反义寡核苷酸vMO调控Bcl-x剪接对人乳腺癌细胞侵袭转移的影响  

Effect of an antisense oligonucleotide targeting Bcl-xsplicing on the invasion and metastasis of human breast cancer cells

作　　者：李清祥 廖志华 张静[3,4] 刘勇 白羽 蒋沁炆 朱春龙 邬艳 王小中[3,4] 刘静[3,4] LI Qing-xiang;LIAO Zhi-hua;ZHANG Jing;LIU Yong;BAI Yu;JIANG Qin-wen;ZHUChun-long;WU Yan;WANG Xiao-zhong;LIU Jing(Clinical Laboratory,Nanchang Third Hospital,Nanchang,China 330009;Clinical Laboratory,Jiangxi Provincial Maternal and Child Health Care Hospital,Nanchang China 330006;Clinical Laboratory,The Second Hospital Affiliated with Nanchang University,Nanchang,China 330006;Jiangxi Province Key Laboratory of Laboratory Medicine,Nanchang,China 330006;Nanchang Donghu District Center for Disease Control and Prevention,Nanchang,China 330000)

机构地区：[1]南昌市第三医院检验科,江西南昌330009 [2]江西省妇幼保健院检验科 [3]南昌大学第二附属医院检验科 [4]江西省检验医学重点实验室 [5]南昌市东湖区疾病预防控制中心

出　　处：《中国病原生物学杂志》2020年第5期527-532,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81560033,No.81860034);江西省科技厅科技计划项目(No.20171BAB205067);江西省教育厅课题(No.GJJ170158)。

摘　　要：目的探讨靶向纠正Bcl-x基因异常剪接对人乳腺癌细胞侵袭转移等生物学行为的影响。方法人乳腺癌MDA-MB-231细胞和正常NIH3T3细胞均设转染组和空白对照组。实验组采用靶向Bcl-x基因的特异性Vivo-Morpholino Antisense Oligomer(vMO)进行转染,空白对照组常规培养。采用qRT-PCR和Western Blot分别检测各组Bcl-x剪接体Bcl-xL和Bcl-xS mRNA和蛋白表达水平,通过流式细胞仪(FCM)、平板克隆形成试验、Transwell侵袭试验及划痕试验检测vMO对MDA-MB-231和NIH3T3细胞增殖、凋亡、侵袭及转移的影响。结果VMO能稳定高效转染细胞,荧光阳性率达99.17%,并能持续稳定存在7天以上;VMO能在胞内有效调节Bcl-x的剪接模式,MDA-MB-231细胞中Bcl-xL mRNA和蛋白较对照组显著降低,而Bcl-xS mRNA和蛋白较对照组显著升高(P<0.05);平板克隆形成试验和FCM结果显示,vMO转染前后,NIH3T3细胞克隆形成率分别为43%和35%,凋亡率分别为0.27%和0.53%,差异均无统计学意义;vMO转染MDA-MB-231细胞前后,穿膜细胞数分别为(67±1)和(47±1)个,发生迁移的细胞数分别为(106±2)和(78±1)个,差异具有统计学意义(P<0.05);划痕试验显示,转染组MDA-MB-231细胞划痕愈合能力较对照组减弱(P<0.05),平均迁移距离分别为(218±21)mm和(389±12)mm。结论vMO靶向结合Bcl-x pre-mRNA促使Bcl-xL向Bcl-xS转化,在抑制乳腺癌细胞侵袭及转移方面具有重要作用。Objective To investigate the effects of targeted correction of aberrant splicing of the Bcl-xgene on the invasion and metastasis of human breast cancer cells.Methods MDA-MB-231 human breast cancer cells and normal NIH3 T3 cells were divided into an experimental group and a blank control group.The experimental group was transfected with Vivo-Morpholino Antisense Oligomer(vMO),which specifically targeted the Bcl-x gene,and the blank control group was cultured routinely.Real-time fluorescence quantitative PCR(qRT-PCR)and Western blotting were respectively used to determine the levels of expression of Bcl-xL and Bcl-xS mRNA and protein in each group.The effects on proliferation and apoptosis were observed using a colony formation assay and flow cytometry(FCM),and the effects on the migration and invasion of breast cancer cells were observed using a wound healing assay and Transwell assay.ResultsCells were stably and efficiently transfected with VMO,with 99.17%fluorescing,and cells fluoresced for more than 7 days.VMO effectively regulated the mode of Bcl-x splicing in cells.Levels of Bcl-xL mRNA and protein in MDA-MB-231 cells were significantly lower than those in the control group,while levels of Bcl-xS mRNA and protein were significantlyhigher than those in the control group(P<0.05).The plate clone formation test and FCM indicated that clone formation by NIH3 T3 cells was 43%before transfection with vMO and 35%afterwards,and the rate of apoptosis was 0.27%before and 0.53%afterwards.Clone formation and the rate of apoptosis did not differ significantly.The number of invading cells was 67±1 before transfection of MDA-MB-231 cells with vMO afterwards and 47±1 afterwards,and the number of invading cells was 106±2 before and 78±1 afterwards.The number of invading and migrating cells differed significantly(P<0.05).The scratch test indicated that the scratch healing ability of MDA-MB-231 cells in the transfection group decreased compared to that of the control group(P<0.05),and the average distance of migration was 21

关 键 词：乳腺癌细胞 BCL-X 反义寡核苷酸 侵袭 转移 

分 类 号：R737.9[医药卫生—肿瘤]