Figure 1.
miR-221 expression and miR-221 oligonucleotides transfection in osteosarcoma cells.
A, the expression of miR-221 was analyzed by qRT-PCR and normalized to RNU24 in five osteosarcoma cell lines. The expression of miR-221 in hFOB1.19 osteoblasts was used as a control. For B–D, SOSP-9607 and MG 63 cells were transfected with miR-221 mimic (221 Mi), miR-221 inhibitor (221 In), or scramble oligonucleotides (Scr). The blank (not transfected) cells were used as control. 24 h after transfection, the cells were observed and miR-221 expression levels were evaluated by qRT-PCR analysis. B, SOSP-9607 cells (top) and MG 63 cells (bottom) were observed with white bright (left) and green fluorescence assay (right) in the same vision using fluorescence microscopy (200×). C, miR-221 expression levels were evaluated by qRT-PCR analysis in transfected SOSP-9607 cells. D, miR-221 expression levels were assayed in transfected MG63 cells. RNU24 was used as an internal loading control to normalize the results. For C and D, data was presented as means ± SD. Columns, mean of three independent experiments; bars, SD; ***, p<0.001.
Figure 2.
miR-221 induces cell proliferation in both SOSP-9607 and MG63 cell lines.
Both SOSP-9607 and MG 63 cells were transfected with miR-221 mimic (221 Mi), miR-221 inhibitor (221 In) or scramble oligonucleotide (Scr). The non-transfected cells (blank) were used as control. After transfection, every 24 h, MTT assay was performed on four groups of SOSP-9607 (A) and MG 63 cells (B). The viable cell number was evaluated as the value of the absorbance at 490 nm with a reference wavelength of 630 nm. Values of optical density (OD) are expressed as means ± SD. Point symbol, mean of three independent experiments; bars, SD; *, p<0.05, **p<0.01. C, 48 h after transfection, cell cycle distribution of treated SOSP-9607 (top) and MG 63 (bottom) was measured using flow cytometry analysis.
Figure 3.
miR-221 regulates apoptosis in both SOSP-9607 and MG63 cells.
A, C SOSP-9607 cells were transfected with 100 nM miR-221 mimic (221 Mi), miR-221 inhibitor (221 In) or scramble oligonucleotide (Scr) for 48 h. The blank cells were used as control. Apoptosis was measured by FACS with Annexin V and propidium iodide staining. B, D Apoptosis of MG 63 was measured by FACS with Annexin V and propidium iodide staining. The data were presented as the means ± SD in panel C and panel D. Columns, mean of three independent experiments; bars, SD; **, p<0.01, ***, p<0.001.
Figure 4.
Involvement of miR-221 in cisplatin resistance in both SOSP-9607 and MG63 cells.
Cells were transfected with miR-221 mimic (221 Mi), miR-221 inhibitor (221 In) or scramble oligonucleotide (Scr). The blank cells were used as control. Twenty four hours after transfection, treated cells were seeded into 96-well plates at the density of 5×103 per well. A and B, twenty four hours later, SOSP-9607 and MG 63 cells were treated with or without 10 uM cisplatin for 24 h, 48 h or 72 h. Viability evaluation were examined using MTT assay. Then viability inhibition was calculated. C and D, twenty four hours later, SOSP-9607 and MG 63 cells were treated with or without the indicated concentrations of cisplatin for 48 h. Then, viability evaluation were examined using MTT assay. E and F, twenty four hours later, transfected SOSP-9607 and MG 63 cells were treated with or without 10 uM cisplatin for 24 h. Caspase3 activity was measured using Caspase-Glo® 3/7 assay kit (Promega). The data were presented as the means ± SD. Columns, mean of three independent experiments; bars, SD. **, p<0.01, ***, p<0.001.
Figure 5.
miR-221 targets PTEN leading to activation of the Akt pathway.
A, putative miR-221-binding sites (red) in the 3′-UTRs of PTEN (top). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B, SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C, SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D, similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p<0.01.
Figure 6.
Introduction of PTEN cDNA lacking 3′-UTR or PI3K/Akt inhibitor LY294002 abrogates miR-221-induced cisplatin resistance.
A, SOSP-9607 cells were transfected with miR-221 mimic (221 Mi), scramble oligonucleotide (Scr) or cotransfected with miR-221 mimic and pcDNA-PTEN. The scramble group was used as a control. The expression of PTEN and phospho-Akt (p-Akt) were analyzed by western blot. β-actin was used as a loading control. B, SOSP-9607 cells were transfected with miR-221 mimic, scramble oligonucleotide or cotransfected with miR-221 mimic and LY294002. The scramble group was used as a control. The expression of PTEN and phospho-Akt (p-Akt) were analyzed by western blot. β-actin was used as a loading control. C, SOSP-9607 and MG 63 cells were treated as described in panel A. Then the cells were treated with or without cisplatin for 48 h and viability evaluation were examined. Data were presented as the percentage of viability inhibition measured in cells treated without cisplatin. D, SOSP-9607 and MG 63 cells were all treated as described in panel B. Then the cells were treated with or without cisplatin for 48 h and viability evaluation were examined. Data were presented as the percentage of viability inhibition measured in cells treated without cisplatin. E, both SOSP-9607 and MG 63 cells treated as described in panel A were seeded into 96-well plates and 24 h later were treated with or without cisplatin for 24 h. Caspase3 activity was measured using Caspase-Glo® 3/7 assay kit (Promega) according to the manufacturer's instructions. F, both SOSP-9607 and MG 63 cells treated as described in panel B were seeded into 96-well plates and 24 h later were treated with or without cisplatin for 24 h. Caspase3 activity was measured using Caspase-Glo® 3/7 assay kit. Columns, mean of three independent experiments; bars, SD; **, p<0.01, ***, p<0.001.
Figure 7.
Inverse correlation of expression of miR-221 and PTEN in human osteosarcoma samples.
A, expression of miR-221 was analyzed by qRT-PCR in normal adjacent tissues (NAT), primary osteosarcoma tissues (POT) and recurrent osteosarcoma tissues (ROT). U24 was used as internal control. The average expression values of adjacent normal tissues is set at 1. Horizontal bars in the box represent median. *, p<0.05, ***, p<0.001. B, expression of PTEN was analyzed in osteosarcoma tissues with immunohistochemical staining. C, Chisquare test analysis of miR-221 and PTEN expression. The inverse correlation is significant (p<0.001).