way analysis among multiple groups. The correlations were analyzed using Spearman’s rank test.
Results
MiR-26b is down-regulated in CRC and 5-FU resistant cells
Drug resistance is a major challenge in the chemotherapy of CRC. More than half of CRC patients develop chemo-resistance with the mechanisms unknown [16,17]. Firstly, we found that the expression levels of miR-26b were down-regulated not only in our own CRC tissues but also in GEO dataset GSE35843 (Figure 1A and 1B). Meanwhile, we found that the expression levels of miR-26b in colorectal cancer cells were significantly lower compared with levels in normal colorectal epithelial cells (FHC) (Figure 1C). Together, our results proved that miR-26b was down-regulated in colorectal cancer specimens and cancer cell lines. Furthermore, we analyzed the miR-26b expression levels in CRC 5-FU resistant cells, and the results showed that the expression levels of miR-26b were significantly down-regulated in 5-FU resistant cells compared to sensitive cells (Figure 1D). These data indicated the important roles of miR-26b in CRC as well as in CRC chemo-resistance.
Figure 1.
miR-26b is down-regulated in CRC and 5-FU resistance cells. A. The expression levels of miR-26b in normal (n = 16) and CRC tissues (n = 36) were analyzed by qRT-PCR and normalized to U6 expression levels. Data were presented as the means ± SD from three independent experiments with triple replicates per experiment. **P<0.01 indicates significant difference. B. The expression levels of miR-26b in GEO dataset GSE35843 with 23 normal tissues and 55 CRC tumors. **P<0.01. C. The expression of miR-26b in colorectal cancer cell lines (HT-29 and LOVO) compared with normal colorectal epithelial cells (FHC). *P<0.05. D. The expression levels of CRC 5-FU sensitive cells (HT-29 and LOVO) and resistant cells (HT-29/5-FU and LOVO/5-FU) miR-26b were analyzed by qRT-PCR and normalized to the values of sensitive cells. U6 levels were used as an internal control. **P<0.01.
MiR-26b suppresses the drug resistance of 5-FU in CRC
Little is known about the effects of miR-26b in CRC chemo-resistance. To evaluate the functions of miR-26b in CRC chemotherapy sensitivity, we down-regulated miR-26b in HT-29 and LOVO cells, and then tested the drug sensitivities by CCK-8 assay. The results indicated that miR-26b knockdown enhanced the chemo-resistance of CRC cells (Figure 2A and 2B). Besides, apoptosis assays also showed that lower expression of miR-26b decreased the apoptosis rate of CRC cells with the treatment of 5-FU (Figure 2C and 2D). Next, we overexpressed miR-26b in CRC 5-FU resistant cells (HT-29/5-FU and LOVO/5-FU), and then tested the drug sensitivities by CCK-8 assay. The results indicated that enforced miR-26b reversed the chemo-resistance of CRC cells (Figure 2E and 2F). As expected, apoptosis assays showed that high expression of miR-26b increased the apoptosis rate of CRC resistant cells with the treatment of 5-FU (Figure 2G and 2H). These data highlighted the significant functions of miR-26b in increasing the sensitivity of CRC to 5-FU and overcoming drug resistance.
Figure 2.
miR-26b suppresses the drug resistance of 5-FU in CRC. A, B. The CRC cells (HT-29 and LOVO) were transfected with anti-miR-26b or negative control (anti-miR-NC), and then were treated with different concentrations of 5-FU. After 48 h, the cell viabilities were analyzed by CCK-8. Data were presented as the means ± SD from three independent experiments with triple replicates per experiment. **P<0.01. C, D. The HT-29 and LOVO were transfected with anti-miR-26b or anti-miR-NC, then were treated with 5 μM 5-FU. After 48 h, the apoptosis cells were analyzed by flow cytometry (FCM). **P<0.01. E, F. The CRC resistance cells (HT-29/5-FU and LOVO/5-FU) were transfected with miR-26b mimics or negative control (miR-NC), and then were treated with different concentrations of 5-FU. After 48 h, the cell viabilities were analyzed by CCK-8. Data were presented as the means ± SD from three independent experiments with triple replicates per experiment. **P<0.01. G, H. The HT-29/5-FU and LOVO/5-FU were transfected with miR-26b mimics or miR-NC, then were treated with 5 μM 5-FU. After 48 h, the apoptosis cells were analyzed by flow cytometry (FCM). **P<0.01.
