CAJ | 학술논문

目的探讨缩短型脾酪氨酸激酶[SYK(S)]对人肝癌细胞侵袭转移的影响及其分子机制.方法采用实时定量逆转录聚合酶链反应(RT-qPCR)和Western blot方法检测7株人肝癌细胞株和人永生化肝细胞L02中SYK(S)的表达；分别构建SYK(S)过表达的稳定细胞株SMMC7721-SYK(S)和MHCC-97H-SYK(S)；Transwell侵袭小室检测稳定细胞株中SYK(S)对肝癌细胞侵袭能力的影响.裸鼠肝脏原位移植瘤模型检测SYK(S)表达对肿瘤肺转移的影响,每组裸小鼠6只；干扰SYK(S)表达后,Western blot检测肝癌细胞中上皮细胞-间充质转化(EMT)主要标志蛋白E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的变化,以及免疫组织化学方法检测裸鼠皮下移植瘤中SYK(S)对E-cadherin和Vimentin蛋白表达的影响.结果在具有较高转移潜能的肝癌细胞株MHCC-97H和MHCC-97L中,SYK(S)相对高表达；SYK(S)可增强肝癌细胞穿透Transwell小室基底膜的能力(P＜0.05).体内实验表明,SYK(S)可促进原位肝移植瘤的肺转移(P＜0.05)；在小鼠皮下移植瘤中,SYK(S)导致E-cadherin蛋白表达降低,而增加Vimentin蛋白表达水平.相反,干扰SYK(S)表达后,肝癌细胞中E-cadherin蛋白表达升高,伴Vimentin蛋白表达降低.结论 SYK(S)可通过EMT促进肝癌的侵袭转移.
목적 탐토축단형비락안산격매[SYK(S)]대인간암세포침습전이적영향급기분자궤제.방법 채용실시정량역전록취합매련반응(RT-qPCR)화Western blot방법검측7주인간암세포주화인영생화간세포L02중SYK(S)적표체；분별구건SYK(S)과표체적은정세포주SMMC7721-SYK(S)화MHCC-97H-SYK(S)；Transwell침습소실검측은정세포주중SYK(S)대간암세포침습능력적영향.라서간장원위이식류모형검측SYK(S)표체대종류폐전이적영향,매조라소서6지；간우SYK(S)표체후,Western blot검측간암세포중상피세포-간충질전화(EMT)주요표지단백E-개점단백(E-cadherin)화파형단백(Vimentin)적변화,이급면역조직화학방법검측라서피하이식류중SYK(S)대E-cadherin화Vimentin단백표체적영향.결과 재구유교고전이잠능적간암세포주MHCC-97H화MHCC-97L중,SYK(S)상대고표체；SYK(S)가증강간암세포천투Transwell소실기저막적능력(P＜0.05).체내실험표명,SYK(S)가촉진원위간이식류적폐전이(P＜0.05)；재소서피하이식류중,SYK(S)도치E-cadherin단백표체강저,이증가Vimentin단백표체수평.상반,간우SYK(S)표체후,간암세포중E-cadherin단백표체승고,반Vimentin단백표체강저.결론 SYK(S)가통과EMT촉진간암적침습전이.
Objective To investigate the possible effect of the short form of spleen tyrosine kinase [SYK(S)],on the invasion and metastasis of human hepatocellular carcinoma (HCC),and the underlying mechanism.Methods Immunoblotting and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to examine the expression of SYK(S) in an immortalized normal liver cell line LO2 and seven human HCC cell lines.Two HCC cell lines,SMMC7721 with no SYK(S) expression and MH-CC-97H with high SYK(S) expression,were used to generate stable cell lines by retroviral transduction.In vitro Matrigel invasion assays were performed to explore the effects of SYK(S) on HCC invasion.Both subcutaneous and orthotopic models (n =6) were used for animal studies,and metastatic nodules in the lung were calculated.Endogenous SYK(S) expression was blocked by small interfering RNA (siRNA) in the SYK(S)-positive HCC cell lines.Epithelial-mesenchymal transition (EMT) related markers,E-cadherin and Vimentin were analyzed by using immunoblotting.In addition,the expression of E-cadherin and Vimentin was also detected in xenograft tissues of SMMC7721-SYK(S) by using immunohistochemistry.Results MHCC-97H and MHCC-97L cells expressed relative higher SYK(S) protein and mRNA,which are the most metastatic among all cell lines used in this study.As compared with parental cells,the invasive ability of MHCC-97H and SMMC7721 cells expressing SYK(S) was obviously increased (P ＜0.05).More importantly,SYK (S) expression facilitated lung metastasis in vivo (P ＜ 0.05).Knockdown of SYK (S) expression led to the increased expression of E-eadherin and reduced expression of Vimentin.On the contrary,the decreased expression of E-cadherin and increased expression of Vimentin were detected in the xenograft tumors of SMMC7721-SYK(S) stable cells by using immunohistochemistry,indicating that SYK(S) promoted HCC metastasis via inducing EMT.Conclusion SYK (S) promotes invasion and metastasis of HCC via inducing EMT.