The Role And Mechanism Of MicroRNA-184 In Doxorubicin Resistance Of Osteosarcoma Cells

BackgroundOsteosarcoma (OS) is malignant tumor in which the cancerouscells produce osteoid, the organic extracellular matrix of bone. It is the most common primary tumor of the bone and is an especially aggressive, predominantly pediatric cancer. Currently, the use of doxorubicin based chemotherapeutics in combination with aggressive surgery remians the goleden standard for the treatment of OS. [5-7] Since the advent of chemotherapy, the 5-year survival rate for osteosarcoma has been much improved. However, the prognosis of osteosarcoma is still poor and the long-term cure rate of OS patients has reached a plateau over the last 30 years. Therapeutic failure is mainly due to tumor recurrence, metastasis and resistance to chemotherapy including intrinsic or acquired doxorubicin resistance. Therefore, in tumor research, it is an important direction to elucidate the underlying mechanism of doxorubicin resistance in osteosarcoma and to search for the drug targets for reversing drug resistance.MicroRNA(miRNA) is a class of intrinsic noncoding small RNA,which can combine its complementary sequence with the 3 ’the translation section (3’utr) mRNA targets and cause inhibition of protein translation or degradation of target messenger RNA. MiRNA can be involved in various biological activities [8-10]. Currently, it has been suggested that mirnas participate widely in in the tumor development, tumor cell metastasis, proliferation and chemoresistance. Anomalous miRNAs expression can impact chemoresistance by modulating gene expression of a number of diferent target genes, such as miRNA-133b, miR-31, miRNA-196a, and miR-506. However, few studies have focused on the involvement of miRNA-184 in the development of doxorubicin resistance in osteosarcoma patients.MicroRNA-184 functions as a tumor suppressor in carcinomas such as ma-lignant gliomas and non-small cell lung cancers and all these antitumor functions are involved in cell proliferation and metastasis. However, little is known about the contribution of miR-184 to chemoresistance of cancers. In Our previous study, high expression of microRNA-184 has already been discovered in drug-resistance cell strain of osteosarcoma. Plenty of evidence shows that tumor metastasis and doxorubicin resistance are closely related. Up to now, it has been not shown about research of microRNA-184 in modulating doxorubicin resistance of osteosarcoma. Therefore, we predict that microRNA-184 might participate in regulating doxorubicin resistance of osteosarcoma and the mechanism will be discussed in this study.AimsTo ascertain the expression of miRNA-184 and regulatory role in doxorubicin resistance of osteosarcoma; to investigate the molecular mechanism of miRNA-184 in doxorubicin resistanceMethods1. Cell culture and transfectionU-2 OS and MG-63 were purchased from the Cell Bank of theInstitute of Biochemistry and Cell Biology, China Academy ofSciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS. For transfection, cells were cultured to 80% confuence and transfected with 50 nM miR-184 agomir or 100 nM antagomir according to the manufacturer’s protocol. Further treatment was done several hours after transfection.2.qRT-PCR(quantitative real-time polymerase chain reaction)Quantitative real-time polymerase chain reaction was conducted to measure the expression levels of microRNA-184 in OS cell line U-2 OS and MG-63 treated with doxorubicin. Total RNA, including miRNA, was extracted from the cell lines and tissue samples using TRIzol reagent. RNA was synthesized into cDNA using a reverse transcriptase kit. Amplifcation of cDNA was performed according to the manufacturer’s protocol using SYBR(?)Premix Ex TaqTM. PCR was carried out in triplicate and analyzed using the ABI 7500 Fast Realtime PCR system. The relative gene expression level was calculated by the comparative CT method 2-ΔΔCtusing U6 as an internal reference. The RT and PCR primers for miR-184 and U6 were purchased from RiBoBio.3. Apoptosis analysisCell apoptosis was analyzed in the U-2 OS cells transfected with miR-184 agomir or miR-184 antagomir. After 48 h of transfection in 6-well plates, cells were exposed to the freshly prepared medium containing diferent concentrations of 0.5 nM doxorubicin for 24 h. Cells were then harvested and washed with ice-cold phosphate-bufered saline (PBS) and then subjected to Annexin V Apoptosis Detection kit (BD Pharmingen, USA) for staining, which was followed by fow cytometry analysis. The test was repeated 3 times per experiment.4. Luciferase assayBCL2L1 was predicted to be target gene of miR-184 by TargetScan. The wide-type (WT) or mutant (MT) 3’UTR of BCL2L1 was cloned into the Renilla luciferase gene. A mutant 3’UTR of BCL2L1 was cloned by use of GeneChem (Shanghai GeneChem Co., Ltd, Shanghai, China). The U-2 OS cells were cotransfected with the vectors carrying WT 3’UTR or MT 3’UTR and miR-184 agomir. Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega Corporation, Fitchburg, USA). Luciferase activity values were normalized relative to that of the Renilla luciferase internal control.5. Expression analysis of BCL2L1Western blot was conducted to measure the protein expression of BCL2L1. Protein quantifcation was performed according to the Bradford method using the Bio-Rad protein assay kit. A total of 30 μg of samples were run on 10% SDS-PAGE. The proteins were then transferred to NC flter membranes. The membranes were blocked overnight in 5% skim milk in Tris-bufered saline. For immunoblotting, the membranes were incubated at 4℃ overnight with the BCL2L1 antibody or GAPDH antibody, fol-lowed by the incubation with the appropriate IRDye 800CW-conjugated secondary antibody. The infrared fuorescence image was obtained using the Odyssey infrared imaging system6. Gene interference testTo clarify the relation between BCL2L1 and miR-184, we further assayed the efect of miR184 on the expression of BCL2L1 in cells treated with or without doxorubicin. After 48 h of transfection in 6-well plates, cells were exposed to the freshly prepared medium containing concentration of 0.5 nM doxorubicin for 24 h. Cells were then harvested and washed with ice-cold phosphate-bufered saline (PBS). Western blot was further conducted to measure the protein expression of BCL2L1.7. Statistical analysisAll data were analyzed in SPSS 20.0; ONE-WAY ANOVA was applied in statistical evaluation, this method can diferentiate the means of diferent groups. A p-value of <0.05 was considered statistically signifcant.Results1. We first assessed the efects of doxorubicin on the expression of miR-184. Exposing MG-63 to 0.5 nM or 1.0 nM doxorubicin signifcantly promoted time-dependent expression of miR-184. After being treated with 0.5 nM doxo-rubicin for 12 h, the expression of miR-184 reached a peak and then declined. Moreover,1.0 nM doxorubicin-induced miR-184 expression mimicked the efect of 0.5 nM doxorubicin. Consistently, the efect of doxorubicin on expression of miR-184 in U-2 OS was further confrmed. These data suggest the key role of miR-184 in chemoresistance of OS cells.2. Up-regulated miR-184 significantly inhibited the cell apoptosis induced by doxorubicin, whereas down-regulated miR-184 facilitated doxorubicin-induced cell apoptosis in U-2 OS. Similar results were also found in MG-63. These data suggest that miR-184 overexpression promotes resistance of OS cells to doxorubicin.3. BCL2L1 was predicted to be target gene of miR-184 by TargetScan. To verify the prediction, we constructed the luciferase reporter vector containing wild-type or mutant 3’UTRof BCL2L1. MiR-184 agomir induced he luciferase activity of vector carrying wild-type 3’UTR of BCL2L1 compared to that carrying mutant 3’UTR of BCL2L1.4. We assayed the effect of doxorubicin on the expression of BCL2L1. Doxorubicin inhibited BCL2L1 expression in a time-dependent manner.5. To clarify the relation between BCL2L1 and miR-184, we further assayed the effect of miR184 on the expression of BCL2L1 in cells treated with or without doxorubicin. We found that up-regulated miR-184 led to a marked increase in the level of BCL2L1 when U-2 OS was treated with doxorubicin, whereas down-regulated miR-184 suppressed BCL2L1 expression.ConclusionsGiven these data, it was likely that miR-184 promotes BCL2L1 expression, which might be related to the doxorubicin-induced up-regulation of miR-184. Then, it was necessary to confirm whether miR-184 amplified BCL2L1 expression to protect OS cells from doxorubicin-induced apoptosis. Thus, we next examined the effects of up-regulated or down-regulated miR-184 on cell death in the presence or absence of doxorubicin. The data showed that miR-184 significantly reduced the number of apoptotic cells from doxorubicin-induced cell death, but down-regulated miR-184 promoted doxorubicin-induced cell apoptosis. These findings suggest that up-regulation of miR-184 made OS cells resistant to doxorubicin.