MiR-26b directly targets Pgp
To discover the mechanism of miR-26b in sensitizing CRC resistant cells to 5-FU, we employed the TargetScan program (http://www.targetscan.org/vert_72/) to find out the targets of miR-26b and Pgp caught our attention (Figure 3A). Next, we performed the luciferase reporter assay to ensure the binding effects of miR-26b in the 3’UTR of Pgp, and the data suggested that miR-26b could suppress the luciferase activities of the reporter through binding to the predicted site (Figure 3B). Down-regulation of miR-26b in CRC cells could increase Pgp protein levels. And, the increased level of Pgp due to miR-26b knockdown was reversed by down-regulation of Pgp (Figure 3C, upper). Furthermore, the overexpression of miR-26b inhibited the expression levels of Pgp in drug resistant CRC cells (Figure 3C, lower). Consistently, Pgp expression levels were higher in CRC tissues compared to normal tissues. Meanwhile, there were significant correlation between miR-26b and Pgp expression levels (Figure 3D and 3E). The data above demonstrated that miR-26b repressed the drug resistance of CRC via targeting Pgp.
Figure 3.
miR-26b directly targets Pgp. A. The target and the binding sites of miR-26b were analyzed by TargetScan. The wide type (WT) and mutation (mut) luciferase reporter vectors were conducted as the schema graph showed. B. Luciferase reporter assay was used on HT-29 to detect the relative luciferase activities of WT and mut reporters. **P<0.01. C. Upper: the LOVO and HT-29 were transfected with anti-miR-NC, anti-miR-26b and anti-miR-26b + si-Pgp respectively and were subjected to western blotting and detected for Pgp expression levels. Lower: the LOVO/5-FU and HT-29/5-FU were transfected with miR-NC, miR-26b mimics and miR-26b mimics + Pgp respectively and were subjected to western blotting and detected for Pgp expression levels. D. The expression levels of Pgp in GEO dataset GSE35843. **P<0.01. E. According to the GEO dataset GSE35843, the spearman’s correlation test was employed to analyze the correlation between the expression levels of Pgp and miR-26b.
Over-expression of Pgp reverses the suppression of miR-26b in drug resistance
To identify the roles of Pgp in miR-26b-repressed CRC drug resistance progress, we reversed the expression of Pgp in miR-26b overexpressing cells to analyze the sensitivities of HT-29/5-FU and LOVO/5-FU to 5-FU. The results suggested that compensation of Pgp reversed the suppression of miR-26b in chemo-resistance (Figure 4A and 4B). Besides, overexpression of Pgp also reduced the apoptosis rates of the resistant cells caused by miR-26b with the treatment of 5-FU (Figure 4C and 4D). Furthermore, the western blotting assay showed that overexpression of miR-26b increased the expression levels of apoptosis related proteins while Pgp could reverse it (Figure 4E). These results showed that overexpression of Pgp could block the suppression of miR-26b in drug resistance, which suggested that the effects of miR-26b in CRC drug resistance depended on Pgp.
Figure 4.
Overexpression of Pgp reverses the suppression of miR-26b in drug resistance. A, B. The HT-29/5-FU and LOVO/5-FU were co-transfected with miR-NC mimics or miR-26b or Pgp, and then were treated with different concentrations of 5-FU. After 48 h, the cell viabilities were analyzed by CCK-8. C, D. Cells were transfected as above, then were treated with 5 μM 5-FU. After 48 h, the apoptosis cells were analyzed by FCM. E. Cells were transfected as above, and were subjected to western blotting and detected for Pgp, Cleaved-PARP, Cleaved-Caspae-3, Cleaved-Caspase-9 and GAPDH expression levels after 48 h. **, P<0.01 indicates significant difference of miR-26b group compared to miR-NC group; ##, P<0.01 indicates significant difference of miR-26b + Pgp group compared to miR-26b group; $, P<0.05 indicates significant difference of miR-NC + Pgp group compared to miR-NC group.
