雌激素相关受体α在骨肉瘤阿霉素耐药中的表达及功能
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摘要
目的研究雌激素相关受体α(ERRα)在骨肉瘤患者及阿霉素耐药细胞株中的表达情况及其功能。方法收集广州中医药大学第一附属医院9例骨肉瘤患者的标本(其中4例为原发,5例为复发)及正常人骨组织标本3例,行免疫组织化学染色,检测ERRα的表达;浓度梯度法建立骨肉瘤U2OS的阿霉素耐药株(ADM-U2OS),并行qRT-PCR、Western blot检测耐药细胞株中ERRα的表达;利用慢病毒感染U2OS亲本细胞后建立U2OS-ERRα超表达细胞株,检测不同浓度ADM处理后,超表达细胞的死亡率(锥虫蓝拒染实验),分析ERRα表达增高后是否增加骨肉瘤细胞对ADM的耐药性。所有数据用SPSS 17.0统计软件分析。结果免疫组织化学染色发现ERRα在9例骨肉瘤标本中的表达量比正常骨组织中的表达量明显增高,在复发患者肿瘤组织中的表达量更高;qRT-PCR及Western blot检测分别发现ERRα在ADM-U2OS耐药细胞株中的基因及蛋白表达水平均增高;不同浓度ADM处理后,U2OS-ERRα超表达细胞死亡率低,ERRα表达增高能增加骨肉瘤细胞对ADM的耐药。结论 ERRα在骨肉瘤及阿霉素耐药细胞株中的表达量增高;ERRα的高表达能增加骨肉瘤细胞对阿霉素的耐药性;ERRα有可能成为治疗骨肉瘤及克服骨肉瘤阿霉素耐药的新靶标。
Objective To investigate the expression and function of estrogen-related receptor alpha(ERRα),an orphan nuclear receptor,in adriamycin(ADM)-resistant osteosarcoma(OS)cells.Methods Nine samples of OS(4 primary,5 recurrent)and 3 normal bones were collected from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.Immunohistochemical staining was performed to detect the expression of ERRα.The ADM-resistant strain of OS U2 OS was established by concentration gradient method(ADM 10 mmol/L-U2 OS)and the expression of ERRαwas evaluated by qRTPCR and Western blotting in U2 OS cells.The cells were infected with the lentiviruses to establish the U2 OS-ERRαcell strain that could overexpress ERRα.The death rate of U2 OS-ERRαcells was detected by trypanosomal blue staining after treatment with different concentrations of ADM in order to observe the ADM resistance in cells with ERRαoverexpression.The data were analyzed using SPSS 17.0.Results Immunohistochemistry showed that the expression of ERRαwas significantly increased in OS specimens as compared to that in the normal bones,with its expression much higher in relapsed patients(P<0.01).qRTPCR and Western blotting revealed that the expression of ERRαwas profoundly enhanced in U2 OS-ERRαcells.The survival rate of U2 OS-ERRαcells treated with different concentrations of ADM was higher and the death rate lower,suggesting that ERRαoverexpression could increase the ADM resistance of OS cells.Conclusion ERRαwas markedly expressed in OS tissues and ADM-resistant OS cells.ERRαoverexpression could increase the ADM resistance of OS cells,which indicated that ERRαcould be a novel target for osteosarcoma therapy.
