Cells from the DLD-1 human colon cancer cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 (Sigma-Aldrich, Hong Kong, China) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1X penicillin-streptomycin-glutamine (10378-016; Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified incubator with 95% air and 5% CO₂.

The 5-FU-resistant cell line from DLD-1 human colon cancer cells was generated as described previously (17). Briefly, DLD-1 cells were treated with gradually increasing concentrations of 5-FU in regular cell culture conditions in order to select resistant cells. Following successive treatments for a duration of up to three months, resistant cell clones were pooled and used for all subsequent experiments in this study. Resistant cells were treated with 5-FU each month for elimination of the cells which may have regained sensitivity to 5-FU.

Rabbit polyclonal LDHA and β-actin antibodies were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA; #2012 and #4967, respectively) and 5-FU antibodies were obtained from Sigma-Aldrich.

Whole cells were lysed in 1X SDS sample buffer, resolved by electrophoresis using SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with primary antibodies overnight and incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit immunoglobulin G secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 3 h, prior to detection using a Super Signal Enhanced Chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA). For sequential blotting, membranes were stripped with Stripping Buffer (Pierce Biotechnology, Inc.) and re-probed with other primary antibodies.

A colorimetric assay using the tetrazolium salt, MTT (EMD Millipore, Billerica, MA, USA), was used to assess the cytotoxicity of the anticancer agent, 5-FU. Single-cell suspensions were prepared and cell density was measured. MTT assays were performed according to the manufacturer’s instructions. Briefly, an equal number of cells were added into each well with culture medium containing normal concentrations of either 5-FU or phosphate-buffered saline for the untreated control. Following four days of culture, 0.1 mg MTT was added to each well and was incubated at 37°C for an additional 4 h. Plates were centrifuged at 450 × g for 5 min at room temperature, and the medium was discarded. Dimethyl sulfoxide (0.15 ml) was added to each well to solubilize the crystals, and the plates were immediately read at 540 nm on a scanning multiwell spectrometer (Bio-Tek instruments, Inc., Burlington, VT, USA). All experiments were performed three times.

miRNA precursors (pre-miRNAs) and pre-miR negative controls were purchased from Applied Biosystems Life Technologies (Foster City, CA, USA). Lipofectamine^® 2000 (Invitrogen Life Technologies) was used for the transfection of pre-miRNAs. At 48 h following transfection, the expression of miR-34a was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the expression of LDHA, a target of miR-34a, was measured using western blotting.

Transfection was performed using the Lipofectamine 2000 Transfection reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Overexpression vectors containing wild type LDHA (Myc-DDK-tagged) were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). At 48 h following transfection, cells were collected or whole-cell lysates were prepared for further analysis.

RNA was extracted from cancer cells using TRIzol reagent (Invitrogen Life Technologies). cDNA synthesis was performed using a SuperScript First-Standard Synthesis system for RT-qPCR (Invitrogen Life Technologies) according to the manufacturer’s instructions. qPCR analyses were performed using Assay-on-Demand primers and the TaqMan Universal PCR Master Mix reagent (Applied Biosystems Life Technologies). Samples were analyzed using an ABI Prism 7700 Sequence Detection system (Applied Biosystems Life Technologies). The following primers were used: LDHA, forward 5′-TGGAGTGGAATGAATGTTGC-3′, reverse 5′-ATAGCCCAGGATGTGTAGCC-3′; and β-actin, forward 5′-AGGCACCAGGGCGTGAT-3′, and reverse 5′-GCCCACATAGGAATCCTTCTGAC-3′. LDHA expression levels were normalized to those of β-actin. For miRNA expression analysis, RT-qPCR was conducted using the TaqMan microRNA reverse transcription kit (Applied Biosystems) and TaqMan microRNA assays kit (Applied Biosystems) according the manufacturer’s instructions. All reactions were performed in triplicate. Human U6 served as an internal control. The relative quantities of mRNA were calculated using the comparative C_T method (17). Experiments were performed three times.

Lactate production was detected using a Lactate assay kit (BioVision, Inc., Milpitas, CA, USA). Results were normalized to the quantity of total protein in the control cells.

Total LDH activity in cell lysates was examined using the LDH cytotoxicity assay kit (BioVision, Inc.) according to the manufacturer’s instructions. Briefly, 2×10⁵ cells were seeded in a 24-well plate one day prior to the assay and all samples were analyzed in triplicate. Cells were collected and washed, and protein was extracted in order to measure LDH activity. Results were normalized to the quantity of total protein in the control cells.

The DLD-1 cells were plated at 5×10⁴ cells/well in 24-well plates. The following day, the cells were co-transfected with luciferase reporter plasmids (pMIR-REPORT™ miRNA Expression Reporter Vector System; Invitrogen Life Technologies; AM5795); with wild type 3′-UTR or mutant 3′-UTR of LDHA, and pre-miR-34a or pre-miR-negative (control miR; Applied Biosystems), using Lipofectamine^® 2000 reagent (Invitrogen Life Technologies; 11668019). Forty-eight hours post-transfection the cells were harvested and lysed using passive lysis buffer (Dual-Luciferase^® Reporter Assay System; Promega; E1910). Luciferase (LUC) activity was measured using a dual luciferase reporter assay (Dual-Luciferase^® Reporter Assay System; Promega; E1910). The pRL-TK vector was used as an internal control. The results are expressed as relative LUC activity (firefly LUC/renilla LUC).

