MiR-30a-5p confers cisplatin resistance by regulating IGF1R expression in melanoma cells

Human melanoma cancer cells (M8, M8/DDP, SK-Mel-19 and SK-Mel-19/DDP) and HEK293T cells were obtained from Jennio Biotech. (Jennio, China). All cells were cultured in Dulbecco Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 IU/ml penicillin (Gibco, USA), and 100 μg/ml streptomycin (Gibco, USA) in a humidified atmosphere containing 5% CO₂ at 37 °C, and subcultured every 3 days at 10–30% confluence. To establish cisplatin-resistant M8/DDP and SK-Mel-19/DDP cells, M8 and SK-Mel-19 cells were first treated with 0.52 μmol/L (μM) of cisplatin (Hansoh, China), and then were treated with increased concentrations of cisplatin in a stepwise manner during each passage. To maintain the drug-resistance phenotype, cisplatin (with the final concentration of 33.4 μM) was added to the culture media for M8/DDP and SK-Mel-19/DDP cells.

Human melanoma cancer cells M8, M8/DDP, SK-Mel-19 and SK-Mel-19/DDP were plated in 6-well plates (3 × 10⁵ cells/well) and 100 pmol of the miR-30a-5p mimic or mimic control were transfected into M8 and SK-Mel-19 cells, while 100 pmol of the miR-30a-5p inhibitor or inhibitor control were transfected into the M8/DDP and SK-Mel-19/DDP, using Lipofectamine-2000 (Invitrogen, USA) according to the manufacturer’s protocol. 24 h after medium changed, the cells were seeded in 96-well plates (M8 and M8/DDP were 4 × 10³ cells/well; SK-Mel-19 and SK-Mel-19/ DDP were 5 × 10³ cells/well) for the following experiment. After cell adhesion, cisplatin was added at a final concentration of 4.18, 8.35, 16.7, 33.4, 66.8 μM to cells. 72 h after the addition of the drug, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS, Promega, USA) assay according to the manufacturer’s protocol. The absorbance at 490 nm (A490) of each well was read on iMark Microplate Absorbance Reader (Bio-Rad, USA).

Total RNA was isolated from cells at the logarithmic phase using Trizol (Sigma, USA) according to the manufacturer’s protocol. First-strand cDNA was synthesised using GoScript™ Reverse Transcription System Kit (Promega, USA). Real-time PCR was performed with GoTaq qPCR Master Mix (Promega, USA) using a C1000 Thermal Cycler apparatus (Bio-Rad) in 20 μl reaction volume according to manufacturer’s protocols. The procedure was as follows: 95 °C-3 min; 39 × (95 °C-15 s, 60 °C-60 s,72 °C-30 s, for mRNA; 95 °C-15 s, 60 °C-60 s for miRNA); 95 °C-10 s, followed by a melt curve analysis (60 °C to 95 °C, increment 0.5 °C for 20 s) to confirm specificity of the PCR primers. Threshold cycle (Ct)-values for mRNA and miRNA species were normalized to housekeeping genes: GADPH for mRNAs and U6 for mature miRNAs (Primers were listed in Table 1). The fold change was calculated using the 2^-ΔΔCt Method.

Cells were lysed in SDS lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Takara, China). Total protein was quantified by BCA Protein Assay Kit (Beyotime, China), and proteins (10 μg/per lane) were separated by SDS–PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Pall, USA). The membranes were blocked in bovine serum albumin (BSA) (3% w/v in PBS + 0.1% w/v in Tween 20) for 30 min at room temperature and incubated with diluted antibodies at 4 °C overnight. Proteins were detected by enhanced chemiluminescence system (Pierce, USA) according to the manufacturer’s instructions. Data were normalized to GAPDH.

To knock down miR-30a-5p expression, TuD-miR-30a-5p was constructed based on the Tough Decoy (TuD) design [17]. Oligonucleotides of the Tough Decoy RNA were annealed and cloned into BamHI and MluI site of lentiviral vector pLent-U6-GFP-puro (ViGene, China), resulting in TuD-miR-30a-5p being driven by polymerase III promoter U6. Lentivirus was produced by transfecting HEK293T cells with each lentiviral construct together with the packaging vectors psPAX2 and pMD2.G (ViGene, China) using Lipofectamine-2000 (Introvigen, USA) according to the instructions of the manufacturer. The supernatant was collected 72 h after transfection and was centrifuged (4000 g for 5 min at room temperature) to remove cell debris; the supernatant was used for M8/DDP and SK-Mel-19/DDP cells infection. The infected cells were then selected by supplementing the culture medium with 6 μg/ml of puromycin 48 h after infection. The efficiency of the inhibition of miRNAs was confirmed by real-time PCR analysis.