Objective To investigate the expression and function of estrogen-related receptor alpha(ERRα),an orphan nuclear receptor,in adriamycin(ADM)-resistant osteosarcoma(OS)cells.Methods Nine samples of OS(4 primary,5 recurrent)and 3 normal bones were collected from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.Immunohistochemical staining was performed to detect the expression of ERRα.The ADM-resistant strain of OS U2 OS was established by concentration gradient method(ADM 10 mmol/L-U2 OS)and the expression of ERRαwas evaluated by qRTPCR and Western blotting in U2 OS cells.The cells were infected with the lentiviruses to establish the U2 OS-ERRαcell strain that could overexpress ERRα.The death rate of U2 OS-ERRαcells was detected by trypanosomal blue staining after treatment with different concentrations of ADM in order to observe the ADM resistance in cells with ERRαoverexpression.The data were analyzed using SPSS 17.0.Results Immunohistochemistry showed that the expression of ERRαwas significantly increased in OS specimens as compared to that in the normal bones,with its expression much higher in relapsed patients(P<0.01).qRTPCR and Western blotting revealed that the expression of ERRαwas profoundly enhanced in U2 OS-ERRαcells.The survival rate of U2 OS-ERRαcells treated with different concentrations of ADM was higher and the death rate lower,suggesting that ERRαoverexpression could increase the ADM resistance of OS cells.Conclusion ERRαwas markedly expressed in OS tissues and ADM-resistant OS cells.ERRαoverexpression could increase the ADM resistance of OS cells,which indicated that ERRαcould be a novel target for osteosarcoma therapy.
引文
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[25]Lam S S,Mak A S,Yam J W,et al.Targeting estrogen-related receptor alpha inhibits epithelial-to-mesenchymal transition and stem cell properties of ovarian cancer cells[J].Mol Ther,2014,22(4):743-751.
[26]Mirebeau-Prunier D,Le Pennec S,Jacques C,et al.Estrogenrelated receptor alpha modulates lactate dehydrogenase activity in thyroid tumors[J].PLoS One,2013,8(3):1-8.
[27]Deblois G,Smith H W,Tam I S,et al.ERRalpha mediates metabolic adaptations driving lapatinib resistance in breast cancer[J].Nat Commun,2016,7(1):1-14.
[28]Festuccia N,Owens N,Navarro P.Esrrb,an estrogen-related receptor involved in early development,pluripotency,and reprogramming[J].FEBS Lett,2018,592(6):852-877.
[2]Cheng D,Qiu X,Zhuang M,et al.MicroRNAs with prognostic significance in osteosarcoma:a systemic review and meta-analysis[J].Oncotarget,2017,8(46):81062-81074.
[3]Meshkini A,Oveisi H.Methotrexate-F127conjugated mesoporous zinc hydroxyapatite as an efficient drug delivery system for overcoming chemotherapy resistance in osteosarcoma cells[J].Colloids Surf B Biointerfaces,2017,(158):319-330.
[4]Chen L,Wong C.Estrogen-related receptor alpha inverse agonist enhances basal glucose uptake in myotubes through reactive oxygen species[J].Biol Pharm Bull,2009,32(7):1199-1203.
[5]Zhang Y,Ma K,Sadana P,et al.Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4gene expression[J].J Biol Chem,2006,281(52):39897-39906.
[6]Villena J A,Kralli A.ERRalpha:a metabolic function for the oldest orphan[J].Trends Endocrinol Metab,2008,19(8):269-276.
[7]Chen P,Wang H,Duan Z,et al.Estrogen-related receptor alpha confers methotrexate resistance via attenuation of reactive oxygen species production and P53mediated apoptosis in osteosarcoma cells[J].Biomed Res Int,2014,2014:616025.
[8]Fradet A,Sorel H,Bouazza L,et al.Dual function of ERRalpha in breast cancer and bone metastasis formation:implication of VEGF and osteoprotegerin[J].Cancer Res,2011,71(17):5728-5738.
[9]Ariazi E A,Jordan V C.Estrogen-related receptors as emerging targets in cancer and metabolic disorders[J].Curr Top Med Chem,2006,6(3):203-215.
[10]Ding S,Tang Z,Jiang Y,et al.IL-8is involved in estrogen-related receptor alpha-regulated proliferation and migration of colorectal cancer cells[J].Dig Dis Sci,2017,62(12):3438-3446.
