Cell culture and induction of drug-resistant cell lines
Caki-2 cells, a human RCC cell line, were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured in the McCoy’s 5A medium (Thermo Fisher scientific, Massachusetts, USA) supplied with 10% FBS and 100 µg/ml double-antibody at 37 °C with the humidified 5% CO2. Caki-2/DOX (doxorubicin-resistant) and Caki-2/VBL (vinblastine-resistant) cells, the drug-resistant RCC cell lines, were constructed via Caki-2 cell lines (their independent parental cell lines) being exposed to the IC50 concentration of DOX and VBL for 3 months, and then exposed to tenfold higher dose of IC50 for 6 months with the same cultural conditions as Caki-2 cell lines [12].
Cell transfection
The RCC cell lines (Caki-2, Caki-2/DOX, and Caki-2/VBL) were seeded in the 6-well plates with the density of 2 × 105 cells/ml, and then maintained for 24 h. Caki-2 cells were transfected with miR-210-3p mimic/pre-NC (20 nM) or miR-210-3p inhibitor (50 nM) + si-ABCC1/si-control (20 nM) using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s protocol. Caki-2/DOX and Caki-2/VBL cells were transfected with miR-210-3p mimic/NC or miR-210-3p mimic + pcDNA-ABCC1/pcDNA by Lipofectamine 2000 (Invitrogen). The transfected RCC cells were maintained for 48 h, followed by harvested for the next experiments. The detailed sequence information was as follows: miR-210-3p mimic, 5-CUGUGCGUGUGACAGCGGGUGA-3; miR-210-3p inhibitor, 5-UCAGCCGCUGUCACACGCACAG-3; si-ABCC1, 5-GUUCCAAGGUGGAUGCGAATT-3. ABCC1 overexpression construct (pcDNA-ABCC1) was synthetized by Guangzhou RiboBio Co., Ltd (Guangzhou, China). The ABCC1 sequence was amplified with forward (F, 5-GTCGACACCATGGCCTGCTATTGC-3) and reverse (R, 5-GATGGATCCGCAGCAGAATGCCCAG-3) primers. After sequence validation, the sequences were subcloned into pcDNA3.1 vector.
Quantitative real-time PCR
The levels of miR-210-3p expression and ABCC1 mRNA expression were determined by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from RCC cell lines using TRIzol (Invitrogen). The extracted RNAs were reverse transcribed to complementary DNA with the PrimeScript® RT reagent kit (TaKaRa). The levels of miR-210-3p expression and ABCC1 mRNA expression were quantified by SYBR® Premix DimerEraser kit (TaKaRa) with 7500 Fast Real-Time PCR System in the ABI Prism 7500 (Applied Biosystems). U6 was used as the internal control for miR-210-3p, and GAPDH acted as the internal control for ABCC1. Comparative CT method, 2−ΔΔCt, serves as the calculation method of relative gene expression. The primer sequences used in qPCR were as follows: miR-210-3p, forward 5-GTGCAGGGTCCGAGGT-3, reverse 5-TATCTGTGCGTGTGACAGCGGCT-3; MDR1, forward 5-CCCATCATTGCAATAGCAGG-3, reverse 5-TGTTCAAACTTCTGCTCCTGA-3; ABCC1, forward 5-ATGTCACGTGGAATACCAGC-3, reverse 5-GAAGACTGAACTCCCTTCCT-3; U6, forward 5-CTCGCTTCGGCAGCACA-3, reverse 5-AACGCTTCACGAATTTGCGT-3.
Western blotting
The levels of ABCC1 and MDR-1 protein were assessed by Western blot assays in accordance with previous report [13]. Total protein from RCC cell lines was extracted with RIPA lysis buffer and then separated by SDS-PAGE on 10% acrylamide gels, followed by transferred into PVDF membrane. Afterwards, the membrane was incubated with primary antibodies against ABCC1 (1:1000 dilution, Abcam, Cat. no. ab99531), MDR-1 (1:500 dilution, Abcam, Cat. no. ab129450) and β-actin (1:3000 dilution, Abcam, Cat. no. ab8226) (4 °C overnight) and then maintained with HRP-conjugated secondary antibody for 1 h. Protein bands were visualized with Amersham ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA).
