2021 Volume 44 Issue 6 Pages 861-868
MicroRNA-221 (miRNA-221) is upregulated in several malignant tumors and is associated with poor patient prognosis. Therefore, the present study aimed to investigate the role and underlying mechanism of miRNA-221 in doxorubicin (DOX) resistance in osteosarcoma cells. We constructed DOX-resistant Saos-2/DOX cells and treated them with DOX. Cell viability was determined by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were transfected with either miRNA-221 mimic or miRNA-221 inhibitor; quantitative (q)RT-PCR was performed to detect the expression of miRNA-221. Flow cytometry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) staining were used to detect cell apoptosis. The immunofluorescence method was also used to detect cell signal transduction and activator of transcription 3 (Stat3) protein expression distribution. In addition, Western blotting was used to detect changes in the expression of each protein. We found that miRNA-221 was upregulated in Saos-2/DOX cells. Moreover, the miRNA-221 mimic induced DOX resistance in Saos-2 cells, whereas the miRNA-221 inhibitor enhanced DOX sensitivity in Saos-2/DOX cells. The miRNA-221 mimic upregulated the expression of phosphorylated-Stat3, P-glycoprotein (P-gp), and B-cell lymphoma-2 (Bcl-2) proteins in Saos-2 cells and induced the entry of Stat3 into the nucleus, whereas the miRNA-221 inhibitor exerted the opposite effect. Pretreatment with the Stat3 chemical inhibitor, STAT3-IN-3, significantly inhibited the upregulation of P-gp and Bcl-2 protein expression induced by the miRNA-221 mimic in Saos-2 cells; it also caused the Saos-2 cells to overcome DOX resistance induced by the miRNA-221 mimic. Thus, miRNA-221 increased the expression of P-gp and Bcl-2 by activating the Stat3 pathway to promote DOX resistance in osteosarcoma cells, indicating a potential use of miRNA-221 in osteosarcoma treatment.
Osteosarcoma is the most common primary malignant bone tumor, generally occurring in adolescents aged 10–20 years.1,2) Conventionally, its treatment involves surgery combined with radiotherapy and chemotherapy. In particular, preoperative chemotherapy is a key factor in the prognosis of patients with osteosarcoma.3,4) The use of chemotherapy drugs has been reported to prolong the survival time of patients to a certain extent, but the emergence of acquired drug resistance eventually leads to treatment failure.5) Doxorubicin (DOX) is an antibiotic chemotherapeutic drug that can intercalate between DNA base pairs, preventing DNA replication and transcription by inhibiting RNA synthesis and ultimately killing tumor cells.6,7) DOX is used as a first-line treatment for acute leukemia, acute lymphoblastic leukemia, and myeloid leukemia.8,9) It is also effective against other malignant tumors such as osteosarcoma, malignant lymphoma, breast cancer, lung cancer, and bladder cancer.10,11) Therefore, investigating and addressing DOX resistance in the clinical setting is important.
A microRNA (miRNA) is a non-coding RNA with a size of approximately 21–25 nucleotides. Since Lee et al.12) discovered these molecules in 1993, extensive research on miRNAs has been performed internationally. miRNAs regulate gene expression and participate in cell differentiation and tissue development.13) Under pathological conditions, miRNAs are involved in the occurrence and development of malignant, neurological, and cardiovascular diseases.14–16) Abnormal miRNA expression can lead to abnormal activation of signaling proteins to promote cancer development.16) miRNA-221, a recently discovered miRNA, is upregulated in osteosarcoma,17) glioma,18) hepatocellular carcinoma,19) and ovarian cancer.20) The expression of miRNA-221 has been associated with the malignant biological behavior of tumors and poor prognosis of patients.17–20) However, the correlation between miRNA-221 and drug resistance in osteosarcoma needs to be further investigated. This study therefore aimed to investigate the role of miRNA-221 in DOX-resistance in osteosarcoma by constructing DOX-resistant cells, observing the effect of miRNA-221 activity on DOX resistance, and exploring the mechanisms underlying this effect.
