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

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。

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

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 218±21 mm in the transfection group and 389±12 mm in the control group.Conclusion Regulation of Bcl-x splicing promoted the conversion of Bcl-xL to Bcl-xS,which played an important role in inhibiting the invasion and metastasis of breast cancer cells.