[11]Ning Y,Chen H,Du Y,et al.A novel compound LingH2-10inhibits the growth of triple negative breast cancer cells in vitro and in vivo as a selective inverse agonist of estrogen-related receptor alpha[J].Biomed Pharmacother,2017,(93):913-922.
[12]Cai H,Lin L,Cai H,et al.Prognostic evaluation of microR-NA-210expression in pediatric osteosarcoma[J].Med Oncol,2013,30(2):499-504.
[13]Mittal N,Kent P M,Ording J.Metastatic and recurrent bone primary bone cancers[J].Curr Probl Cancer,2013,37(4):215-224.
[14]Luetke A,Meyers P A,Lewis I,et al.Osteosarcoma treatment-where do we stand?A state of the art review[J].Cancer Treat Rev,2014,40(4):523-532.
[15]Buondonno I,Gazzano E,Jean S R,et al.Mitochondria-targeted doxorubicin:A new therapeutic strategy against Doxorubicin-resistant osteosarcoma[J].Mol Cancer Ther,2016,15(11):2640-2652.
[16]Yang N,Shigeta H,Shi H,et al.Estrogen-related receptor,hERR1,modulates estrogen receptor-mediated response of human lactoferrin gene promoter[J].J Biol Chem,1996,271(10):5795-5804.
[17]Baranski Z,de Jong Y,Ilkova T,et al.Pharmacological inhibition of Bcl-xL sensitizes osteosarcoma to doxorubicin[J].Oncotarget,2015,6(34):36113-36125.
[18]Zhao D,Yuan H,Yi F,et al.Autophagy prevents doxorubicininduced apoptosis in osteosarcoma[J].Mol Med Rep,2014,9(5):1975-1981.
[19]Wang Z,Liu Z,Wu S.Long non-coding RNA CTA sensitizes osteosarcoma cells to doxorubicin through inhibition of autophagy[J].Oncotarget,2017,8(19):31465-31477.
[20]Bonnelye E,Kung V,Laplace C,et al.Estrogen receptor-related receptor alpha impinges on theestrogen axis in bone:potential function in osteoporosis[J].Endocrinology,2002,143(9):3658-3670.
[21]Murray J,Huss J M.Estrogen-related receptor alpha regulates skeletal myocyte differentiation via modulation of the ERK MAP kinase pathway[J].Am J Physiol Cell Physiol,2011,301(3):C630-C645.
[22]Bonnelye E,Laurin N,Jurdic P,et al.Estrogen receptor-related receptor-alpha(ERR-alpha)is dysregulated in inflammatory arthritis[J].Rheumatology(Oxford),2008,47(12):1785-1791.
[23]Delhon I,Gutzwiller S,Morvan F,et al.Absence of estrogen receptor-related-alpha increases osteoblastic differentiation and cancellous bone mineral density[J].Endocrinology,2009,150(10):4463-4472.
[24]Deblois G,Hall J A,Perry M C,et al.Genome-wide identification of direct target genes implicates estrogen-related receptor alpha as a determinant of breast cancer heterogeneity[J].Cancer Res,2009,69(15):6149-6157.
[25]Lam S S,Mak A S,Yam J W,et al.Targeting estrogen-related receptor alpha inhibits epithelial-to-mesenchymal transition and stem cell properties of ovarian cancer cells[J].Mol Ther,2014,22(4):743-751.
[26]Mirebeau-Prunier D,Le Pennec S,Jacques C,et al.Estrogenrelated receptor alpha modulates lactate dehydrogenase activity in thyroid tumors[J].PLoS One,2013,8(3):1-8.
[27]Deblois G,Smith H W,Tam I S,et al.ERRalpha mediates metabolic adaptations driving lapatinib resistance in breast cancer[J].Nat Commun,2016,7(1):1-14.
[28]Festuccia N,Owens N,Navarro P.Esrrb,an estrogen-related receptor involved in early development,pluripotency,and reprogramming[J].FEBS Lett,2018,592(6):852-877.