微RNA-129-5p下调PAK5表达并调控骨肉瘤细胞增殖和凋亡
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摘要
目的 :探讨微RNA-129-5p(microRNA-129-5p,miR-129-5p)对骨肉瘤细胞增殖和凋亡的影响,以及其可能的分子作用机制。方法:采用实时荧光定量PCR法检测骨肉瘤细胞MG63和成骨细胞hFOB1.19中miR-129-5p表达的差异。将miR-129-5p模拟物转染至骨肉瘤MG63细胞,并采用实时荧光定量PCR法验证转染效率后,CCK-8和平板克隆形成实验检测骨肉瘤细胞增殖;FCM法检测细胞凋亡;双荧光素酶报告基因检测实验验证miR-129-5p和靶基因p21活化激酶5(p21-activated kinase 5,PAK 5)的相互作用;实时荧光定量PCR和蛋白质印迹法检测PAK5 mRNA和蛋白水平的变化;蛋白质印迹法检测磷酸化糖原合成酶激酶3β(phospho-glycogen synthase kinase-3β,p-GSK-3β)和β-连环蛋白(β-catenin)表达水平的变化。将siRNA-PAK5、PAK5重组质粒以及miR-129-5p mimics+PAK5重组质粒分别转染骨肉瘤MG63细胞后,采用上述方法进一步验证miR-129-5p对骨肉瘤细胞增殖和凋亡的分子作用机制。结果 :miR-129-5p在骨肉瘤MG63细胞中低表达(P <0.05)。转染miR-129-5p模拟物后,骨肉瘤细胞的增殖和克隆形成能力降低(P <0.001,P <0.01),而细胞凋亡率升高(P <0.05)。mi R-129-5p负性调控PAK5基因表达(P <0.05)。转染miR-129-5p模拟物或siRNA-PAK5后,PAK5mRNA和蛋白表达水平明显降低(P值均<0.01),而且p-GSK-3β和β-catenin蛋白表达水平也明显降低(P <0.05,P <0.01);而过表达PAK5可以恢复miR-129-5p对骨肉瘤细胞增殖、克隆形成和凋亡的影响(P值均<0.05)。结论 :miR-129-5p通过下调PAK5基因表达,抑制骨肉瘤细胞增殖,促进细胞凋亡;其作用机制可能与调控p-GSK-3β和β-catenin蛋白表达相关。
Objective: To investigate the effects of microRNA-129-5 p(miR-129-5 p) on the proliferation and apoptosis of osteosarcoma cells, and to explore the possible molecular mechanism.Methods: The expression of miR-129-5 p in osteosarcoma MG63 cells and osteoblasts hFOB1.19 cells was determined by real-time fluorescent quantitative PCR(RFQ-PCR). After miR-129-5 p mimics were transfected into osteosarcoma MG63 cells, the expression of miR-129-5 p was detected by RFQ-PCR to verify the transfection efficiency. CCK-8 assay and plate clony formation experiment were used to detect the cell proliferative ability, while FCM was performed to analyze the cell apoptosis. Double luciferase reporter gene system was used to verify the interaction between miR-129-5 p and the target gene p21-activated kinase 5(PAK 5). The expressions of PAK5 mRNA and protein in MG63 cells with miR-129-5 p overexpression were tested by RFQ-PCR and Western blotting, while the expression levels of phospho-glycogen synthase kinase-3β(p-GSK3β) and β-catenin were detected by Western blotting. After siRNA-PAK5, PAK5 overexpressed plasmids and miR-129-5 p mimics+PAK5 overexpressed plasmids were separately transfected into osteosarcoma MG63 cells, the above methods were used to further verify the molecular mechanism of miR-129-5 p regulating the proliferation and apoptosis of osteosarcoma cells.Results: The level of miR-129-5 p in MG63 cells was lower than that in hFOB1.19 cells(P < 0.05). After the transfection with miR-129-5 p mimics, the proliferation and clony formation abilities of MG63 cells were decreased(P < 0.001, P < 0.01), while the apoptosis rate was increased(P < 0.05). miR-129-5 p regulated negatively the expression of PAK 5 gene(P < 0.05). After the transfection with miR-129-5 p mimics or siRNA-PCK5, the expression levels of PAK5 mRNA and protein were decreased(all P < 0.01), while the expressions of p-GSK-3β and β-catenin proteins were also decreased(both P < 0.05, both P < 0.01). The overexpression of PAK5 could restore the effects of miR-129-5 p on proliferation and apoptosis of osteosarcoma cells(both P < 0.05).Conclusion: miR-129-5 p can inhibit the proliferation ability of osteosarcoma cells, and promote apoptosis by down-regulating the expression of PAK 5 gene. The mechanism may be associated with the expression regulation of p-GSK-3β and β-catenin proteins.
