Abstract
BackgroundMicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. Specifically, microRNA-221 (miR-221) is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-221 in human osteosarcoma has not been totally elucidated. In the present study, the effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated.Methodology/Principal FindingsReal-time quantitative PCR analysis revealed miR-221 was significantly upregulated in osteosarcoma cell lines than in osteoblasts. Both human osteosarcoma cell lines SOSP-9607 and MG63 were transfected with miR-221 mimic or inhibitor to regulate miR-221 expression. The effects of miR-221 were then assessed by cell viability, cell cycle analysis, apoptosis assay, and cisplatin resistance assay. In both cells, upregulation of miR-221 induced cell survival and cisplatin resistance and reduced cell apoptosis. In addition, knockdown of miR-221 inhibited cell growth and cisplatin resistance and induced cell apoptosis. Potential target genes of miR-221 were predicted using bioinformatics. Moreover, luciferase reporter assay and western blot confirmed that PTEN was a direct target of miR-221. Furthermore, introduction of PTEN cDNA lacking 3′-UTR or PI3K inhibitor LY294002 abrogated miR-221-induced cisplatin resistance. Finally, both miR-221 and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR and immunohistochemistry. High miR-221 expression level and inverse correlation between miR-221 and PTEN levels were revealed in osteosarcoma tissues.Conclusions/SignificanceThese results for the first time demonstrate that upregulation of miR-221 induces the malignant phenotype of human osteosarcoma whereas knockdown of miR-221 reverses this phenotype, suggesting that miR-221 could be a potential target for osteosarcoma treatment.
Highlights
Osteosarcoma is the most primary bone tumor and occurs predominantly in adolescents and young adults [1]
The cells transfected with nothing were used as control. These oligonucleotides could be observed with fluorescence microscopy (OLYMPUS) because there was a FAM fluorescent label in their 59 oligonucleotide structure. 24 hours after transfection, transfection efficiency of miR-221 mimic in these two cells were observed by fluorescence microscopy analysis (Figure 1B). miR-221 inhibitor and scramble group had the similar transfection efficiency in these two cells
Increasing evidences indicate that miRNAs may exert functions as oncogenes or tumor suppressors in human cancers depending on the role of the their targets [6,7,8,9,10,11,12,13]
Summary
Osteosarcoma is the most primary bone tumor and occurs predominantly in adolescents and young adults [1]. CDNA lacking 39-UTR or PI3K/Akt inhibitor LY294002 abrogates miR-221-induced cisplatin resistance Because miR-221 directly targets PTEN through interaction between PTEN 39-UTR and miR-221 (Figure 5), we reasoned that ectopic expression of PTEN by transfection of the cDNA that only contains the open reading frame of PTEN (PTEN -ORF) laking 39-UTR should escape the regulation by miR-221 and attenuate or decrease miR-221 function. To this end, we cotransfected pcDNA3.1-PTEN (only contains the coding region of PTEN and laking 39-UTR) and miR-221 mimic into SOSP-9607 cells and treated them with or without 10 uM cisplatin. The effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated
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