The data were analyzed using GraphPad 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Unpaired Student’s t-test was used for the data analysis. All data are shown as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.

Since miR-34a has been reported to act as a tumor suppressor in a number of tumor types (18) and is associated with 5-FU resistance (17), the present study investigated the role of miR-34a in 5-FU resistance in human colon cancer cells. A 5-FU-resistant cell line was generated using DLD-1 cells, by selection with gradually increasing concentrations of 5-FU in a cell culture medium. Following successive treatments for a duration of three months, several 5-FU-resistant cell clones were developed and pooled for use in the subsequent experiments. To verify resistance, parental cells and resistant pool cells were treated with 5-FU at various concentrations for 72 h. As hypothesized, cell viability assays demonstrated that DLD-1 5-FU-resistant cells tolerated markedly higher concentrations of 5-FU compared with sensitive cells, which exhibited significant inhibition of viability at 4, 20 and 50 μM (Fig. 1A). The IC₅₀ was ~5 μM for 5-FU-sensitive cells and 45 μM for resistant cells. As hypothesized, the expression of miR-34a was significantly downregulated in 5-FU-resistant cells compared with sensitive cells (Fig. 1B), suggesting that miR-34a may act as a tumor suppressor in human colon cancers and that it is involved in the development of resistance to 5-FU.

The initial results showed that miR-34a is downregulated in 5-FU-resistant cells. Potential targets of miR-34a were then investigated. An miRNA database (www.targetscan.org) was searched for predicted targets of miR-125b that may contribute to 5-FU resistance. The public miRNA database, TargetScan, predicted that LDHA may be a target for miR-34a, and showed that the 3′-UTR of LDHA contains a highly conserved binding site for miR-34a (Fig. 2A). To the best of our knowledge, thus far no publication has reported that LDHA is a direct target of miR-34a in colon cancer cells. To determine whether miR-34a targets LDHA in colon cancer cells, pre-miR-34a was transfected into DLD-1 cells. Overexpression of miR-34a significantly downregulated expression of the LDHA protein (Fig. 2B). The following experiment sought to investigate whether miR-34a directly targets the 3′-UTR of LDHA mRNA. A luciferase reporter analysis was performed by co-transfecting a vector containing pMIR reporter-luciferase fused with either the original 3′-UTR sequence or a sequence with a mutation in the predicted binding site of the 3′-UTR of LDHA mRNA, and with either pre-miR-34a or control microRNA. Overexpression of miR-34a decreased the luciferase activity of the reporter containing the wild type 3′-UTR of LDHA by ~60% in DLD-1 cells (Fig. 2C). However, no such inhibitory effects of miR-34a on the activity of the reporter fused with the 3′-UTR of LDHA with the mutation in the predicted binding site were detected (Fig. 2C), These results demonstrate that LDHA is a direct target of miR-34a in colon cancer cells.

Previous studies have shown that dysregulated cellular metabolism is associated with 5-FU resistance in cancer cells (19). LDHA catalyzes the final step in the glycolytic pathway, which is the conversion of pyruvate and nicotinamide adenine dinucleotide dehydrogenase to lactate and nicotinamide adenine dinucleotide, and is known to be involved in tumor maintenance. The present study aimed to investigate whether LDHA is involved in miR-34a-mediated 5-FU resistance in colon cancer cells. Notably, the expression of LDHA was upregulated at protein and mRNA levels in 5-FU-resistant cells (Fig. 3A and B), suggesting that the downregulation of miR-34a in 5-FU-resistant cells may contribute to LDHA upregulation. Consistent with this, the activity of LDH and the levels of lactate were increased in 5-FU-resistant cells compared with 5-FU-sensitive cells (Fig. 3C and D). These results demonstrate that LDHA is associated with 5-FU resistance in colon cancer cells and may be a promising therapeutic target.

To investigate the mechanism underlying the association between miR-34a-mediated downregulation of LDHA and 5-FU resistance in colon cancer cells, miR-34a was exogenously overexpressed in DLD-1 5-FU-sensitive and resistant cells through transient transfection. These cells were then treated with increasing concentrations of 5-FU for 72 h. Transfection with miR-34a significantly inhibited cell viability in sensitive and resistant cancer cells (Fig. 4A and B). Compared with control microRNA, overexpression of miR-34a in 5-FU-sensitive cells led to a decrease in IC₅₀ from 4 to 1 μM. The IC₅₀ of 5-FU-resistant cells in response to 5-FU decreased from 45 to 8 μM. To verify whether overexpression of miR-34a sensitizes colon cancer cells to 5-FU treatment through inhibition of LDHA, LDHA was transiently transfected into DLD-1 5-FU-sensitive cells (Fig. 5A) and the sensitivity to increasing concentrations of 5-FU for 72 h was measured (Fig. 5B). The results demonstrated that exogenous overexpression of LDHA rendered DLD-1 cells resistant to 5-FU, indicating that overexpression of miR-34a results in cells that are susceptive to 5-FU via the inhibition of LDHA.