| BackgroundsOsteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Despite improved prognosis following polichemotherapy introduction, the metastatic and relapsing forms continue to be fatal. Novel chemotherapeutic drugs failed to improve overall survival.Therapeutic alternatives are urgently needed.Solid tumors, including osteosarcoma, are characterized by oncogenic signaling and hypoxic microenvironments that promote HIFa accumulation. HIF-la and HIF-2a promote adaptation of tumor cells to hypoxic stress by regulating the expression of over1000genes involved in angiogenesis, metabolism, erythropoiesis, artocrine growth/survival signaling, apoptosis, epithelial-mesenchymal transition, immortalization, invasion, metastasis, stem cell maintenance, and resistance to radiation and chemotherapy. Despite of approximately48%amino-acid sequence homology, HIF-la and HIF-2α fulfill distinct roles in gene regulation. HIF-la is found to be expressed at a higher level in dysplastic nodules and implicates a malignant transformation. However, much less is known about the role of HIF-2a isoform in cancers, including osteosarcoma.Vascular endothelial growth factor (VEGF) is a multifunctional vascular growth factor, the most impotant endothelial cell splitting factor, and the most important regulating factor of tumor angiogenesis. Clinic study of relapsing osteosarcoma patients suggested that, the expression of VEGF, a target gene of HIF, was a cue of pulmonary metastasis and worse prognostication. ERK pathway is at the hinge of cell signaling, and responsible for cell division, growth, development, differentiation and apoptosis, including tumor cell survival. Myeloid cell leukaemia-1(Mcl-1), a Bcl-2family member, is an intensive antiapoptotic protein. Knowles demonstrated a preferential utilisation of HIF-2a in osteosarcoma cells in mediating expression of VEGF, Glut-1and PGK, which were HIF target genes. Moreover, in vitro study demonstrated that both HIF-la and HIF-2a were involved in regulating the proliferation and migration of osteosarcama cells under hypoxia condition.RNA interference (RNAi) is a phenomenon mediated by endogenous or exogenous double-strand RNA (dsRNA) aiming at distinguished nucleotide sequence, is a post-transcriptional genesilencing(PTGS) effect inducing a gene silencing cell type, and an important protective mechanism fighting against infection.In this study, we took advantage of RNAi technique, with siRNA retrovirus as carrier, to respectively transfected siRNA-HIF-2a and the control siRNA-scramble into MG-63cells so as to investigate the effect of HIF-2a gene silence on human osteosarcoma MG-63cells. In vitro studies demonstrated that HIF-2a gene silence significantly inhibited cell proliferation, migration and clone formation in MG-63cells under hypoxia condition, moreover, HIF-2a silence down-regulated expression of VEGF、P-ERK/ERK and Mcl-1. In vivo study, HIF-2a silence significantly inhibited subcutaneous xenograft tumors. All the above results suggested HIF-2a played an important role in the development of osteosarcoma, supporting the future study of oncotherapy aimed at HIF-2a.Objective1.To investigate whether hypoxia-induced HIF-2a plays a significant role in osteosarcoma tumor formation; 2. To investigate the effect of HIF-2a silence by transfection of siRNA on MG-63cells under hypoxia.Methods1. Human osteosarcoma MG-63cells were cultured under hypoxia to mimic the micro-environment in tumors in vivo. MG-63cells were divided into four groups: group Oh, group6h, group12h and group24h, which were cultured under hypoxia for0hour,6hours,12hours and24hours, respectively. Expressions of HIF-2a, VEGF, P-ERK/ERK and Mcl-1were determined by Western Blot.2. HIF-2a gene silence was with small interfering RNA (siRNA) to create MG-63/siHIF-2a (siHIF-2a) cells and control MG-63/scramble (NC) cells. Fluorescence microscope and flow cytometry were used to observe and determine the expression of green fluorescence protein (GFP), so as to evaluate the efficience of siRNA. MG-63cells, NC cells and siHIF-2a cells were cultured under hypoxia for24hours, the expression of HIF-2aprotein was