The human osteosarcoma Saos-2 cell line was purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai, China). Cell lines were maintained in a 5% CO2 atmosphere at 37 °C in McCoy’s 5A medium (HyClone, Logan, UT, U.S.A.) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin (HyClone), and 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, U.S.A.). Saos-2 cells were exposed to gradually increasing drug concentrations of 0.001–5 µM DOX (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 5 months. Finally, a DOX-resistant cell line, Saos-2/DOX, was successfully constructed. Saos-2/DOX cells were cultured with 1 µM DOX for 1 week before use in experiments to maintain drug resistance.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) AssayCells were seeded in a 96-well plate, with 5000 cells per well and subsequently treated with gradient concentrations of DOX (0.001–10 µM) for 48 h. After this, 10 µL MTT (5 mg/mL; Solarbio, China) was added to each well, and plates were further incubated for 4 h at 37 °C. Subsequently, the culture medium was discarded, and 100 µL dimethyl sulfoxide was added to each well. Absorbance at 490 nm was measured with an xMark™ microplate reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
Quantitative (q)RT-PCRTotal RNA was extracted from the cells using TRIZOL (Solarbio). TaqMan MicroRNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA, U.S.A.) was used to reverse transcribe the RNA into cDNA. TaqMan Universal Master Mix II (Invitrogen) was used to perform qPCR using the ABI 7300 real-time fluorescence quantitative PCR system (Applied Biosystems, Foster City, CA, U.S.A.). U6 (Applied Biosystems) was used for normalization. The expression level of miR-221 was calculated using the 2−ΔΔCt method. The following primers were used: miR-221 forward, 5′-GAA GTT CGT CCA GCT ACA TTG TCT-3′; miR-221 reverse, 5′-TAT GGT TGT TCT CGT CTC TGT GTC-3′; U6 forward, 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′; and U6 reverse, 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′.
Cell TransfectionAfter the cells had grown to the logarithmic phase, 5 × 105 cells per well were seeded in a 6-well plate. Transfection was started when the cells grew to 90% confluence. Saos-2 cells were transfected with the negative control (NC) and a miRNA-221 mimic (15 nM; Genepharma, China). Saos-2/DOX cells were transfected with the negative control inhibitor (NC inhibitor) and miRNA-221 inhibitor (80 nM, Genepharma), in accordance with the transfection instructions provided for Lipofectamine 2000 Reagents (Genepharma). After 48 h of transfection, the cells were used for subsequent experiments.
Immunofluorescence AnalysisCells (6 × 106) were plated in a cell culture dish equipped with a sterile glass slide and cultured for a specified time. The glass slide was removed, and the cells were fixed with 4% paraformaldehyde. The membrane structure was permeated with 0.5% Triton X-100 (Solarbio), and the cells were blocked with goat serum for 30 min. Cells were incubated with primary anti-signal transduction and activator of transcription 3 (Stat3) monoclonal antibodies (1 : 800; Cell Signaling Technology, Beverly, MA, U.S.A.) overnight at 4 °C, followed by incubation with fluorescent secondary antibody (1 : 50) for 1 h in a dark room at room temperature and with 0.5 µg/mL 4′,6-diamidino-2-phenylindole (Solarbio) for 5 min in the dark. The slides were mounted with glycerol and visualized and photographed using an IX73 fluorescence microscope (Olympus, Japan).
Flow CytometryAn annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Absin, China) was used to detect cell apoptosis. According to the instructions, cells were suspended in 1× binding buffer at a density of 1 × 106 cells/mL; 100 µL of this cell suspension was transferred to a 1.5-µL Eppendorf tube, and 5 µL each of FITC-Annexin V and propidium iodide were added. Samples were incubated for 15 min in a dark room at room temperature. Cell apoptosis was analyzed using an Accuri C6 Plus flow cytometer (BD Bioscience, San Jose, CA, U.S.A.).