Cell viability
For drug-resistant analysis, the cell viability assays were performed. RCC cells (2 × 104 cells/well) were planted in 96-well plates and cultured at 37 °C with a 5% CO2 humidified atmosphere for 24 h. Afterwards, DOX with various concentrations (0, 10, 50, 100, 200, 400 μg/ml and 0, 1, 2, 3, 4, 5 μg/ml) were respectively administrated to Caki-2/DOX and Caki-2 cell culture, and VBL at various concentrations (0, 10, 50, 100, 200, 300 and 0, 1, 2, 3, 4, 5 μg/ml) were respectively administrated to Caki-2/VBL and Caki-2 cell culture for 24 h of incubation. The MTT assays were applied to analyze the viabilities of each RCC cell line in accordance with previous report [14]. MTT solutions (20µl, 5 mg/ml) was added to each well for 4 h at 37 °C.
Dual Luciferase assay
The bind site of miR-210-3p and ABCC1 were predicted and the wild type (WT) and mutant (Mut; the bind site was mutant) fragment of ABCC1 was shown in Fig. 3a. Two fragments were amplified by PCR using the primers: for the WT segment, 5′-AATTAGATCTAAAGAAAAGCGAGAGCAGCA-3′ (forward) and 5′-AATTAGATCTGCTCTCTGGGTTTGAAGTCG-3′ (reverse); for the Mut segment, 5′-AATTAGATCTGCTGTGAAGCACACGGAGAG-3′ (forward) and 5′-AATTAGATCTCAGACATTCGCGGTCAGAGA-3′ (reverse). Two ABCC1 fragments were respectively cloned into the downstream of the luciferase gene of pGL3 Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to synthesize the recombinant reporter vector named pGL3-ABCC1-WT and pGL3-ABCC1-Mut. The reporter vector pGL3-ABCC1-WT/pGL3-ABCC1-Mut and miR-210-3p inhibitor/NC were co-transfected into Caki-2 cells using Lipofectamine 2000 (Invitrogen). The reporter vector pGL3-ABCC1-WT/pGL3-ABCC1-Mut and miR-210-3p mimic/pre-NC were co-transfected into Caki-2/DOX and Caki-2/VBL cells using Lipofectamine 2000 (Invitrogen). After positive lysis of the cells, multimode detector with the Dual-Luciferase Reporter Assay System (Promega) was used to evaluate the activities of luciferase.
Xenograft model
In order to analyze the effects of miR-210-3p on drug-resistant renal tumor growth in vivo, a nude mouse tumor xenograft model was established. The present study was approved by Animal Care and Experimentation Committee of The First Affiliated Hospital of Zhengzhou University. Nude mice were transplanted subcutaneously with 2.5 × 106 Caki-2/DOX cells with/without miR-210-3p over-expression into the right flank [pre-NC group (n = 8) and miR-210-3p mimic group (n = 8)]. After 10 days, the mice of two groups were treated with DOX (2 mg/kg/day) via intraperitoneal injections. Every 3 days, the length (L) and width (W) of tumor in mice were measured, and the tumor volume was calculated using the following equation: (L × W2)/2. After 30 days, the mice were killed and tumor tissues were removed for the following study.
In the following experiment, Mice were transplanted subcutaneously with 2.5 × 106 Caki-2 cells with/without miR-210-3p knockdown into the right flank [NC group (n = 8) and miR-210-3p inhibitor group (n = 8)]. After 10 days, the mice of two groups were treated with DOX (2 mg/kg/day) via intraperitoneal injections. Every 3 days, the length (L) and width (W) of tumor in mice were measured, and the tumor volume was calculated using the following equation: (L × W2)/2. After 30 days, the mice were killed and tumor tissues were removed for the following study.
Statistical analysis
All data from three independent repeated experiments were exhibited as the mean ± SD and statistically analyzed with Student’s t test for single comparison between two groups and one-way ANOVA for comparison of multiple groups on SPSS 17.0 (SPSS Inc., Chicago, IL, USA). A value of P less than 0.05 was considered statistically significant.