靶向p65基因的miRNA对人三阴性乳腺癌细胞裸鼠移植瘤生长的抑制作用

Suppression of human triple-negative breast cancer in nude mice by p65miRNA

背景与目的:三阴性乳腺癌(triple-negative breast cancer,TNBC)是乳腺癌中预后较差的一个亚型,如何防治TNBC的快速生长成为近几年临床研究的热点之一。NF-κB信号通路在肿瘤发生、发展的各个环节中扮演重要角色,有望成为肿瘤基因治疗新的方向。本研究通过建立人TNBC裸鼠移植瘤模型,观察靶向沉默NF-κB p65亚基的微小RNA(microRNA,miRNA)治疗对TNBC裸鼠移植瘤生长及凋亡的影响,并初步探讨其可能的作用机制。方法:建立人TNBC细胞株MDA-MB-231裸鼠移植瘤动物模型,瘤旁注射p65miRNA质粒(p65miRNA组),同时以注射Neg-miRNA质粒和PBS作为Neg-miRNA对照组和空白对照组。监测肿瘤生长变化,测量肿瘤质量。流式细胞术(flow cytometry,FCM)检测肿瘤细胞凋亡的变化。免疫组化法检测肿瘤组织中p65的表达。Western blot法检测肿瘤组织中Bcl-2和Bax蛋白的表达水平。结果:经p65miRNA处理后,裸鼠肿瘤的生长受到明显抑制。FCM结果表明,p65miRNA组肿瘤细胞凋亡率为(31.08±3.52)%,明显高于Neg-miRNA组(5.76±1.02)%和空白对照组(4.29±0.86)%(P<0.05)。此外,p65miRNA组裸鼠肿瘤组织p65和Bcl-2的蛋白表达明显下调,Bax的蛋白表达显著上调。结论:p65miRNA能抑制人TNBC裸鼠皮下移植瘤的生长,且在体内可诱导肿瘤细胞凋亡。

Background and purpose：Triple-negative breast cancer（TNBC） often grows rapidly and has the worst outcomes,with a high recurrence rate and a short interval between recurrence and death among all subtypes of breast cancer.It is expected that more effective strategies to prevent the rapid growth of TNBC will become available in the near future.NF-κB signaling plays critical roles in tumor development and progression,suggesting that it might become a potential therapeutic target for cancer treatment.Thus,the aim of this study is to evaluate the possible role of p65miRNA on the growth and apoptosis of xenografted TNBC nude mice,and elucidate its potential mechanism.Methods：Human TNBC MDA-MB-231 cells were subcutaneously inoculated into BALB/c nude mice to establish the xenografted model.The tumors injected with control miRNA（Neg-miRNA） or PBS served as control.Tumor growth and tumor weight were observed after treatment with p65miRNA.Apoptotic rate was measured by flow cytometry（FCM）.Protein expression of p65 was detected by immunohistochemical staining.Expressions of apoptosis-related genes,Bcl-2 and Bax,were detected by Western blotting.Results：Tumor growth was obviously suppressed and tumor volume was significantly smaller in the group treated with p65miRNA than in control groups.FCM result revealed that p65miRNA evidently evoked cell apoptosis.The apoptotic rate of p65miRNA group was（31.08±3.52）%,which was obviously higher than that of Neg-miRNA [（5.76±1.02）%] and control group [（4.29±0.86）%].The expressions of p65 and Bcl-2 in p65miRNA group were significantly downregulated,but the expression of Bax was markedly upregulated in vivo.Conclusion：p65miRNA inhibits the tumorigenesis and induces cell apoptosis of MDA-MB-231 cell xenograft in nude mice.