determined by Western Blot.3. MG-63cells, NC cells and siHIF-2a cells were cultured under hypoxia for24hours, the expression of VEGF, P-ERK/ERK and Mcl-1protein was determined by Western Blot.4. siHIF-2a cells and NC cells (5,000) were plated in96-well plates, cultured under hypoxia for12hours and24hours, OD was measured using the MTT assay as per the manufacturer’s instructions according to the manufacturer’s instructions.5. siHIF-2a cells and NC cells (5,000) were plated in six-well plates. A scratch was made through cells at90%confluence using a20ul pipette tip. Specific points of the scratch were photographed before and after exposure to hypoxia conditions for12hours. Wound width was measured in Image J and migration expressed as fraction wound closure.6. siHIF-2a cells and NC cells (1000,2000,3000,4000,5000, respectively) were plated in six-well plates, cultured under hypoxia. At the end day of three weeks, cells were fixed with formalin, stained with crystal blue and colonies of more than50cells were counted under a contrast-phase microscope. Clone formation rate (%)=(clone count number/cell count number)×100%.7. BALB/c male nude mice aged4-6weeks were purchased from Beijing WeiTongLiHa Corporation and kept under pathogen-free conditions, fed with sterile food and given free access to sterilized water. The stably transfected NC cells (5×106) or siHIF-2a cells in100μl of PBS were injected subcutaneously over each side of the shoulder region. Each group included six mice (Group A:NC tumor protocol; Group B:siHIF-2a tumor protocol). At the end of observation (four weeks after injection of cells), the mice were sacrificed, the tumors were excised, measured, and weighed.Results1. When MG-63cells were cultured under hypoxia for0hours,6hours,12hours and24hours, the relative expression of HIF-2a, VEGF, P-ERK/ERK and Mcl-1were increasing(F=2037.412, P<0.001; F=61.276, P<0.001; F=582.596, P<0.001; F=128.516, P<0.001).2. HIF-2a gene was successfully silenced with small interfering RNA (siRNA). The GFP expression was observed with fluorescence microscope, while the GFP expression rates of NC cells and siHIF-2a cells were (79.1±1.9)%and (82.7±2.1)%, the difference was not significant (P>0.05)3. The expressions of HIF-2α、VEGF、P-Erk/Erk、Mcl-1proteins were significant inhibited in siHIF-2a cells, compared with those in NC cells(F=817.462, P<0.001; F=87.098, P<0.001; F=58.672, P<0.001; F=1280.843, P<0.001).4. The OD values of NC cells and siHIF-2a cells under hypoxia for12h and24h were1.73±0.05and1.27±0.04(1=12.74, P=0.001),1.61±0.03and1.03±0.05(t=16.62, P<0.001). The difference of OD values of NC cells and siHIF-2α cells was more obovius at24h.5. The relative widths of NC cells and siHIF-2a cells under hypoxia for12h were 0.21±0.01and0.78±0.02(t=44.15, P<0.001).When cell counts were1000,2000,3000,4000and5000, the clone formation rates (%) of siHIF-2a and NC cells were1.02±0.15and2.57±0.25(t=9.208, P=0.0027);1.17±0.27and2.75±0.18(t=8.433, P=0.0038);1.35±0.22and2.76±0.28(t=6.858, P=0.0063);1.44±0.19and2.93±0.39(t=5.949, P=0.0276);1.98±0.27and3.91±0.40(t=6.927, P=0.0062)6. The sizes (cm3), weight (g) and density (g/cm3) of siHIF-2a tumors and NC tumors were0.196±0.071and0.486±0.108(t=3.886, P=0.006);0.049±0.009and0.384±0.108(t=5.354, P<0.001);0.251±0.001and0.790±1.002(t=0.9317, P<0.001)Conclusions1. Hypoxia induces the expression of HIF-2a, VEGF, P-Erk/Erk and Mcl-1in a notably time-dependent manner.2. Nucleotide sequences of siRNA-HIF-2a and siRNA-scramble were stably expressed in siHIF-2a cells and NC cells.3. HIF-2a gene silence inhibited the expression of HIF-2a, VEGF, P-ERK/ERK and Mcl-1, indicating the possibility that HIF-2a had great effect on the expression of VEGF, P-ERK/ERK and Mcl-1.4. HIF-2a gene silence significantly inhibited the cell viability of MG-63cells under hypoxia.5. HIF-2a gene silence significantly inhibited the migration of MG-63cells under hypoxia.6. HIF-2a gene silence significantly inhibited the clone formation of MG-63cells under hypoxia.7. HIF-2a gene silence significantly inhibited the development of osteosarcoma in vivo. |
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