Objective: To investigate the effects of microRNA-129-5 p(miR-129-5 p) on the proliferation and apoptosis of osteosarcoma cells, and to explore the possible molecular mechanism.Methods: The expression of miR-129-5 p in osteosarcoma MG63 cells and osteoblasts hFOB1.19 cells was determined by real-time fluorescent quantitative PCR(RFQ-PCR). After miR-129-5 p mimics were transfected into osteosarcoma MG63 cells, the expression of miR-129-5 p was detected by RFQ-PCR to verify the transfection efficiency. CCK-8 assay and plate clony formation experiment were used to detect the cell proliferative ability, while FCM was performed to analyze the cell apoptosis. Double luciferase reporter gene system was used to verify the interaction between miR-129-5 p and the target gene p21-activated kinase 5(PAK 5). The expressions of PAK5 mRNA and protein in MG63 cells with miR-129-5 p overexpression were tested by RFQ-PCR and Western blotting, while the expression levels of phospho-glycogen synthase kinase-3β(p-GSK3β) and β-catenin were detected by Western blotting. After siRNA-PAK5, PAK5 overexpressed plasmids and miR-129-5 p mimics+PAK5 overexpressed plasmids were separately transfected into osteosarcoma MG63 cells, the above methods were used to further verify the molecular mechanism of miR-129-5 p regulating the proliferation and apoptosis of osteosarcoma cells.Results: The level of miR-129-5 p in MG63 cells was lower than that in hFOB1.19 cells(P < 0.05). After the transfection with miR-129-5 p mimics, the proliferation and clony formation abilities of MG63 cells were decreased(P < 0.001, P < 0.01), while the apoptosis rate was increased(P < 0.05). miR-129-5 p regulated negatively the expression of PAK 5 gene(P < 0.05). After the transfection with miR-129-5 p mimics or siRNA-PCK5, the expression levels of PAK5 mRNA and protein were decreased(all P < 0.01), while the expressions of p-GSK-3β and β-catenin proteins were also decreased(both P < 0.05, both P < 0.01). The overexpression of PAK5 could restore the effects of miR-129-5 p on proliferation and apoptosis of osteosarcoma cells(both P < 0.05).Conclusion: miR-129-5 p can inhibit the proliferation ability of osteosarcoma cells, and promote apoptosis by down-regulating the expression of PAK 5 gene. The mechanism may be associated with the expression regulation of p-GSK-3β and β-catenin proteins.
引文
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[16]CHEN D,WANG H,CHEN J,et al.MicroRNA-129-5p regulates glycolysis and cell proliferation by targeting the glucose transporter SLC2A3 in gastric cancer cells[J].Front Pharmacol,2018,9:502.
[17]DIAO Y,JIN B,HUANG L,et al.MiR-129-5p inhibits glioma cell progression in vitro and in vivo by targeting TGIF2[J].J Cell Mol Med,2018,22(4):2357-2367.
[18]ZENG H,WANG L,WANG J,et al.microRNA-129-5p suppresses adriamycin resistance in breast cancer by targeting SOX2[J].Arch Biochem Biophys,2018,651:52-60.
[19]XIAO L,ZHOU X,LIU F,e t a l.MicroRNA-129-5p modul ates epithelial-to-mesenchymal transition by targeting SIP1 and SOX4 during peritoneal dialysis[J].Lab Invest,2015,95(7):817-832.
[20]WANG F,XU C,REECE EA,et al.Protein kinase C-alpha suppresses autophagy and induces neural tube defects via miR-129-2 in diabetic pregnancy[J].Nat Commun,2017,8:15182.