Western BlottingThe cells were treated with radio immunoprecipitation assay lysis buffer (Solarbio) for 10 min for lysis. Protein lysis products were then centrifuged at 12000 rpm at 4 °C and for 20 min. The supernatant contained the total protein fraction. A bicinchoninic acid protein quantification kit (Solarbio) was used to determine the protein concentration. Thirty micrograms of protein was loaded per well for conducting gel electrophoresis. The proteins were separated using polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with 5% skim milk for 4 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies against B-cell lymphoma-2 (Bcl-2; ab32124, 1 : 2000), signal transduction and activator of transcription 3 (Stat3; ab68153, 1 : 2000; Abcam, Cambridge, MA, U.S.A.), P-glycoprotein (P-gp; #13342, 1 : 2000), phospho-Stat3 (#9145, 1 : 2000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174, 1 : 3000; Cell Signaling Technology). The membranes were washed with Tris-buffered saline with Tween solution (TBST). Anti-rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP)-linked antibodies (7074S, Cell Signaling Technology) were added, and the cells were incubated for 1 h at room temperature in the dark. The membrane was washed with TBST, developed by using an enhanced chemiluminescence (ECL) chemiluminescence kit (Merck Millipore Company, Billerica, CA, U.S.A.), and imaged using an iBright Intelligent Imaging System (Invitrogen).
Statistical AnalysisAll data were analyzed with SPSS 17.0 for Windows independently at least three times. All data were tested for normal distribution. Normally distributed data were represented as x̄ ± s, and non-normal data were converted into normal distribution data for analysis by natural logarithms. One-way ANOVA and Fisher’s multiple comparison test were performed, with p < 0.05 considered statistically significant.
A DOX-resistant osteosarcoma cell line, Saos-2/DOX, was successfully constructed. MTT results showed that the IC50 of DOX in Saos-2 and Saos-2/DOX cells was 226.3 ± 13.5 nM and 10.6 ± 0.9 µM, respectively, and the resistance index of Saos-2/DOX cells was 47.0 (Fig. 1A). Compared to Saos-2 cells, the Saos-2/DOX cells had significantly higher levels of miRNA-221 expression (Fig. 1B). To further explore the role of miRNA-221 in DOX resistance, we transfected Saos-2 cells with a miRNA-221 mimic, which was found to significantly enhance the expression levels of miRNA-221 in Saos-2 cells (Fig. 1C). Notably, transfection with the miRNA-221 mimic significantly increased the IC50 of DOX in Saos-2 cells (p < 0.01) (Fig. 1D). Conversely, transfection with a miRNA-221 inhibitor significantly downregulated the expression levels of miRNA-221 in Saos-2/DOX cells and significantly reduced the IC50 of DOX (p < 0.01; Figs. 1E, F).
(A) Saos-2 and Saos-2/DOX cells were treated with gradient concentrations of DOX (1 nM–100 µM) for 48 h. Cell viability was detected via MTT assay, and the IC50 value was calculated. (B) RT-qPCR was performed to detect the relative expression levels of miRNA-221 in Saos-2 and Saos-2/DOX cells. (C) After transfection with the negative control (NC) or miRNA-221 mimic, the relative expression level of miRNA-221 in cells was detected using RT-qPCR. (D) Control, NC, and miRNA-221 mimic Saos-2 cells were treated with gradient concentrations of DOX (1 nM–100 µM) for 48 h; cell viability was measured using MTT, and the IC50 values were calculated. (E) After transfection of the NC inhibitor and miRNA-221 inhibitor, RT-qPCR was used to detect the relative expression levels of miRNA-221 in the Saos-2/DOX cells. (F) Saos-2/DOX cells in the control, NC inhibitor, and miRNA-221 inhibitor groups were treated with gradient concentrations of DOX (1 nM–100 µM) for 72 h. Cell viability was detected by MTT, and the IC50 was calculated. ** p < 0.01. For all experiments, n = 6.
