摘要
目的探讨显性负性突变ALK5(DNALK5)是否可通过抑制转化生长因子-β(TGF-β)/SMAD信号通路来抑制人乳腺癌癌细胞MDA-MB-231增殖。方法采用荧光定量PCR检测乳腺癌细胞中ALK5受体表达;通过流式细胞术和MTT检测DNALK5对三阴性乳腺癌MDA-MB-231增殖和周期的改变;采用western blot检测DNALK5感染MDA-MB-231后对乳腺癌细胞中TGF-β/SMAD信号通路中SMAD2/3总蛋白和磷酸化蛋白表达及其下游靶基因ID1表达的影响。结果乳腺癌细胞系中存在ALK5的普遍表达,其中MDA-MB-231表达最高;DNALK5腺病毒感染MDA-MB-231后,MTT显示第3天DNALK5组细胞吸光度值(0.35±0.04)显著低于RFP组(0.58±0.06),流式细胞术显示其周期阻滞于G+0期;荧光定量PCR和western blot显示ID1和CyclinD1表达降低,进一步研究发现TGF-β/SMAD信号通路中关键蛋白SMAD2/3磷酸化蛋白表达降低。结论 DNALK5抑制乳腺癌MDA-MB-231增殖,可通过抑制TGF-β/SMAD信号通路的激活,下调肿瘤增殖相关基因ID1和CyclinD1表达来实现。
Objective To investigate whether dominant negative mutation ALK5 can inhibit the proliferation of human breast cancer cell line MDA-MB-231 by inhibiting the TGF-β/SMAD signaling pathway.Methods The expression of ALK5 in breast cancer cells was detected by fluorescence quantitative PCR.The change of proliferation and cycle of breast cancer cells were determined by MTT assay and flow cytometry.The expression of ID1,phosphorylation protein and total protein of SMAD2/3 on TGF-β/SMAD signaling pathway were detected by the western blot while MDA-MB-231 cells were infected with Adenovirus dominant negative ALK5.Results The results showed that ALK5 mRNA was detected in all kinds of breast cancer cells and the the highest expression breast cancer cell was MDA-MB-231;MTT and flow cytometry showed the absorbance of DNALK5 group cell(0.35±0.04)was significantly lower than RFP group(0.58±0.06)and cell cycle was blocked in G 0 stage after Ad DNALK5 infection 3 days.The expression of mRNA and protein of ID1 and CyclinD1 were decreased,by real time PCR and western blot.further study found that SMAD2/3 phosphorylation protein on TGF-β/SMAD signaling pathway were reduced.Conclusion DNALK5 can inhibit the proliferation of breast cancer cell line MDA-MB-231 through down regulation ID1 and CyclinD1 expression and inhibit the activation of TGF-β/SMAD signaling pathway.
作者
方娟
李文鲜
黄小兰
FANG Juan;LI Wenxian;HUANG Xiaolan(Department of Clinical Laboratory,Yubei People′s Hospital,Chongqing 401120,China)
出处
《检验医学与临床》
CAS
2018年第13期1903-1905,1909,共4页
Laboratory Medicine and Clinic
关键词
显性负性突变
ALK5
转化生长因子-Β
SMAD
乳腺癌
增殖
dominant negative mutation
ALK5
transforming growth factor-β
SMAD
breast cancer
tumor proliferation
