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目的:探讨microRNA-130b(miR-130b)的表达差异对三阴性乳腺癌(triple-negative breast cancer,TNBC)MDA-MB-231细胞增殖、迁移及侵袭能力的影响及机制。方法 :通过Lipofectamine TM 2000将miR-130b抑制剂或模拟物及阴性对照转染到MDA-MB-231细胞中。应用实时定量聚合酶链反应(quantitative real time-polymerase chain reaction,qRT-PCR)检测miR-130b表达和转染效率。应用MTT实验、克隆形成实验、划痕实验以及transwell实验,检测miR-130b模拟物或抑制剂对MDA-MB-231细胞功能影响。应用生物信息学网站寻找miR-130b可能的靶基因。应用双荧光素酶载体实验验证预测的靶基因,并通过qRT-PCR以及Western印迹实验进一步证实。结果:MDA-MB-231细胞转染miR-130b抑制剂或模拟物。与阴性对照组相比,模拟物组miR-130b的表达显著升高,抑制剂组miR-130b的表达显著下降,差异有统计学意义(P<0.01)。与阴性对照组相比,模拟物组细胞增殖(P<0.05)、克隆形成(P<0.05)、侵袭(P<0.01)及迁移(划痕试验P<0.05,transwell试验P<0.05)能力明显增高。相反,抑制剂组细胞增殖(P<0.05)、克隆形成(P<0.05)、侵袭及迁移(划痕试验P<0.05,transwell试验P<0.01)能力较阴性对照组明显受抑制。生物信息学技术预测CYLD为miR-130b的潜在靶基因。双荧光素酶载体实验结果显示转染miR-130b模拟物+psciCHECK2-CYLD 3′UTR(野生型)后荧光素酶活性较转染阴性对照+psciCHECK2-CYLD 3′UTR(野生型)降低57.21%(P<0.001)。此外,与阴性对照组相比,CYLD基因m RNA水平在抑制剂组或模拟物组均无明显改变(P>0.05);但CYLD蛋白表达在抑制剂组明显上调,模拟物组的表达明显下降(P<0.001)。结论:miR-130b可促进TNBC MDA-MB-231细胞的增殖、侵袭及迁移,其作用至少部分通过抑制CYLD基因实现。
OBJECTIVE: To investigate the effect of the differential expression of microRNA-130b (miR-130b) on the proliferation, migration and invasion of triple negative breast cancer (TNBC) MDA-MB-231 cells and its mechanism. Methods: miR-130b inhibitors or mock and negative controls were transfected into MDA-MB-231 cells by Lipofectamine ™ 2000. The expression of miR-130b and transfection efficiency were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of miR-130b mimics or inhibitors on the function of MDA-MB-231 cells were detected by MTT assay, clone formation assay, scratch assay and transwell assay. Application of bioinformatics website for miR-130b potential target genes. The predicted target genes were verified by dual luciferase vector assays and further confirmed by qRT-PCR and Western blotting. Results: MDA-MB-231 cells were transfected with miR-130b inhibitor or mimic. Compared with the negative control group, the expression of miR-130b in the mock group was significantly increased, and the expression of miR-130b in the inhibitor group was significantly decreased (P <0.01). Compared with the negative control group, the mock cells showed significant proliferation (P <0.05), colony formation (P <0.05), invasion (P <0.01) and migration (P <0.05 in scratch test, P <0.05 in transwell test) Increase. On the contrary, the cell proliferation (P <0.05), clonality (P <0.05), invasion and migration (P <0.05 in scratch test and P <0.01 in transwell test) were significantly inhibited in the inhibitor group compared with the negative control group. Bioinformatics techniques predict CYLD as a potential target gene for miR-130b. The results of dual luciferase assay showed that the luciferase activity of miR-130b mock + psciCHECK2-CYLD 3’UTR (wild type) was 57.21% lower than that of transfected negative control + psciCHECK2-CYLD 3’UTR (wild type) (P <0.001). In addition, compared with the negative control group, the m RNA level of CYLD gene in the inhibitor group or the mock group had no significant change (P> 0.05); however, the expression of CYLD protein was significantly up-regulated in the inhibitor group and the mock group was significantly decreased (P <0.001). Conclusion: miR-130b can promote the proliferation, invasion and migration of TNBC MDA-MB-231 cells. The effect of miR-130b is at least partly achieved by inhibiting CYLD gene.