首页|ISLR通过活化PI3K-AKT通路促进上皮-间质转化影响骨肉瘤细胞恶性进展研究

ISLR通过活化PI3K-AKT通路促进上皮-间质转化影响骨肉瘤细胞恶性进展研究

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目的 研究含免疫球蛋白超家族亮氨酸丰富重复蛋白(immunoglobulin superfamily containing leucine-rich repeat protein,ISLR)参与骨肉瘤细胞恶性进展的作用及其潜在调节机制.方法 通过实时定量聚合酶链反应(qRT-PCR)检测骨肉瘤组织和细胞中ISLR mRNA水平.通过转染ISLR短发夹RNA(short hairpin RNA,shRNA)序列或阴性对照 shRNA(negative-control shRNA,NC shRNA)序列至 U2OS 细胞,后用磷脂酰肌醇 3 激酶(phosphatidylinositol 3 kinase,PI3K)激活剂740 Y-P处理细胞.通过CCK-8法、Transwell实验和流式细胞术分别检测细胞活力、侵袭能力和细胞凋亡率.蛋白印迹实验(Western blot)检测ISLR蛋白、上皮-间质转化(epithelial-mesenchymal transition,EMT)相关蛋白[上皮钙黏蛋白(epitheia-cadherin,E-cadherin)、神经钙黏蛋白(nerve-cadherin,N-cadherin)、波形蛋白(Vimentin),Snail]、PI3K/蛋白激酶B(protein kinase B,AKT)通路相关蛋白、细胞凋亡相关蛋白[半胱天冬氨酸蛋白酶 3(cysteinyl aspartate-specific proteinase-3,Caspase-3),B 淋巴细胞瘤-2(B cell lymphoma/leukemia-2,Bcl-2),Bcl-2相关X蛋白(Bcl-2 associated X,Bax)]和肿瘤增殖标志物Ki67蛋白表达.采用慢病毒转染的U2OS细胞注射裸鼠构建异种移植瘤模型,监测肿瘤生长情况.结果 与癌旁组织(1.01±0.02)相比,骨肉瘤组织(5.14±1.63)中ISLRmRNA水平显著上调,差异具有统计学意义(t=-14.332,P<0.001).与正常人成骨细胞hFOB1.19(1.01±0.01)相比,骨肉瘤细胞 MG63(3.05±0.57),U2OS(4.55±0.79),HOS(2.46±0.41),Saos-2(2.62±0.44)和 143B(3.62±0.51)中ISLR mRNA相对表达均显著升高,差异具有统计学意义(t=4.883,8.473,3.471,3.854,6.247,均P<0.05).与对照组和NC shRNA组比较沉默ISLR明显抑制了 U2OS细胞增殖(t=6.593,6.835)及侵袭(t=8.621,8.448),促进细胞凋亡(t=25.505,25.574),差异具有统计学意义(均P<0.05).沉默ISLR明显促进U2OS细胞中Caspase-3 活性(t=13.489,13.366)及 Bax 蛋白(t=8.628,8.524)表达,抑制 Bcl-2 蛋白(t=10.948,10.775)表达,差异具有统计学意义(均P<0.05).沉默ISLR显著促进EMT相关蛋白E-cadherin(t=15.168,15.087)表达,抑制N-cadherin(t=10.220,10.058),Vimentin(t=8.303,8.164)和 Snail(t=9.211,9.384)蛋白表达,降低 PI3K/AKT 通路关键蛋白PI3K和AKT磷酸化水平(t=17.441,14.452),差异具有统计学意义(均P<0.05).740 Y-P处理可逆转ISLR沉默对U2OS细胞的影响.裸鼠体内实验显示敲低ISLR显著抑制了肿瘤生长.结论 ISLR可能通过激活PI3K/AKT通路促进骨肉瘤EMT及细胞增殖、侵袭,抑制细胞凋亡,从而促进骨肉瘤进展.
ISLR Promotes Epithelial-mesenchymal Transition Through Activating PI3K-AKT Pathway and Influences the Malignant Progression of Osteosarcoma Cells
Objective To investigate the role of immunoglobulin superfamily containing leucine-rich repeat protein(ISLR)in the malignant progression of osteosarcoma cells and its potential regulatory mechanism.Methods ISLR mRNA levels in osteosarcoma tissues and cells were detected by quantitative real time polymerase chain reaction(qRT-PCR).U2OS cells were transfected with ISLR short hairpin RNA(shRNA)sequence or negative-control shRNA(NC shRNA)sequence,thus the cells were treated with phosphatidylinositol 3 kinase(PI3K)activator 740 Y-P.The cell viability,invasion ability and apoptosis rate were detected by CCK-8 assay,Transwell