缺氧诱导因子-1α诱导隧道纳米管形成并促进膀胱肿瘤细胞的侵袭能力  

Hypoxia inducible factor-1αinduces tunneling nanotube formation and promotes the invasive ability of bladder tumor cells

作　　者：陆金金 李国灏[1,2] Lu Jinjin;Li Guohao(Department of Urology,the Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China;The Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Key Laboratory for Molecular Diagnosis of Hubei Province,Wuhan 430014,China)

机构地区：[1]华中科技大学同济医学院附属武汉中心医院泌尿外科,武汉430014 [2]华中科技大学同济医学院附属武汉中心医院分子诊断湖北省重点实验室,武汉430014

出　　处：《中华实验外科杂志》2024年第12期2717-2720,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金资助项目(2021CFB196)。

摘　　要：目的探讨缺氧诱导因子-1α(HIF-1α)促进膀胱肿瘤细胞侵袭能力以及诱导细胞间隧道纳米管(TNTs)形成的机制。方法分别将T24细胞和RT4细胞(购自ATCC公司)培养24 h后, 用Mito-Tracker Deep Red标记线粒体, Actin-Tracker Green标记细胞骨架, 使用激光共聚焦显微镜观察并计数细胞间的TNTs。将HIF-1α转染T24细胞, 使用激光共聚焦显微镜观察过表达HIF-1α的T24细胞间TNTs数量的变化, 通过划痕实验及Transwell实验观察转染后T24细胞的侵袭转移能力的变化。蛋白质印迹法(Western blot)检测雷帕霉素靶蛋白(mTOR)/4EBP1/P70S6K-eIF4e/S6RP信号通路蛋白的表达差异。两组间比较采用t检验, 多组间比较采用单因素方差分析。结果过表达HIF-1α基因的T24细胞(HIF-1α-T24细胞)间TNTs的数量多于转染空载体的T24细胞(NC-T24细胞)(84.80±2.91比60.40±0.91, t=8.01, P<0.01)。划痕实验结果显示HIF-1α-T24细胞的愈合速度高于NC-T24细胞[(43.39±0.57)%比(25.76±0.53)%,t=22.93,P<0.01], Transwell实验发现通过基质迁移的HIF-1α-T24细胞高于NC-T24细胞(55.00±1.53比32.00±1.16, t=12.01, P<0.01)。分子学研究显示HIF-1α-T24细胞mTOR的磷酸化水平高于NC-T24细胞(0.58±0.12比0.26±0.06, t=4.47, P<0.01), 下游分子4EBP1和P70S6K的磷酸化水平上调(0.51±0.06比0.32±0.06, t=5.74, P<0.01;0.44±0.02比0.27±0.03, t=5.21, P<0.01), 从而效应分子eIF4E和S6RP的磷酸化水平上调(0.45±0.02比0.22±0.03, t=11.61, P<0.01;0.52±0.12比0.29±0.06, t=3.32, P<0.01)。结论 HIF-1α通过激活mTOR信号通路诱导膀胱肿瘤细胞间TNTs的形成进而促进膀胱肿瘤细胞的侵袭能力。ObjectiveTo explore the mechanism by which hypoxia inducible factor-1α(HIF-1α)promotes the invasive ability of bladder tumor cells and induces the formation of intercellular tunneling nanotubes(TNTs).MethodsT24 cells and RT4 cells(purchased from ATCC)were cultured for 24 hours,mitochondria were labeled with Mito-Tracker Deep Red,and cytoskeletal was labeled with Actin-Tracker Green.Laser confocal microscopy was used to observe and count intercellular TNTs.HIF-1αwas transfected into T24 cells,and the changes in the number of TNTs between T24 cells overexpressing HIF-1αwere observed using laser confocal microscopy.The changes in the invasion and metastasis ability of T24 cells after transfection were observed by scratch test and Transwell test.The expression difference of mammalian target of rapamycin(mTOR)/4EBP1/P70S6K-eIF4e/S6RP signaling pathway proteins was detected by Western blotting.ResultsThe number of TNTs between T24 cells overexpressing HIF-1αgene(HIF-1α-T24 cells)was greater than that between T24 cells transfected with empty vector(NC-T24 cells)(84.80±2.91 vs.60.40±0.91,t=8.01,P<0.01).The results of scratch test showed that the healing speed of HIF-1α-T24 cells was faster than that of NC-T24 cells[(43.39±0.57)%vs.(25.76±0.53)%,t=22.93,P<0.01].Transwell assay found that more HIF-1α-T24 cells migrated through the matrix than NC-T24 cells(55.00±1.53 vs.32.00±1.16,t=12.01,P<0.01).Molecular studies showed that the phosphorylation level of mTOR in HIF-1α-T24 cells was higher than that in NC-T24 cells(0.58±0.12 vs.0.26±0.06,t=4.47,P<0.01),and the phosphorylation levels of downstream molecules 4EBP1 and P70S6K were upregulated(0.51±0.06 vs.0.32±0.06,t=5.74,P<0.01;0.44±0.02 vs.0.27±0.03,t=5.21,P<0.01),thereby upregulating the phosphorylation levels of effector molecules eIF4E and S6RP(0.45±0.02 vs.0.22±0.03,t=11.61,P<0.01;0.52±0.12 vs.0.29±0.06,t=3.32,P<0.01).ConclusionHIF-1αinduces the formation of TNTs between bladder tumor cells by activating the mTOR signaling pathway,thereby promot

关 键 词：缺氧诱导因子-1Α 隧道纳米管 膀胱肿瘤 侵袭能力 

分 类 号：R737.14[医药卫生—肿瘤]