CircBIRC6调节miR-138-5p/RRM2轴对乳腺癌细胞恶性生物学行为的影响

Impact of CircBIRC6 on the malignant biological behavior of breast cancer cells by regulating miR-138-5p/RRM2 axis

目的探讨环状非编码RNA(Circ)BIRC6调节微小RNA(miR)-138-5p/核糖核苷酸还原酶小亚基M2(RRM2)轴对乳腺癌(BC)细胞恶性生物学行为的影响。方法qRT-PCR检测BC组织及癌旁组织、人乳腺上皮细胞系(MCF-10A)以及BC细胞系(MCF-7、MDA-MB-231、MDA-MB-468和MDA-MB-453)中CircBIRC6、miR-138-5p、RRM2 mRNA表达水平;免疫组化法检测RRM2表达;以MCF-7细胞为研究对象,分别转染干扰CircBIRC6质粒(si CircBIRC6)、阴性对照(si NC)至细胞,记为si CircBIRC6组、si NC组;将si CircBIRC6分别与miR-138-5p抑制剂(miR-138-5pinhibitor)、阴性对照(inhibitorNC)共转染至细胞,标记为siCircBIRC6+miR-138-5pinhibitor组、si CircBIRC6+inhibitor NC组,同时以未转染的MCF-7细胞作为对照组,CCK-8、划痕实验及Transwell实验分别检测增殖、迁移率及侵袭变化;qRT-PCR检测各组MCF-7细胞中CircBIRC6、miR-138-5p、RRM2 mRNA表达水平;Western blot检测各组MCF-7细胞中基质金属蛋白酶(MMP-9)、增殖蛋白Ki-67、RRM2表达;双萤光素酶验证CircBIRC6、RRM2与miR-138-5p靶向关系。结果BC组织及细胞中CircBIRC6、RRM2mRNA表达显著增加,miR-138-5p表达显著降低(P<0.05)。双萤光素酶实验验证了CircBIRC6、RRM2与miR-138-5p的靶向关系。RRM2阳性表达在BC组织增加(P<0.05);与siNC组、对照组相比,siCircBIRC6组增殖率、迁移率、侵袭数、CircBIRC6、MMP-9、Ki-67、RRM2蛋白及mRNA表达显著降低,miR-138-5p表达显著增加(P<0.05);与siCircBIRC6+inhibitorNC组相比,si CircBIRC6+miR-138-5pinhibitor组增殖率、迁移率、侵袭数、MMP-9、Ki-67、RRM2蛋白及mRNA表达显著增加,miR-138-5p表达显著降低(P<0.05),CircBIRC6表达无差异(P>0.05)。结论干扰CircBIRC6可抑制BC细胞恶性生物学行为,可能通过miR-138-5p/RRM2轴实现。

Objective To investigate the impact of circular non coding RNA(Circ)BIRC6 on the malignant biological behavior of breast cancer(BC)cells by regulating the microRNA(miR)-138-5p/ribonucleotide reductase small subunit M2(RRM2)axis.Methods The expression levels of CircBIRC6,miR-138-5p and RRM2 mRNA in BC tissues and adjacent tissues,human breast epithelial cell lines(MCF-10A)and BC cell lines(MCF-7,MDA-MB-231,MDA-MB-468 and MDA-MB-453)were detected by qRT-PCR.The expression of RRM2 was detected by immunohistochemistry.MCF-7 cells were studied,the interfering CircBIRC6 plasmid(si CircBIRC6)and negative control(si NC)were transfected into the cells respectively,and were recorded as si CircBIRC6 group and si NC group.si CircBIRC6 was co-transfected with miR-138-5p inhibitor(miR-138-5p inhibitor)or negative control(inhibitor NC)into cells,and label them as si CircBIRC6+miR-138-5p inhibitor group,si CircBIRC6+inhibitor NC group respectively,meanwhile,the non-transfected MCF-7 cells were used as the control group,CCK-8,scratch test and Transwell test were used to respectively detect proliferation,mobility and invasion.the expression levels of CircBIRC6,miR-138-5p and RRM2 mRNA in MCF-7 cells were detected by qRT-PCR.Western blot was applied to detect the expression of matrix metalloproteinase(MMP-9),proliferative protein Ki-67 and RRM2 in MCF-7 cells of each group;double luciferase was applied to verify the targeting relationship between CircBIRC6,RRM2 and miR-138-5p.Results The expression of CircBIRC6 and RRM2 mRNA was obviously higher in BC tissues and cells,and the expression of miR-138-5p was obviously lower(P<0.05).The targeting relationship between CircBIRC6,RRM2 and miR-138-5p was verified by double luciferase experiment.The positive expression of RRM2 was increased in BC tissues(P<0.05),Compared with the si NC group and the control group,the proliferation rate,migration rate,invasion number,and the expression of CircBIRC6,MMP-9,Ki-67,RRM2 protein and mRNA in the si CircBIRC6 group were obviously decreased,the expression of miR-138-5p was obviously increased(P<0.05).Compared with the si CircBIRC6+inhibitor NC group,the proliferation rate,migration rate,invasion number,the expression of MMP-9,Ki-67,RRM2 protein and mRNA in the si CircBIRC6+miR-138-5p inhibitor group were obviously increased,the expression of miR-138-5p was obviously decreased(P<0.05),there was no difference in the expression of CircBIRC6(P>0.05).Conclusion Down-regulating CircBIRC6 can inhibit the malignant biological behavior of BC cells,possibly by the miR-138-5p/RRM2 axis.