Flow cytometry results showed that transfection with the miRNA-221 mimic significantly increased resistance to DOX (200 nM)-induced apoptosis in Saos-2 cells (Fig. 2A). Meanwhile, transfection with the miRNA-221 inhibitor enhanced the sensitivity of Saos-2/DOX cells to DOX (1 µM; Fig. 2B). Western blot results showed that compared to NC Saos-2 cells, miRNA-221 mimic-transfected Saos-2 cells had significantly increased expression of P-gp and Bcl-2 proteins (p < 0.01; Fig. 3A). In addition, transfection with the miRNA-221 inhibitor significantly downregulated the expression of P-gp and Bcl-2 proteins in Saos-2/DOX cells (p < 0.01; Fig. 3B).
(A) Flow cytometric analysis after DOX treatment of control, NC, miRNA-221 mimic group Saos-2 cells for 48 h showing the apoptosis level of each group. ** p < 0.01. (B) Flow cytometric analysis after DOX treatment of control, NC inhibitor, and miRNA-221 inhibitor Saos-2/DOX cells for 48 h showing the level of apoptosis of cells in each group. ** p < 0.01. For all experiments, n = 3.
(A) Western blot detection of P-gp and Bcl-2 protein expression in Saos-2 cells in the control, NC, and miRNA-221 mimic groups compared to the NC group, ** p < 0.01. (B) Western blot detection of P-gp and Bcl-2 protein expression in Saos-2/DOX cells in the control, NC inhibitor, and miRNA-221 inhibitor groups, compared with the NC inhibitor, ** p < 0.01. For all experiments, n = 3.
Western blot results showed that the miRNA-221 mimic significantly upregulated the expression of the p-Stat3 protein in Saos-2 cells, whereas the miRNA-221 inhibitor downregulated the expression of the p-Stat3 protein in Saos-2/DOX cells (Fig. 4B). Immunofluorescence results showed that after miRNA-221 mimic treatment, the expression of Stat3 in the nucleus of Saos-2 cells increased, whereas treatment with a miRNA-221 inhibitor significantly reduced the expression of Stat3 in the nucleus of Saos-2/DOX cells (Fig. 4A). We pretreated Saos-2 cells transfected with the miRNA-221 mimic with the Stat3 chemical inhibitor STAT3-IN-3 (1 µM) for 4 h. Western blot results showed that inhibiting Stat3 significantly inhibited the upregulation of P-gp and Bcl-2 protein expression induced by the miRNA-221 mimic (Fig. 4C).
(A) An immunofluorescence assay was used to detect the expression and distribution of Stat3 in Saos-2 and Saos-2/DOX cells. (B) Western blotting was used to detect the expression of p-Stat3 in Saos-2 and Saos-2/DOX cells. ** p < 0.01. (C) Western blotting was used to detect the effect of STAT3-IN-3 on the expression of P-gp and Bcl-2 in Saos-2 cells. (D) ** p < 0.01, △△ p < 0.01. For all experiments, n = 3.
Flow cytometric analysis showed that STAT3-IN-3 pretreatment of Saos-2 cells reversed the DOX resistance induced by the miRNA-221 mimic (Fig. 5A).Terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling (TUNEL) staining also showed similar results (Fig. 5B).
(A) Representative graph of the apoptosis level in each group of Saos-2 cells as detected by flow cytometry. ** p < 0.01. (B) TUNEL staining-based detection of apoptosis in Saos-2 cells in each group. ** p < 0.01. For all experiments, n = 3.
