解整合素金属酶结构域8调控软骨肉瘤的机制  

The role of adad metallopeptidase domain 8 in hondrosarcoma

作　　者：毛洪刚 陈宝林 刘岩[1] Mao Honggang;Chen Baolin;Liu Yan(Department of Orthopedics,Inner Mongolia Baogang Hospital,the Third Affiliated Hospital of Inner Mongolia Medical University,Baotou 014010,China)

机构地区：[1]内蒙古包钢医院(内蒙古医科大学第三附属医院)骨科,包头014010

出　　处：《中华实验外科杂志》2023年第11期2208-2211,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82160424);内蒙古自然科学基金(2019MS08180)。

摘　　要：目的:探讨解整合素金属酶结构域8(ADAM8)通过磷酸肌醇-3激酶(PI3K)/丝氨酸-苏氨酸激酶(Akt)/雷帕霉素哺乳动物靶点(mTOR)通路调控软骨肉瘤的作用机制。方法:取人软骨肉瘤细胞株(SW1353及HCS-2/8)及人正常软骨细胞(HC)(美国模式培养物集存库),通过免疫印迹法检测细胞中ADAM8及PI3K/Akt/mTOR通路蛋白。根据随机数字表法将HCS-2/8细胞分为3组:阴性对照(NC)短发卡RNA(shRNA)组,转染NC shRNA;shADAM8组,转染ADAM8 shRNA;shADAM8+SC79组,转染ADAM8 shRNA 48 h后进行10.96 M Akt激动剂(SC79)给药。观察各组细胞PI3K/Akt/mTOR通路、增殖、迁移、侵袭、凋亡的变化。取20只BALB/c裸鼠,建立软骨肉瘤体内模型后根据随机数字表法分为NC shRNA组10只及ADAM8 shRNA组10只,验证ADAM8对Akt/mTOR通路的作用机制。通过独立样本t检验或单因素方差分析进行统计学分析。结果:免疫印迹结果表明,SW1353及HCS-2/8组ADAM8、p-PI3K、p-Akt、mTOR的表达均高于HC组(ADAM8:3.40±0.53及2.01±0.20比1.00,F=27.260,P<0.05;p-PI3K:0.52±0.07及0.53±0.04比0.21±0.05,F=22.120,P<0.05;p-Akt:0.58±0.05及0.61±0.08比0.14±0.04,F=38.540,P<0.05;mTOR:0.52±0.04及0.65±0.04比0.21±0.03,F=40.640,P<0.05)。sh-ADAM8组p-Akt及mTOR低于NC shRNA组(p-Akt:0.11±0.03比0.75±0.03,F=0.578,P<0.05;mTOR:0.11±0.02比0.79±0.07,F=69.600,P<0.05),差异均有统计学意义。sh-ADAM8组细胞增殖、细胞迁移率、侵袭细胞数均低于NC shRNA组(72 h时细胞活性:0.81±0.01比1.56±0.01,F=255.200,P<0.05);迁移:[(47.67±2.62)%比(78.67±1.70)%,F=131.400,P<0.05];侵袭:[(127.67±3.68)个/视野比(249.30±3.29)个/视野,F=819.000,P<0.05],而sh-ADAM8组细胞凋亡率高于NC shRNA组[(16.66±0.90)%比(4.34±0.61)%,F=158.300,P<0.05],差异均有统计学意义。SC79逆转shADAM8对软骨肉瘤细胞的调控作用。在体内模型中,sh-ADAM8组p-Akt及mTOR分别低于NC shRNA组(p-Akt:0.03±0.01比0.29±0.10,t=3.092,P<0.05;mTOR:0.03±0.01比0.24±0.08,t=3.622,P<0.05),差异有统Objective Our study aims to explore the mechanism of ADAM metallopeptidase domain 8(ADAM8)in chondrosarcoma based on phosphoinositol 3 kinase(PI3K)/serine threonine kinase(Akt)/rapamycin mammalian target(mTOR).Methods The expressions of ADAM8 and proteins in PI3K/Akt/mTOR pathway in chondrosarcoma cell lines(SW1353 and HCS-2/8)and normal chondrocytes,purchased from American Type Culture Collection,were detected using Western blotting.According to a table of random number,HCS-2/8 cells were divided into NC short hairpin RNA(shRNA)group including cells transfected with negative control(NC)shRNA;shADAM8 group containing cells transfected with ADAM8 shRNA;shADAM8+SC79 group containing cells transfected with ADAM8 shRNA for 48 h and then incubated with 10.96 M SC79.The changes in proliferation,migration,invasion and apoptosis in cells were observed.To verify the role of ADAM8 in Akt/mTOR pathway,20 BALB/c nude mice mimicking chondrosarcoma in vivo were randomly divided into NC shRNA(n=10)and shADAM8(n=10)groups.Independent t test,one-way analysis of variance were used for statistical analysis.It indicated significance when P<0.05.Results ADAM8,p-PI3K,p-Akt and mTOR in chondrosarcoma cells was higher than these in HC cells(ADAM8:3.40±0.53 and 2.01±0.20 vs.1,F=27.260,P<0.05;p-PI3K:0.52±0.07 and 0.53±0.04 vs.0.21±0.05,F=22.120,P<0.05;p-Akt:0.58±0.05 and 0.61±0.08 vs.0.14±0.04,F=38.540,P<0.05;mTOR:0.52±0.04 and 0.65±0.04 vs.0.21±0.03,F=40.640,P<0.05).p-Akt,mTOR,proliferation,migration and invasion in shADAM8 group were lower than these in NC shRNA group(p-Akt:0.11±0.03 vs.0.75±0.03,F=0.578,P<0.05;mTOR:0.11±0.02 vs.0.79±0.07,F=69.600,P<0.05;proliferation at 72 h:0.81±0.01 vs.1.56±0.01,F=255.200,P<0.05:migration:(47.67±2.62)%vs.(78.67±1.70)%,F=131.400,P<0.05;invasion:127.67±3.68 vs.249.30±3.29,F=819.000,P<0.05),apoptosis rate in sh-ADAM8 group was higher than that in NC shRNA group[(16.66±0.90)%vs.(4.34±0.61)%,F=158.300,P<0.05].SC79 revised the role of ADAM8 silence in chondrosarcoma cells.In vivo,bot

关 键 词：软骨肉瘤 解整合素金属酶结构域8 丝氨酸-苏氨酸激酶/雷帕霉素哺乳动物靶点通路 

分 类 号：R738.1[医药卫生—肿瘤]