The attenuations of miR-26b in drug resistance cells are induced by methylation in the promoter region
Methylation is an important regulatory mechanism of gene expression, including miRNAs [18,19]. We argued that whether the down-regulations of miR-26b in 5-FU resistant cells were caused by hyper-methylation in miR-26b promoter region. Depended on which, we analyzed the methylation rate of miR-26b promoter in 5-FU resistant cells and sensitive cells by MSP, and the data indicated that there were more methylation modification in 5-FU resistant cells’ promoters (Figure 5A and 5B). Furthermore, the expression levels of miR-26b were upregulated with the inhibitor of methylation in 5-FU resistant cells, which suggested the repressed expression levels of miR-26b were induced by hyper-methylation (Figure 5C and 5D).
Figure 5.
The attenuations of miR-26b in drug resistance cells are induced by methylation in the promoter region. A, B. The Methylation specific PCR (MSP) was used to analyze the methylation levels of miR-26b promoter region in CRC 5-FU sensitive cells (LOVO and HT-29) and resistant cells (LOVO/5-FU and HT-29/5-FU). The relative densities were normalized as the ratio of the sensitive cells group. M means the PCR products were amplified by methylation-specific primers and U means the PCR products were amplified by unmethylation-specific primers. **P<0.01. C, D. The LOVO/5-FU and HT-29/5-FU were treated with 5-Aza-dC for 5 days, and then the expression levels of miR-26b were analyzed by qRT-PCR. **P<0.01.
MiR-26b represses CRC drug resistance in vivo
To evaluate whether miR-26b could suppress the drug resistance of CRC in vivo, the tumor formation assay was employed. The miR-26b and miR-NC overexpressing HT-29/5-FU cells were subcutaneously injected into the nude mice and then treated with 5-FU. As expected, 5-FU suppressed the tumor growth in both miR-NC and miR-26b group. Meanwhile, overexpression of miR-26b not only inhibited the tumor growth but also enhanced the drug sensitivity to 5-FU (Figure 6A-C). These data indicated miR-26b could repress CRC drug resistance in vivo.
Figure 6.
miR-26b represses CRC drug resistance in vivo. The HT-29/5-FU miR-26b- and miR-NC-overexpressing cells were conducted by lentivirus. The cells were dispersed in 100 μl of serum-free medium and subcutaneously injected into the sides of posterior flank of nude mice. A. The mice were daily intraperitoneal administration of 5-FU when the tumors were detected at day 8, and the tumors were measured every two days. **Indicates the significant difference of group: miR-26b compared to group: miR-NC. $$indicates the significant difference of group: miR-26b + 5-FU compared to group: miR-NC + 5-FU. ##Indicates the significant difference of group: miR-NC + 5-FU compared to group: miR-NC. B, C. The mice were sacrificed and the tumors weighed after 18 days, and the representative photos of tumors were showed (Bar = 10 mm). Data were presented as the means ± SD from four tumor samples. **P<0.01, $$P<0.01, ##P<0.01.
Discussion
Mounting evidence has demonstrated that miRNAs play critical roles in developing chemo-resistance based on their functions and targets [20-22]. Previous reports suggested that miR-26b played an important role in the development and progression of various cancers, including CRC [23]. Previous study suggested miR-26b suppressed NF-κB signaling and sensitized HCC cells to the doxorubicin-induced apoptosis by targeting TAK1 and TAB3 [24]. However, whether and how miR-26b might be involved in 5-FU chemo-resistance of CRC has not been studied. In this study, we focused on the role of miR-26b in the emergence of chemo-resistance and investigated the targeted signaling that mediates CRC escaping from drug toxicity. Consistent with previous studies, we also found a significantly reduced miR-26b expression of CRC tissues compared with normal tissues. Meanwhile, the expression level of miR-26b was downregulated in the CRC resistant cells: Lovo/5-FU and HT-29/5-FU when compared with maternal cells. More importantly, we found that overexpression of miR-26b inhibited cell proliferation and induced apoptosis in vitro and in vivo. Western blot analysis showed that miR-26b overexpression elevated the key apoptosis-related protein levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP. Our study is first to identify the role of miR-26b in 5-FU chemo-resistance.
