miR-155靶向SOCS1对骨肉瘤Saos2细胞的增殖、侵袭和迁移能力的影响
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摘要
目的:探讨microRNA-155(miR-155)对骨肉瘤Saos2细胞增殖、侵袭和迁移的影响以及其作用机制。方法:利用实时荧光定量(qRT-PCR)实验检测miR-155在正常成骨细胞与骨肉瘤Saos2细胞中的表达水平,以及miR-155-mimic、miR-155-inhibitor的转染效率。采用CCK-8实验检测细胞的增殖能力,Transwell实验和划痕实验分别检测Saos2细胞的侵袭和迁移能力,Western blot检测细胞内的STAT3磷酸化水平以及SOCS1表达水平,双荧光素酶报告基因实验进行靶基因验证。结果:miR-155在骨肉瘤Saos2细胞中表达明显高于正常成骨细胞(P<0.001)。在分别转染miR-155-mimic和miR-155-inhibitor后,Saos2细胞内miR-155表达水平明显上调和下降(P<0.001)。过表达miR-155可促进Saos2细胞增殖、侵袭和迁移,降低SOCS1的蛋白水平,上调STAT3的磷酸化水平,差异均具有统计学意义。相反,降低miR-155水平可抑制Saos2细胞的增殖、侵袭和迁移能力,差异均具有统计学意义。结论:骨肉瘤Saos2细胞中高表达的miR-155可以通过抑制SOCS1表达来激活STAT3信号通路进而促进细胞的增殖、侵袭和迁移,因此,靶向抑制miR-155表达可以作为潜在治疗骨肉瘤的途径。
Objective: To investigate the effects and the mechanism of action of microRNA-155(miR-155) on proliferation,invasion and migration of osteosarcoma Saos2 cells. Methods: The expression of miR-155 in normal osteoblasts and osteosarcoma Saos2 cells was detected by real-time PCR, and the transfection efficiency of miR-155-mimic and miR-155-inhibitor was measured by qRT-PCR. CCK-8 assay was used to evaluate the cell proliferation. Transwell assay and wound healing assay were used to detect the invasion and migration ability of Saos2 cells, respectively. SOCS1 and p-STAT3 protein expression was examined by using western blot assay. Dual luciferase reporter gene assay was performed to determine the target gene. Results: The expression of miR-155 was significantly higher in osteosarcoma Saos2 cells than normal osteoblasts(P <0.001). After transfection with miR-155-mimic or miR-155-inhibitor, miR-155 expression of Saos2 cells was significantly upregulated or downregulated, respectively(P <0.001).Overexpression of miR-155 promoted proliferation, invasion and migration of Saos2 cells, and decreased the protein level of SOCS1, as well as increased the phosphorylation level of STAT3. The difference was statistically significant(P<0.05). Conversely, downregulation of miR-155 inhibited the proliferation, invasion, and migration of Saos2 cells. The difference was statistically significant(P <0.05).Conclusion: miR-155, which is highly expressed in osteosarcoma Saos2 cells, can activate the STAT3 signaling pathway by inhibiting the expression of SOCS1, thereby promotes cell proliferation, migration and invasion. Therefore, targeting miR-155 therapy may be a potential approach for osteosarcoma.
Objective: To investigate the effects and the mechanism of action of microRNA-155(miR-155) on proliferation,invasion and migration of osteosarcoma Saos2 cells. Methods: The expression of miR-155 in normal osteoblasts and osteosarcoma Saos2 cells was detected by real-time PCR, and the transfection efficiency of miR-155-mimic and miR-155-inhibitor was measured by qRT-PCR. CCK-8 assay was used to evaluate the cell proliferation. Transwell assay and wound healing assay were used to detect the invasion and migration ability of Saos2 cells, respectively. SOCS1 and p-STAT3 protein expression was examined by using western blot assay. Dual luciferase reporter gene assay was performed to determine the target gene. Results: The expression of miR-155 was significantly higher in osteosarcoma Saos2 cells than normal osteoblasts(P <0.001). After transfection with miR-155-mimic or miR-155-inhibitor, miR-155 expression of Saos2 cells was significantly upregulated or downregulated, respectively(P <0.001).Overexpression of miR-155 promoted proliferation, invasion and migration of Saos2 cells, and decreased the protein level of SOCS1, as well as increased the phosphorylation level of STAT3. The difference was statistically significant(P<0.05). Conversely, downregulation of miR-155 inhibited the proliferation, invasion, and migration of Saos2 cells. The difference was statistically significant(P <0.05).Conclusion: miR-155, which is highly expressed in osteosarcoma Saos2 cells, can activate the STAT3 signaling pathway by inhibiting the expression of SOCS1, thereby promotes cell proliferation, migration and invasion. Therefore, targeting miR-155 therapy may be a potential approach for osteosarcoma.
