CAJ | 학술논문

目的研究增殖抑制基因(HSG)对乳腺癌MDA-MB-231细胞(简称231细胞)生物学行为的影响并对其作用机制进行探讨.方法构建HSG全长真核表达载体,通过脂质体瞬时转染法使其在231细胞中高表达,通过MTT比色实验检查肿瘤细胞的体外增殖能力、Matrigel穿膜实验检测肿瘤细胞体外侵袭能力和应用流式细胞术检测肿瘤细胞的细胞周期及凋亡情况;并应用GST-pulldown法检测HSG高表达对乳腺癌细胞Ras蛋白活性的影响.结果将构建好的重组人HSG真核表达质粒pcDNA3-MYC-HSG转染至231细胞中,MTT比色实验结果显示HSG的过表达可以抑制肿瘤细胞的增殖;Matrigel侵袭实验显示转染HSG的肿瘤细胞的穿膜细胞数(HSG/231组,78.5个±5.8个)与转染空载体组(vector/231组,131.1个±14.5个)相比明显减少.流式细胞分析结果显示转染HSG/231组细胞出现了G_0/G_1期阻滞(56.3%±2.3%),且凋亡细胞的百分比与对照组(vector/231组为50.4%±1.9%)相比有所增加.Ras蛋白活性检测发现转染HSG/231组的乳腺癌细胞Ras蛋白活性明显下降.结论 HSG表达上调可抑制高侵袭性231细胞的体外增殖和侵袭能力,使其出现G_0/G_1期细胞周期阻滞,并可促进细胞凋亡.HSG对乳腺癌细胞的抑制作用可能与其抑制细胞Ras活性有关.
목적 연구증식억제기인(HSG)대유선암MDA-MB-231세포(간칭231세포)생물학행위적영향병대기작용궤제진행탐토.방법 구건HSG전장진핵표체재체,통과지질체순시전염법사기재231세포중고표체,통과MTT비색실험검사종류세포적체외증식능력、Matrigel천막실험검측종류세포체외침습능력화응용류식세포술검측종류세포적세포주기급조망정황;병응용GST-pulldown법검측HSG고표체대유선암세포Ras단백활성적영향.결과 장구건호적중조인HSG진핵표체질립pcDNA3-MYC-HSG전염지231세포중,MTT비색실험결과현시HSG적과표체가이억제종류세포적증식;Matrigel침습실험현시전염HSG적종류세포적천막세포수(HSG/231조,78.5개±5.8개)여전염공재체조(vector/231조,131.1개±14.5개)상비명현감소.류식세포분석결과현시전염HSG/231조세포출현료G_0/G_1기조체(56.3%±2.3%),차조망세포적백분비여대조조(vector/231조위50.4%±1.9%)상비유소증가.Ras단백활성검측발현전염HSG/231조적유선암세포Ras단백활성명현하강.결론 HSG표체상조가억제고침습성231세포적체외증식화침습능력,사기출현G_0/G_1기세포주기조체,병가촉진세포조망.HSG대유선암세포적억제작용가능여기억제세포Ras활성유관.
Objective To investigate the effect of over expression of human hyperplasia suppressor gene(HSG)on proliferation,invasion,apoptosis and cell cycle of human breast cancer cells and to determine the relationship between HSG and Ras-dependent signaling pathway.Methods Full length HSG coding sequences were cloned into plasmid pcDNA3.0.The recombinant plasmids were transfected into MDA-MB-231,a highly malignant breast cancer cell line.Vacant pcDNA3.0 was used as the control.MTT,Matrigel transwell assay and flow cytometric analysis were used to test for proliferation,invasion,cell cycle distribution and apoptosis of tumor cells after transient transfection of HSG.GST-pulldown and Western blotting assays were performed to investigate the activity of Ras protein.Results HSG transfection inhibited proliferation of MDA-MB-231 ceils,and significanfly decreased the number of invading cells in Matrigel transwell assay compared with the vector/231 group(78.5±5.8 vs.131.1±14.5)cells.FACS analyses demonstrated that compared with the vector/231 group,up-regulation of HSG promoted breast cancer cell apoptosis[(35.8±4.8)%vs.(25.6±3.5%)]and induced G_0/G_1 phase arrest[(56.3±2.3)%vs.(50.4±1.9%)]after transfection for 18 hours.Furthermore,GST-pulldown assay showed that overexpression of HSG remarkably decreased the activity of Ras(about 65%lower than control).Conclusions HSG exibits multiple anticancer functions in breast cancer cells including inhibition of proliferation and in vitro invasion,G_0/G_1 arrest and promotion of apoptosis.Besides,inhibition of Ras-dependent signaling pathway may be involved in these processes.