So far, miR-26b has been reported to bind to various targets such as COX-2, CDC6, CHD1 and so on [25-27]. However, the function of miR-26b and the association of its target gene with 5-FU chemo-resistance in CRC remained unclear. Mounting studies uncovered that one of the most important mechanisms being involved in chemo-resistance is the expression of Pgp, encoded by the multidrug resistance 1 (MDR1) gene, which acted as an ATP-dependent transport pump capable of effluxing cytotoxic agents [28]. In addition, Pgp also played a role in inhibition of drug accumulation and caspase activation in the multidrug resistance tumor [29,30]. Our study confirmed that miR-26b inhibited Pgp expression by directly targeting the 3’-UTR. Forced expression of Pgp restored miR-26b-inhibited cell effects. Furthermore, Pgp was upregulated in CRC tissue specimens, and the increased levels of Pgp protein expression were inversely correlated with the decreased miR-26b levels, further confirming that Pgp was downregulated by miR-26b in clinical CRC tumor tissues.
The regulatory mechanism of miR-26b in the CRC remains unclear. Increasing evidence demonstrates that epigenetics plays an important role in abnormal miRNA expression [31,32]. RNA methylation of CpG islands in target gene promoters can alter gene expression, and be involved in chemo-resistance; for instance, methylation of miR-7 has been linked to the regulation of MAFG expression involved in Platinum Response [33]. Therefore, we speculated that methylation of miR-26b promoter CpG islands led to a decrease in miR-26b expression in CRC resistant cells; this was confirmed by MSP analysis. We also found that miR-26b expression was upregulated by 5-Aza treatment, suggesting that miR-26b promoter methylation led to down-regulation of miR-26b and an increase in chemo-resistance.
In the present manuscript, we introduce the epigenetic regulation of miR-26b as a mechanism involved in 5-FU-resistance in cancer cell lines directly regulating Pgp, which is overexpressed in resistant phenotypes. To the best of our knowledge, this is the first report linking the regulation of Pgp by miR-26b and its role in chemotherapy response to 5-FU. Moreover, miR-26b methylation and Pgp arise as potential predictive biomarker for the identification of CRC patients that may present worst response to 5-FU-derived treatment.
Acknowledgements
The Scientific and Technologic Development Programme of Changshu (CR 201722).
Disclosure of conflict of interest
None.
References
- 1.Kahouli I, Tomaro-Duchesneau C, Prakash S. Probiotics in colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives. J Med Microbiol. 2013;62:1107–1123. doi: 10.1099/jmm.0.048975-0. [DOI] [PubMed] [Google Scholar]
- 2.Bekaii-Saab T, Wu C. Seeing the forest through the trees: a systematic review of the safety and efficacy of combination chemotherapies used in the treatment of metastatic colorectal cancer. Crit Rev Oncol Hematol. 2014;91:9–34. doi: 10.1016/j.critrevonc.2014.01.001. [DOI] [PubMed] [Google Scholar]
- 3.Folprecht G, Pericay C, Saunders MP, Thomas A, Lopez LR, Roh JK, Chistyakov V, Hohler T, Kim JS, Hofheinz RD, Ackland SP, Swinson D, Kopp M, Udovitsa D, Hall M, Iveson T, Vogel A, Zalcberg JR. Oxaliplatin and 5-FU/folinic acid (modified FOLFOX6) with or without aflibercept in first-line treatment of patients with metastatic colorectal cancer: the AFFIRM study. Ann Oncol. 2016;27:1273–1279. doi: 10.1093/annonc/mdw176. [DOI] [PubMed] [Google Scholar]
- 4.Tominaga T, Iwahashi M, Takifuji K, Hotta T, Yokoyama S, Matsuda K, Higashiguchi T, Oku Y, Nasu T, Yamaue H. Combination of p53 codon 72 polymorphism and inactive p53 mutation predicts chemosensitivity to 5-fluorouracil in colorectal cancer. Int J Cancer. 2010;126:1691–1701. doi: 10.1002/ijc.24929. [DOI] [PubMed] [Google Scholar]