Based on the miRNA databases (microRNA.​org, miRDB and TargetScan databases), IGF1R is a predicted target of miR-30a-5p. Hence, we cloned IGF1R 3’-UTR fragment containing the predicted site (5’-GTTTACA-3′ and 5’-TGTTTAC-3′) or the mutant sequence (5’-CAAATGT-3′ and 5’-ACAAATG-3′) into psiCHECK™-2 luciferase reporter vector (Promega, USA) (Primers were listed in Table 1). For luciferase assay, the reporter plasmid was co-transfected with miR-30a-5p mimic or mimic control in HEK293T cells. After 48 h, cells were lysed and luciferase expression was measured using the Dual-luciferase assay system (Promega, USA) following the manufacturer’s protocol. The renilla luciferase (Rluc) was normalized by the firefly luciferase (Luc).

GraphPad Prism software (La Jolla, CA) was used to plot the curves and statistical analysis. Data were presented as mean ± SD from at least three independent experiments. Statistical analysis was performed by Student’s t test. p values of < 0.05 (*), < 0.01(**), and < 0.001 (***) were considered significant.

Two cisplatin-resistant cell lines M8/DDP and SK-Mel-19/DDP were induced by continuous exposure to cisplatin after 5 months for more than 50 cell passages. The cell lines were used for experiments after culturing in drug-free medium for another 2 months. We then tested the half maximal inhibitory concentration (IC₅₀) and drug resistance indices (RI) of the resistant cells as well as their parental cells by MTS assay. In Fig. 1a and b, the IC₅₀ of M8 cells was 3.97 μM, the IC₅₀ of M8/DDP cells was 21.23 μM, the resistance index was 5.3; the IC₅₀ of SK-Mel-19 cells was 10.16 μM, the IC₅₀ of SK-Mel-19/DDP cells was 31.93 μM, and its resistance index was 3.1. The results indicated that the resistant lines were established successfully. Since the drug-resistant cells differed significantly from their parental cells at cisplatin concentrations of 4.18 μM, 8.35 μM, 16.7 μM, 33.4 μM, and 66.8 μM, these five concentrations were selected for follow-up experiments.

We used microRNA microarray analysis to screen the differential expressed miRNAs (≥2.0 fold or ≤ 0.5 fold) between the resistant cells and their parental cells, and 21 miRNAs were verified by real-time PCR and listed in Table 2. Among them, a cancer-related miRNA, miR-30a-5p, was found to be markedly up-regulated in both M8/DDP and SK-Mel-19/DDP cells. This difference in expression of miR-30a-5p was also validated by real-time PCR analysis (Fig. 1c), the difference multiples of miR-30a-5p were 35.37 and 5.65.

To explore the potential roles of miR-30a-5p in the cisplatin-resistance of melanoma cells, in-vitro studies using miR-30a-5p mimics and inhibitors were performed. As shown in Fig. 2a, the IC₅₀ of M8 cells increased when transfected with miR-30a-5p mimic, which indicated their resistance to cisplatin was enhanced. Similar results were obtained in SK-Mel-19 cells. On the other hand, the IC₅₀ of M8/DDP cells was markedly reduced after transfected with miR-30a-5p inhibitor, which could also be repeated in the SK-Mel-19/DDP cells.

Transfection into a cell can be transient or stable. To further study the relationship between miR-30a-5p and cisplatin resistance in melanoma, we constructed a Tough Decoy (TuD) vector expressing a miR-30a-5p-TuD that could stably suppress the expression of miR-30a-5p in M8/DDP and SK-Mel-19/DDP cells (Fig. 2b). Knockdown efficiency of the stable transfection was validated by the real-time PCR analysis (Fig. 2c), the transfection of TuD-miR-30a-5p reduced the expression of miR-30a-5p by more than 40% in M8/DDP and SK-Mel-19/DDP cells. As shown in Fig. 2d, the IC₅₀ of M8/DDP and SK-Mel-19/DDP cells decreased significantly after stably transfected with TuD-miR-30a-5p. In both transient transfection and stable transfection approaches, the sensitivity of the melanoma cells to cisplatin was influenced by the change of miR-30a-5p expression. These provide the evidence of the association between miR-30a-5p and cisplatin-resistance of the melanoma cells.

