CAJ | 학술논문

目的观察下调膜联蛋白A2 (annxin A2,ANXA2)表达对肝癌(HCC)细胞生物学行为的影响. 方法以定量PCR分析不同肝癌细胞ANXA2 mRNA转录水平,以Western blot 分析ANXA2表达;以免疫荧光检测ANXA2在细胞内表达和分布;以碘化丙啶染色和流式细胞术分析细胞周期;以CCK-8试剂盒分析细胞增殖潜能;以transwell实验分析其侵袭潜能;以创伤愈合试验分析其迁移潜能;以裸鼠移植瘤模型分析其致瘤潜能.两样本均数比较用t检验,率的比较用x2检验,等级资料比较用秩和检验,多样本均数的两两比较采用q检验,组间差异用单因素方差分析. 结果高侵袭潜能MHCC97-H细胞ANXA2表达明显高于HepG2、SMMC-7721和SMMC-7402和L02细胞;特异性shRNA沉默ANXA2效率在80％以上;免疫荧光显示ANXA2定位于细胞膜和细胞质,胞核少见;下调ANXA2表达明显下调HCC细胞S期比例(q=8.001,P=0.002),抑制细胞增殖(q=17.140,P＜0.01)、迁移潜能(q=12.808,P＜0.01)和侵袭潜能(q=9.069,P=0.002);裸鼠移植瘤模型显示干扰组瘤重下降(q=11.968, P＜0.01),瘤体中ANXA2表达下调(Z=2.530,P=0.011).结论下调ANXA2基因转录显著影响肝癌细胞的生物学行为,可望成为肝癌分子治疗的潜在靶目标.
목적 관찰하조막련단백A2 (annxin A2,ANXA2)표체대간암(HCC)세포생물학행위적영향. 방법 이정량PCR분석불동간암세포ANXA2 mRNA전록수평,이Western blot 분석ANXA2표체;이면역형광검측ANXA2재세포내표체화분포;이전화병정염색화류식세포술분석세포주기;이CCK-8시제합분석세포증식잠능;이transwell실험분석기침습잠능;이창상유합시험분석기천이잠능;이라서이식류모형분석기치류잠능.량양본균수비교용t검험,솔적비교용x2검험,등급자료비교용질화검험,다양본균수적량량비교채용q검험,조간차이용단인소방차분석. 결과 고침습잠능MHCC97-H세포ANXA2표체명현고우HepG2、SMMC-7721화SMMC-7402화L02세포;특이성shRNA침묵ANXA2효솔재80％이상;면역형광현시ANXA2정위우세포막화세포질,포핵소견;하조ANXA2표체명현하조HCC세포S기비례(q=8.001,P=0.002),억제세포증식(q=17.140,P＜0.01)、천이잠능(q=12.808,P＜0.01)화침습잠능(q=9.069,P=0.002);라서이식류모형현시간우조류중하강(q=11.968, P＜0.01),류체중ANXA2표체하조(Z=2.530,P=0.011).결론 하조ANXA2기인전록현저영향간암세포적생물학행위,가망성위간암분자치료적잠재파목표.
Objective To investigate the effects of Annexin A2 (ANXA2) deficiency on the malignant biological behaviour of hepatoma cells.Methods The human hepatocellular carcinoma (HCC) cells lines MHCC97-H,HepG2,SMMC-7721,SMMC-7402 and L02 were evaluated.The expression and distribution of ANXA2 were analysed by western blotting,real-time PCR,immunofluorescence and immunohistochemistry.Cell cycle was assessed by flow cytometry and propidium iodide staining.Effects of ANXA2 silencing on invasion and migration potential were assessed by transwell assay and wound healing assay,respectively.Proliferative potential was assessed by CCK-8 kit in vitro and xenograft tumour-growth assay in vivo.The t-test,chi square test,rank sum test,q-test and F-test were used for statistical analyses.Results The expression level of ANXA2 was markedly higher in the MHCC97-H cells with high metastasis potential than in the HepG2,SMMC-7721,SMMC-7402 and L02 cells.The efficiency of shRNA-mediated ANXA2 deficiency was more than 80％.Immunofluorescence analysis of the MHCC97-H cells indicated that ANXA2 expression was mainly localized to the cellular membrane and cytoplasm,with some nuclear localization.Down-regulation of ANXA2 led to S-phase arrest of HCC cells (q =8.001,P =0.002) and an inhibition of proliferation (q =17.140,P ＜ 0.01),migration (q =12.808,P ＜ 0.01) and invasion potential (q =9.069,P =0.002).Xenograft tumour-growth assay indicated that shRNA targeting of ANXA2 led to lower tumour weight (q =11.968,P ＜0.001) and down-regulated ANXA2 expression (Z =2.530,P =0.011).Conclusion Down-regulation of Annexin A2 gene transcription effectively changes the biological behaviours ofhepatoma cells,and may represent a potential target of HCC molecular therapies.