- 5.Liu B, Li J, Cairns MJ. Identifying miRNAs, targets and functions. Brief Bioinform. 2014;15:1–19. doi: 10.1093/bib/bbs075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Wu C, Bardes EE, Jegga AG, Aronow BJ. ToppMiR: ranking microRNAs and their mRNA targets based on biological functions and context. Nucleic Acids Res. 2014;42:W107–W113. doi: 10.1093/nar/gku409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Berindan-Neagoe I, Monroig PC, Pasculli B, Calin GA. MicroRNAome genome: a treasure for cancer diagnosis and therapy. CA Cancer J Clin. 2014;64:311–336. doi: 10.3322/caac.21244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Qiu Z, Guo W, Wang Q, Chen Z, Huang S, Zhao F, Yao M, Zhao Y, He X. MicroRNA-124 reduces the pentose phosphate pathway and proliferation by targeting PRPS1 and RPIA mRNAs in human colorectal cancer cells. Gastroenterology. 2015;149:1587–1598. doi: 10.1053/j.gastro.2015.07.050. [DOI] [PubMed] [Google Scholar]
- 9.Zhao L, Shan Y, Liu B, Li Y, Jia L. Functional screen analysis reveals miR-3142 as central regulator in chemoresistance and proliferation through activation of the PTEN-AKT pathway in CML. Cell Death Dis. 2017;8:e2830. doi: 10.1038/cddis.2017.223. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 10.Si W, Shen J, Du C, Chen D, Gu X, Li C, Yao M, Pan J, Cheng J, Jiang D, Xu L, Bao C, Fu P, Fan W. A miR-20a/MAPK1/c-Myc regulatory feedback loop regulates breast carcinogenesis and chemoresistance. Cell Death Differ. 2018;25:406–420. doi: 10.1038/cdd.2017.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Zhang Y, Talmon G, Wang J. MicroRNA-587 antagonizes 5-FU-induced apoptosis and confers drug resistance by regulating PPP2R1B expression in colorectal cancer. Cell Death Dis. 2016;7:e2525. doi: 10.1038/cddis.2016.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Wilke CM, Hess J, Klymenko SV, Chumak VV, Zakhartseva LM, Bakhanova EV, Feuchtinger A, Walch AK, Selmansberger M, Braselmann H, Schneider L, Pitea A, Steinhilber J, Fend F, Bosmuller HC, Zitzelsberger H, Unger K. Expression of miRNA-26b-5p and its target TRPS1 is associated with radiation exposure in post-Chernobyl breast cancer. Int J Cancer. 2018;142:573–583. doi: 10.1002/ijc.31072. [DOI] [PubMed] [Google Scholar]
- 13.Jin F, Wang Y, Li M, Zhu Y, Liang H, Wang C, Wang F, Zhang CY, Zen K, Li L. MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy. Cell Death Dis. 2017;8:e2540. doi: 10.1038/cddis.2016.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kato M, Goto Y, Matsushita R, Kurozumi A, Fukumoto I, Nishikawa R, Sakamoto S, Enokida H, Nakagawa M, Ichikawa T, Seki N. MicroRNA-26a/b directly regulate La-related protein 1 and inhibit cancer cell invasion in prostate cancer. Int J Oncol. 2015;47:710–718. doi: 10.3892/ijo.2015.3043. [DOI] [PubMed] [Google Scholar]
- 15.Zhang Z, Kim K, Li X, Moreno M, Sharp T, Goodheart MJ, Safe S, Dupuy AJ, Amendt BA. MicroRNA-26b represses colon cancer cell proliferation by inhibiting lymphoid enhancer factor 1 expression. Mol Cancer Ther. 2014;13:1942–1951. doi: 10.1158/1535-7163.MCT-13-1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Longley DB, Allen WL, Johnston PG. Drug resistance, predictive markers and pharmacogenomics in colorectal cancer. Biochim Biophys Acta. 2006;1766:184–196. doi: 10.1016/j.bbcan.2006.08.001. [DOI] [PubMed] [Google Scholar]
- 17.Panczyk M. Pharmacogenetics research on chemotherapy resistance in colorectal cancer over the last 20 years. World J Gastroenterol. 2014;20:9775–9827. doi: 10.3748/wjg.v20.i29.9775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Xhemalce B, Robson SC, Kouzarides T. Human RNA methyltransferase BCDIN3D regulates microRNA processing. Cell. 2012;151:278–288. doi: 10.1016/j.cell.2012.08.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Sump B, Brickner JH. Nup98 regulation of histone methylation promotes normal gene expression and may drive leukemogenesis. Genes Dev. 2017;31:2201–2203. doi: 10.1101/gad.310359.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol. 2005;205:275–292. doi: 10.1002/path.1706. [DOI] [PubMed] [Google Scholar]