Identification of miRNA-regulated gene targets is a necessary step to understand miRNA functions. First, we used the online tools (microRNA.​org, miRDB and TargetScan databases) to screen the potential target genes of miR-30a-5p. Among the genes obtained from screening results, we noticed that a cancer-related gene, IGF1R, might be a tentative target of miR-30a-5p whose 3’-UTR contains two putative miR-30a-5p seed sites (Fig. 3a). We noticed that the mRNA and protein levels of IGF1R in M8/DDP and SK-Mel-19/DDP were significantly lower than those in the parental cells (Fig. 3b & c), which were contrary to the expression levels of miR-30a-5p in the two groups. In the in-vitro approaches, transfection of miR-30a-5p mimics could reduce the protein levels of IGF1R in M8 and SK-Mel-19 cells, while transfection of miR-30a-5p inhibitors could promote the expression of IGF1R in the resistant cells. At the same time, we noticed that the mRNA levels of IGF1R were less influenced by the alteration of miR-30a-5p expression (Fig. 3d and e), which suggested that miR-30a-5p could not induce the degradation of IGF1R mRNA.

To test whether the predicted miR-30a-5p target sites in the 3’-UTR of IGF1R mRNA were responsible for its regulation, we cloned IGF1R 3’-UTR wild type (wt) or 3’-UTR mutant types (mut A, mut A and mut A B) into downstream of the luciferase reporter gene. In the absence of miR-30a-5p, luciferase would be expressed, whereas in the presence of miR-30a-5p, luciferase mRNA would be degraded. As shown in Fig. 3f, when the double luciferase reporter vector containing the wild type of IGF1R 3’-UTR and miR-30a-5p mimic were co-transfected into HEK293T cells, the Renilla luciferase activities were significantly inhibited. Mutations of the IGF1R 3’-UTR binding site in region A drastically abolished ability of miR-30a-5p to regulate luciferase expression, while mutations in the region B partially impaired the luciferase expression. Thus, miR-30a-5p could directly bind to both region A and B on 3’-UTR of IGF1R gene.

We next investigated whether IGF1R itself could influence cisplatin resistance in the two melanoma cell lines. We conducted small interfering RNA (siRNA)-based silencing of IGF1R and assessed its efficiency in M8 and SK-Mel-19 cells. As shown in Fig. 4a, siRNA3 was more efficient in M8 cells and siRNA2 was more efficient in SK-Mel-19 cells. After transfection these two small interfering RNA, the expression of miR-30a-5p reduced more than 40% in M8 and SK-Mel-19 cells. Then, after IGF1R was knockdown in the M8 and SK-Mel-19 cells using the appropriate siRNAs, the sensitivities of both cell lines to cisplatin were reduced (Fig. 4b).This suggested the direct correlationship between IGF1R and cisplatin-resistance in melanoma cells.

As a receptor of insulin-like growth factor, IGF1R has tyrosine kinase activity and plays a critical role in transformation events. Previous studies have shown that activation of the IGF1R signaling pathway is involved in proliferation, survival, and metastasis of cancer cells. In the present study, we noticed that the expression of phosphorylation of AKT (Ser473) was lower while the expression of P53 protein was higher in the resistant cells compared to their parental cells (Fig. 4c). We wondered whether it was associated with the alteration of IGF1R. In the subsequent in-vitro studies, we used the specific siRNA to knockdown the expression of IGF1R in M8 and SK-Mel-19 cells, and found that the phosphorylation of AKT (Ser473) was impaired while the protein levels of P53 were increased (Fig. 4d). At the same time, cell cycle arrests were detected in both cell lines (Fig. 5a). Based on the IC₅₀ of M8 and SK-Mel-19 cells (Fig. 1a and b, Fig. 4b), we selected three concentrations of cisplatin(4.18 μM, 8.35 μM and 16.7 μM) that cover the range of IC₅₀ of the both cell lines for cell cycle examination. Interestingly, we found that cisplatin at concentration of 8.35 μM influence the cell cycle most significantly in the both cell lines. As shown in Fig. 5a, after M8 cells were treated with cisplatin, a G2/M arrest was induced. When the IGF1R was silenced before the cisplatin treatment, the G2/M arrest was impaired. On the other hand, in case of SK-Mel-19 cells, cisplatin treatment induced cell cycle disruption. However, when the IGF1R was silenced before the cisplatin treatment, the cell cycle only varied a little after cisplatin treatment. Thus, IGF1R might play a protective role in cisplatin-mediated DNA damage through regulating cell cycle by influencing AKT/P53 pathway in melanoma cells.