引文
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[2]Bacci G,Longhi A,Bertoni F,et al.Bone metastases in osteosarcoma patients treated with neoadjuvant or adjuvant chemotherapy:the Rizzoli experience in 52 patients[J].Acta Orthopaed,2006,77(6):938-943
[3]Yang N,Zhu S,Lv X,et al.MicroRNAs:Pleiotropic Regulators in the Tumor Microenvironment[J].Front Immunol,2018,9:2491
[4]Faraoni I,Antonetti FR,Cardone J,et al.miR-155 gene:A typical multifunctional microRNA[J].Biochim Biophys Acta,2009,1792(6):497-505
[5]张开亮,任坤,张迎龙,等.miR-155抑制物对骨肉瘤细胞增殖与凋亡的影响[J].现代肿瘤医学,2013,21(6):1189-1192
[6]Neilsen PM,Noll JE,Mattiske S,et al.Mutant p53 drives invasion in breast tumors through up-regulation of miR-155[J].Oncogene,2013,32(24):2992-3000
[7]Geller DS and Gorlick R.Osteosarcoma:a review of diagnosis,management,and treatment strategies[J].Clin Adv Hematol Oncol,2010,8:705-718
[8]Luetke A,Meyers PA,Lewis I,et al.Osteosarcoma treatment-where do we stand?A state of the art review[J].Cancer Treat Rev,2014,40:523-532
[9]Tam W.Identification and characterization of human BIC,a gene on chromosome 21 that encodes a noncoding RNA[J].Gene,2001,274(1-2):157-167
[10]Brown CY,Dayan S,Wong SW,et al.FOXP3 and miR-155cooperate to control the invasive potential of human breast cancer cells by down regulating ZEB2 independently of ZEB1[J].Oncotarget,2018,9(45):27708-27727
[11]Khori V,Alizadeh AM,Gheisary Z,et al.The effects of low-level laser irradiation on breast tumor in mice and the expression of Let-7a,miR-155,miR-21,miR125,and miR376b[J].Lasers Med Sci,2016,31(9):1775-1782
[12]Li Shi-qing,Zhang Tao,Zhou Xiao-qing,et al.The tumor suppressor role of miR-155-5p in gastric cancer[J].Oncol Lett,2018,16(2):2709-2714
[13]Ma Z,Ma Y,Xia Q,et al.MicroRNA-155 expression inversely correlates with pathologic stage of gastric cancer and it inhibits gastric cancer cell growth by targeting cyclin D1[J].J Cancer Res Clin Oncol,2016,142(6):1201-1212
[14]Cuadros M,Sánchez-Martín V,Herrera A,et al.BRG1 regulation by miR-155 in human leukemia and lymphoma cell lines[J].Clin Transl Oncol,2017,19(8):1010-1017
[15]Li XD,Li XM,Gu JW,et al.MiR-155 regulates lymphoma cell proliferation and apoptosis through targeting SOCS3/JAK-STAT3signaling pathway[J].Eur Rev Med Pharmacol Sci,2017,21(22):5153-5159
[16]Papageorgiou SG,Kontos CK,Diamantopoulos MA,et al.MicroRNA-155-5p Overexpression in Peripheral Blood Mononuclear Cells of Chronic Lymphocytic Leukemia Patients Is a Novel,Independent Molecular Biomarker of Poor Prognosis[J].Dis Markers,2017,2017:2046545
[17]Chen N,Feng L,Qu H,et al.Overexpression of IL-9 induced by STAT3 phosphorylation is mediated by miR-155 and miR-21 in chronic lymphocytic leukemia[J].Oncol Rep,2018,39(6):3064-3072
[18]Martin EC,Krebs AE,Burks HE,et al.miR-155 induced transcriptome changes in the MCF-7 breast cancer cell line leads to enhanced mitogen activated protein kinase signaling[J].Genes Cancer,2014,5(9-10):353-364
[19]Qu Ya-jing,Zhang Hai-yang,Sun Wu,et al.MicroRNA-155 promotes gastric cancer growth and invasion by negatively regulating transforming growth factor-βreceptor 2[J].Cancer Sci,2018,109(3):618-628
[20]Wang L,Tang B,Han H,et al.miR-155 Affects Osteosarcoma MG-63 Cell Autophagy Induced by Adriamycin Through Regulating PTEN-PI3K/AKT/mTOR Signaling Pathway[J].Cancer Biother Radiopharm,2018,33(1):32-38
[21]Bartel DP:MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116:281-297
[22]Starr R,Willson TA,Viney EM,et al.A family of cytokine-inducible inhibitors of signaling[J].Nature,1997,387(6636):917-921
[23]Culig Z.Suppressors of cytokine signalling-3 and-1 in human carcinogenesis[J].Front Biosci(Schol Ed),2013,5:277-283
[24]Elliott J,Hookham MB,Johnston JA.The suppressors of cytokine signalling E3 ligases behave as tumour suppressors[J].Biochem Soc Trans,2008,36(3):464-468
[25]Aigner P,Just V,Stoiber D.STAT3 isoforms:Alternative fates in cancer?[J].Cytokine,2018,pii:S1043-4666(18)30300-4
[26]Arora L,Kumar AP,Arfuso F,et al.The Role of Signal Transducer and Activator of Transcription 3(STAT3)and Its Targeted Inhibition in Hematological Malignancies[J].Cancers(Basel),2018,10(9)
[27]Morikawa T,Baba Y,Yamauchi M,et al.STAT-3 expression,molecular features,inflammation patterns,and prognosis in a database of 724 colorectal cancers[J].Clin Cancer Res,2011,17(6):1452-1462
[28]Huang C,Li H,Wu W,et al.Regulation of miR-155 affects pancreatic cancer cell invasiveness and migration by modulating the STAT3 signaling pathway through SOCS1[J].Oncol Rep,2013,30(3):1223-1230
[29]Lei K,Du W,Lin S,et al.3B,a novel photosensitizer,inhibits glycolysis and inflammation via miR-155-5p and breaks the JAK/STAT3/SOCS1 feedback loop in human breast cancer cells[J].Biomed Pharmacother,2016,82:141-150
[30]Zhou X,Yan T,Huang C,et al.Melanoma cell-secreted exosomal miR-155-5p induce proangiogenic switch of cancer-associated fibroblasts via SOCS1/JAK2/STAT3 signaling pathway[J].J Exp Clin Cancer Res,2018,37(1):242