- 21.Chen Y, Jacamo R, Konopleva M, Garzon R, Croce C, Andreeff M. CXCR4 downregulation of let-7a drives chemoresistance in acute myeloid leukemia. J Clin Invest. 2013;123:2395–2407. doi: 10.1172/JCI66553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Xia H, Ooi LL, Hui KM. MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer. Hepatology. 2013;58:629–641. doi: 10.1002/hep.26369. [DOI] [PubMed] [Google Scholar]
- 23.Chen X, Xiao W, Chen W, Liu X, Wu M, Bo Q, Luo Y, Ye S, Cao Y, Liu Y. MicroRNA-26a and -26b inhibit lens fibrosis and cataract by negatively regulating Jagged-1/Notch signaling pathway. Cell Death Differ. 2017;24:1990. doi: 10.1038/cdd.2017.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Zhao N, Wang R, Zhou L, Zhu Y, Gong J, Zhuang SM. MicroRNA-26b suppresses the NF-kappaB signaling and enhances the chemosensitivity of hepatocellular carcinoma cells by targeting TAK1 and TAB3. Mol Cancer. 2014;13:35. doi: 10.1186/1476-4598-13-35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Xia M, Duan ML, Tong JH, Xu JG. MiR-26b suppresses tumor cell proliferation, migration and invasion by directly targeting COX-2 in lung cancer. Eur Rev Med Pharmacol Sci. 2015;19:4728–4737. [PubMed] [Google Scholar]
- 26.Zhang X, Xiao D, Wang Z, Zou Y, Huang L, Lin W, Deng Q, Pan H, Zhou J, Liang C, He J. MicroRNA-26a/b regulate DNA replication licensing, tumorigenesis, and prognosis by targeting CDC6 in lung cancer. Mol Cancer Res. 2014;12:1535–1546. doi: 10.1158/1541-7786.MCR-13-0641. [DOI] [PubMed] [Google Scholar]
- 27.Tan S, Ding K, Li R, Zhang W, Li G, Kong X, Qian P, Lobie PE, Zhu T. Identification of miR-26 as a key mediator of estrogen stimulated cell proliferation by targeting CHD1, GREB1 and KPNA2. Breast Cancer Res. 2014;16:R40. doi: 10.1186/bcr3644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Mahadevan D, List AF. Targeting the multidrug resistance-1 transporter in AML: molecular regulation and therapeutic strategies. Blood. 2004;104:1940–1951. doi: 10.1182/blood-2003-07-2490. [DOI] [PubMed] [Google Scholar]
- 29.Tainton KM, Smyth MJ, Jackson JT, Tanner JE, Cerruti L, Jane SM, Darcy PK, Johnstone RW. Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux. Cell Death Differ. 2004;11:1028–1037. doi: 10.1038/sj.cdd.4401440. [DOI] [PubMed] [Google Scholar]
- 30.Wang JQ, Chen BA, Cheng J, Xu WL, Sun XC. Comparison of reversal effects of 5-bromotetrandrine and tetrandrine on P-glycoprotein-dependent Resistance to adriamycin in human lukemia cell line K562/A02. Ai Zheng. 2008;27:491–495. [PubMed] [Google Scholar]
- 31.Roman-Gomez J, Agirre X, Jimenez-Velasco A, Arqueros V, Vilas-Zornoza A, Rodriguez-Otero P, Martin-Subero I, Garate L, Cordeu L, San JE, Martin V, Castillejo JA, Bandres E, Calasanz MJ, Siebert R, Heiniger A, Torres A, Prosper F. Epigenetic regulation of microRNAs in acute lymphoblastic leukemia. J. Clin. Oncol. 2009;27:1316–1322. doi: 10.1200/JCO.2008.19.3441. [DOI] [PubMed] [Google Scholar]
- 32.Moutinho C, Esteller M. MicroRNAs and epigenetics. Adv Cancer Res. 2017;135:189–220. doi: 10.1016/bs.acr.2017.06.003. [DOI] [PubMed] [Google Scholar]
- 33.Vera O, Jimenez J, Pernia O, Rodriguez-Antolin C, Rodriguez C, Sanchez Cabo F, Soto J, Rosas R, Lopez-Magallon S, Esteban Rodriguez I, Dopazo A, Rojo F, Belda C, Alvarez R, Valentin J, Benitez J, Perona R, De Castro J, Ibanez de Caceres I. DNA methylation of mir-7 is a mechanism involved in platinum response through MAFG overexpression in cancer cells. Theranostics. 2017;7:4118–4134. doi: 10.7150/thno.20112. [DOI] [PMC free article] [PubMed] [Google Scholar]