[2]ISAKOFF MS,BIELACK SS,MELTZER P,et al.Osteosarcoma:current treatment and a collaborative pathway to success[J].JClin Oncol,2015,33(27):3029-3035.
[3]GAO Y,FENG B,HAN S,et al.MicroRNA-129 in human cancers:from tumorigenesis to clinical treatment[J].Cell Physiol Biochem,2016,39(6):2186-2202.
[4]ZHAI J,QU S,LI X,et al.MiR-129suppresses tumor cell growth and invasion by targeting PAK5 in hepatocellular carcinoma[J].Biochem Biophys Res Commun,2015,464(1):161-167.
[5]HA BH,MORSE EM,TURK BE,et al.Signaling,regulation,and specificity of the typeⅡp21-activated kinases[J].J Biol Chem,2015,290(21):12975-12983.
[6]MARTIN H,MALI RS,MA P,et al.Pak and Rac GTPases promote oncogenic KIT-induced neoplasms[J].J Clin Invest,2013,123(10):4449-4463.
[7]WEN YY,ZHENG JN,PEI DS.An oncogenic kinase:putting PAK5forward[J].Expert Opin Ther Targets,2014,18(7):807-815.
[8]COTTERET S,JAFFER ZM,BEESER A,et al.P21-activated kinase 5(PAK5)localizes to mitochondria and inhibits apoptosi s by phosphorylati ng BAD[J].Mol Cell Biol,2003,23(16):5526-5539.
[9]HAN K,ZHOU Y,TSENG KF,et al.PAK5overexpression is associated with lung metastasis in osteosarcoma[J].Oncol Lett,2018,15(2):2202-2210.
[10]王春燕,汤丽娜,胡蓓蓓,等.骨肉瘤肺转移患者不同治疗方法的生存分析[J].肿瘤,2014,34(10):935-940.
[11]TABANIFAR B,ZHAO Z,MANSER E.PAK5 is auto-activated by a central domain that promotes kinase oligomerization[J].Biochem J,2016,473(12):1777-1789.
[12]PAN YJ,WEI LL,WU XJ,et al.MiR-106a-5p inhibits the cell migration and invasion of renal cell carcinoma through targeting PAK5[J].Cell Death Dis,2017,8(10):e3155.
[13]ISMAIL AF,OSKAY HALACLI S,BABTEEN N,et al.PAK5 mediates cell:cell adhesion integrity via interaction with E-cadherin in bladder cancer cells[J].Biochem J,2017,474(8):1333-1346.
[14]ZHU G,LI X,GUO B,et al.PAK5-mediated E47 phosphorylation promotes epithelial-mesenchymal transition and metastasis of colon cancer[J].Oncogene,2016,35(15):1943-1954.
[15]HAN K,ZHOU Y,GAN ZH,et al.p21-activated kinase 7 is an oncogene in human osteosarcoma[J].Cell Biol Int,2014,38(12):1394-1402.
[16]CHEN D,WANG H,CHEN J,et al.MicroRNA-129-5p regulates glycolysis and cell proliferation by targeting the glucose transporter SLC2A3 in gastric cancer cells[J].Front Pharmacol,2018,9:502.
[17]DIAO Y,JIN B,HUANG L,et al.MiR-129-5p inhibits glioma cell progression in vitro and in vivo by targeting TGIF2[J].J Cell Mol Med,2018,22(4):2357-2367.
[18]ZENG H,WANG L,WANG J,et al.microRNA-129-5p suppresses adriamycin resistance in breast cancer by targeting SOX2[J].Arch Biochem Biophys,2018,651:52-60.
[19]XIAO L,ZHOU X,LIU F,e t a l.MicroRNA-129-5p modul ates epithelial-to-mesenchymal transition by targeting SIP1 and SOX4 during peritoneal dialysis[J].Lab Invest,2015,95(7):817-832.
[20]WANG F,XU C,REECE EA,et al.Protein kinase C-alpha suppresses autophagy and induces neural tube defects via miR-129-2 in diabetic pregnancy[J].Nat Commun,2017,8:15182.