In the present study, we showed that the expression level of miRNA-221 was elevated in DOX-resistant Saos-2/DOX cells. We also found that exogenous upregulation of miRNA-221 expression enhanced the resistance of Saos-2 cells to DOX, whereas the suppression of endogenous miRNA-221 expression improved the sensitivity of Saos-2/DOX cells to DOX. These are consistent with results of clinical studies.17)
DOX is an effective, first-line chemotherapy drug used for osteosarcoma. Treatment with DOX initially improves the survival rate of patients, but drug resistance development eventually leads to treatment failure.21,22) miRNAs, which are non-coding RNAs, can inhibit gene expression by inhibiting transcription or inducing the degradation of target genes.23) Some miRNAs are abnormally expressed in osteosarcoma cells and are involved in chemotherapy-resistant osteosarcoma.24,25) Moreover, in osteosarcoma, miRNA-221 regulates cell proliferation, apoptosis, migration, and invasion by targeting CDKN1B/p27.26) Clinical studies have also shown that elevated expression of miRNA-221 in osteosarcoma is associated with tumor staging, metastasis, and response to chemotherapy.17) Moreover, miRNA-221 is involved in the malignant biological behavior of osteosarcoma and in inducing resistance to chemotherapy drugs. Our molecular investigation showed that the miRNA-221 expression level was related to the expression of P-gp and Bcl-2; miRNA-221 mimics upregulated the expression of P-gp and Bcl-2 in Saos-2 cells, whereas application of the miRNA-221 inhibitor repressed P-gp and Bcl-2 expression in Saos-2/DOX cells. P-gp is an ATP-dependent membrane transporter protein encoded by the MDR1 gene.27) P-gp pumps drugs out of the cell, thereby reducing the concentration of the drug in the cell, leading to cell resistance.28,29) The upregulation of Bcl-2, a classic apoptosis inhibitor protein, is related to apoptosis evasion.30) We found that miRNA-221-induced an increase in the expression of P-gp, and Bcl-2 proteins promoted DOX resistance in osteosarcoma cells. We also showed that a miRNA-221 mimic induced the phosphorylation of Stat3 in Saos-2 cells, whereas the miRNA-221 inhibitor inhibited the phosphorylation level of Stat3 in Saos-2/DOX cells.
Stat3 is a major component of many signal transduction pathways and an activator of the transcription complex, which plays an important role in tumor cell proliferation, metastasis, angiogenesis, and drug resistance.31,32) Stat3 phosphorylation leads to the activation and formation of homodimers or heterodimers, and these dimers translocate to the nucleus to activate the transcription of target genes.33,34) Studies have confirmed that the target genes of Stat3 include bcl-2, bcl-xl, and mcl-1.35–37) Recent studies have shown that the activation of Stat3 is related to the upregulation of P-gp expression.38,39) Immunofluorescence experiments showed that the miRNA-221 mimic induced the entry of Stat3 into the nucleus, whereas the miRNA-221 inhibitor had the opposite effect. This indicates that miRNA-221 induced P-gp and Bcl-2 protein expression through the Stat3 signaling pathway, thereby promoting DOX resistance in osteosarcoma cells. For further verification, we evaluated the effect of intervention with the Stat3 chemical inhibitor STAT3-IN-3. STAT3-IN-3 pretreatment significantly inhibited the upregulation of P-gp and Bcl-2 protein expression induced by miRNA-221 mimic. Moreover, STAT3-IN-3 pretreatment reversed the DOX resistance induced by the miRNA-221 mimic.
In conclusion, we showed that microRNA-221 was upregulated in DOX-resistant osteosarcoma cells and was involved in this drug resistance. We also found that microRNA-221 promoted DOX resistance in osteosarcoma cells by upregulating the expression of P-gp and Bcl-2. The mechanism underlying this effect involved inducing the entry of Stat3 into the nucleus to promote transcription (Fig. 6). The findings of this study, after further validation, might help to establish new treatment strategies to reverse DOX resistance in osteosarcoma.
This work was supported by the Hengshui City Science and Technology Plan Project (No. 2016014054Z).
The authors declare no conflict of interest.