assay and flow cytometry,respectively.Western blot was used to detect the expressions of ISLR protein,epithelial-mesenchymal transition(EMT)-related proteins[Epitheia-cadherin(E-cadherin),Nerve cadherin(N-cadherin),Vimentin,Snail],PI3K/protein kinase B(AKT)pathline-related proteins,apoptotic proteins[Cysteinyl aspartate-specific proteinase-3(Caspase-3),B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax)]and proliferation marker Ki67 protein.Lentivirus was used to transfect U2OS cells,and the cells were injected into nude mice to construct a xenograft tumor model,and tumor growth was monitored.Results ISLR mRNA level in osteosarcoma tissue(5.14±1.63)was up-regulated compared with para-cancerous tissue(1.01±0.02),and the difference was significant(t=-14.332,P<0.001).Compared with normal osteoblasts hFOB1.19(1.01±0.01),osteosarcoma cells MG63(3.05±0.57),U2OS(4.55±0.79),HOS(2.46±0.41),the relative expression of ISLR mRNA in Saos-2(2.62±0.44)and 143B(3.62±0.51)were increased,and differences were significant(t=4.883,8.473,3.471,3.854,6.247,all P<0.05).Silencing ISLR inhibited the proliferation of U2OS cells(t=6.593,6.835)and invasion(t=8.621,8.448),but promoted cell apoptosis(t=25.505,25.574),and the differences were significant(all P<0.05).Silencing ISLR promoted Caspase-3 activity in U2OS cells(t=13.489,13.366)and Bax protein(t=8.628,8.524),but inhibited Bcl-2 protein expression(t=10.948,10.775),with significant differences(all P<0.05).Silencing ISLR promoted EMT-related protein E-cadherin(t=15.168,15.087),inhibited N-cadherin(t=10.220,10.058),Vimentin(t=8.303,8.164)and Snail(t=9.211,9.384),but reduced the phosphorylation levels of PI3K and AKT(t=17.441,14.452),with significant differences(all P<0.05).Additionally,740 Y-P treatment reversed the effect of silencing ISLR on U2OS cells.Experimental results in vivo showed that knockdown of ISLR significantly inhibited tumor growth.Conclusion ISLR could promote EMT,proliferation and invasion,but inhibit apoptosis of osteosarcoma cells by activating the PI3K/AKT pathway,there by promoting osteosarcoma progression.

osteosarcomacell proliferationepithelial-mesenchymal transitionimmunoglobulin superfamily containing leucine-rich repeat proteinPI3K/AKT pathway

李青山、郭红生、贾天阳

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邯郸市中心医院骨科,河北邯郸 056001

骨肉瘤 细胞增殖 上皮-间质转化 含免疫球蛋白超家族亮氨酸丰富重复蛋白 PI3K/AKT通路

河北省2022年度医学科学研究课题计划项目

20220582

2024

现代检验医学杂志
陕西省临床检验中心,陕西省人民医院

现代检验医学杂志

CSTPCD
影响因子:0.713
ISSN:1671-7414
年,卷(期):2024.39(5)