中国肿瘤临床
中國腫瘤臨床
중국종류림상
CHINESE JOURNAL OF CLINICAL ONCOLOGY
2014年
9期
551-554
,共4页
孙磊%刘雨清%李小龙%刘菲%张丽娜%李洪利%张宝刚
孫磊%劉雨清%李小龍%劉菲%張麗娜%李洪利%張寶剛
손뢰%류우청%리소룡%류비%장려나%리홍리%장보강
胶质瘤%ARK5%Gab2%LN-229细胞%小RNA干扰%侵袭
膠質瘤%ARK5%Gab2%LN-229細胞%小RNA榦擾%侵襲
효질류%ARK5%Gab2%LN-229세포%소RNA간우%침습
glioma%ARK5%Gab2%LN-229 cell%siRNA%invasiveness
目的：探讨Gab2-Akt-ARK5通路在胶质瘤侵袭中的意义。方法：采用免疫组织化学SP法检测90例胶质瘤组织中ARK5及Gab2表达。采用小RNA干扰转染LN-229细胞株，Western Blot检测瞬时转染后ARK5及Gab2表达。体外侵袭实验检测转染后侵袭能力变化及Western Blot检测Gab2下降后Akt和ARK5的磷酸化。结果：胶质瘤组织中ARK5和Gab2免疫组织化学阳性结果呈正相关且在高级别胶质瘤（WHO分级为Ⅲ、Ⅳ级）中表达明显高于低级别胶质瘤（WHO分级为Ⅰ、Ⅱ级）。转染ARK5、Gab2、ARK5-Gab2及SCR质粒的LN-229细胞分别称siARK5/LN-229、siGab2/LN-229、siARK5-siGab2/LN-229和SCR/LN-229。其中siARK5干扰效率为70%，siGab2的干扰效率为75%。转染后，与SCR/LN-229相比，siARK5/LN-229中ARK5表达降低， siGab2/LN-229中Gab2表达降低，siARK5-siGab2/LN-229中ARK5和Gab2表达均降低。siARK5/LN-229和siGab2/LN-229侵袭并穿透Matrigel膜基质的细胞数均比对照组少（P<0.01），且siARK5-siGab2/LN-229细胞数减少更显著（P<0.01）。在IGF-1刺激下，siGab2/LN-229中Akt和ARK5的磷酸化减弱。结论：应用小RNA干扰技术降低ARK5或Gab2表达使LN-229细胞侵袭转移能力降低，同时Gab2表达降低抑制ARK5和Akt磷酸化，提示Gab2-Akt-ARK5通路参与胶质瘤细胞的侵袭。
目的：探討Gab2-Akt-ARK5通路在膠質瘤侵襲中的意義。方法：採用免疫組織化學SP法檢測90例膠質瘤組織中ARK5及Gab2錶達。採用小RNA榦擾轉染LN-229細胞株，Western Blot檢測瞬時轉染後ARK5及Gab2錶達。體外侵襲實驗檢測轉染後侵襲能力變化及Western Blot檢測Gab2下降後Akt和ARK5的燐痠化。結果：膠質瘤組織中ARK5和Gab2免疫組織化學暘性結果呈正相關且在高級彆膠質瘤（WHO分級為Ⅲ、Ⅳ級）中錶達明顯高于低級彆膠質瘤（WHO分級為Ⅰ、Ⅱ級）。轉染ARK5、Gab2、ARK5-Gab2及SCR質粒的LN-229細胞分彆稱siARK5/LN-229、siGab2/LN-229、siARK5-siGab2/LN-229和SCR/LN-229。其中siARK5榦擾效率為70%，siGab2的榦擾效率為75%。轉染後，與SCR/LN-229相比，siARK5/LN-229中ARK5錶達降低， siGab2/LN-229中Gab2錶達降低，siARK5-siGab2/LN-229中ARK5和Gab2錶達均降低。siARK5/LN-229和siGab2/LN-229侵襲併穿透Matrigel膜基質的細胞數均比對照組少（P<0.01），且siARK5-siGab2/LN-229細胞數減少更顯著（P<0.01）。在IGF-1刺激下，siGab2/LN-229中Akt和ARK5的燐痠化減弱。結論：應用小RNA榦擾技術降低ARK5或Gab2錶達使LN-229細胞侵襲轉移能力降低，同時Gab2錶達降低抑製ARK5和Akt燐痠化，提示Gab2-Akt-ARK5通路參與膠質瘤細胞的侵襲。
목적：탐토Gab2-Akt-ARK5통로재효질류침습중적의의。방법：채용면역조직화학SP법검측90례효질류조직중ARK5급Gab2표체。채용소RNA간우전염LN-229세포주，Western Blot검측순시전염후ARK5급Gab2표체。체외침습실험검측전염후침습능력변화급Western Blot검측Gab2하강후Akt화ARK5적린산화。결과：효질류조직중ARK5화Gab2면역조직화학양성결과정정상관차재고급별효질류（WHO분급위Ⅲ、Ⅳ급）중표체명현고우저급별효질류（WHO분급위Ⅰ、Ⅱ급）。전염ARK5、Gab2、ARK5-Gab2급SCR질립적LN-229세포분별칭siARK5/LN-229、siGab2/LN-229、siARK5-siGab2/LN-229화SCR/LN-229。기중siARK5간우효솔위70%，siGab2적간우효솔위75%。전염후，여SCR/LN-229상비，siARK5/LN-229중ARK5표체강저， siGab2/LN-229중Gab2표체강저，siARK5-siGab2/LN-229중ARK5화Gab2표체균강저。siARK5/LN-229화siGab2/LN-229침습병천투Matrigel막기질적세포수균비대조조소（P<0.01），차siARK5-siGab2/LN-229세포수감소경현저（P<0.01）。재IGF-1자격하，siGab2/LN-229중Akt화ARK5적린산화감약。결론：응용소RNA간우기술강저ARK5혹Gab2표체사LN-229세포침습전이능력강저，동시Gab2표체강저억제ARK5화Akt린산화，제시Gab2-Akt-ARK5통로삼여효질류세포적침습。
Objective:This study aimed to investigate the effect and significance of a binding protein-2 (Gab2)-Akt-ARK5 signaling pathway on the invasion of glioma cells. Methods:Immunohistochemical methods were used to detect the expressions of Gab2 and ARK5 in 45 cases of glioma tissue. siRNA plasmid was used to transfect LN-229 cells, and western blot was performed to analyze the protein expressions of Gab2 and ARK5. In vitro Matrigel invasion assay was conducted to detect variations in the invasiveness of transfected cells. Western blot was also conducted to analyze the protein phosphorylation of Akt and ARK5 in the cells transfected with Gab2 plasmid. Results:Immunohistochemical assay revealed that the expressions of ARK5 and Gab2 in glioma cells were positively correlated, and both expressions were higher in high-grade glioma (WHO gradeⅢ,Ⅳ) than in low-grade glioma (WHO gradeⅠ,Ⅱ). LN-229 cells transfected with ARK5 plasmid, Gab2 plasmid, ARK5 and Gab2 plasmid, and control plasmid were named siARK5/LN-229, siGab2/LN-229, siARK5 and siGab2/LN-229, and SCR/LN-229, respectively. After transfection was performed, the protein expressions of ARK5 and Gab2 were respectively decreased in siARK5/LN-229 and siGab2/LN-229. The protein expressions of ARK5 and Gab2 in siARK5 and siGab2/LN-229 were also respectively decreased. After ARK5 or Gab2 was downregulated, the number of glioma cells, which invaded and penetrated Matrigel, was decreased (P<0.01). The number of glioma cells also decreased significantly after ARK5 and Gab2 were downregulated. The phosphorylation of Akt and ARK5 in siGab2/LN-229 cells was decreased after these cells were stimulated by insulin-like growth factor-1. Conclusion:The silencing of ARK5 or Gab2 impaired glioma cell invasiveness. The decreased protein expression of Gab2 inhibited the phosphorylation of Akt and ARK5. These results suggested that the Gab2-Akt-ARK5 signaling pathway could be relevantly involved in glioma cell invasion.
