FDCs-miR-548m-CDK6轴在套细胞淋巴瘤集落形成中的研究
Role of FDCs-miR-548m-CDK6 axis in clonogenicity of mantle cell lymphoma
-
摘要:目的 探讨FDCs-miR-548m-CDK6轴在套细胞淋巴瘤(mantle cell lymphoma, MCL)集落形成中的作用。方法 分别采用RT-qPCR和Western blot检测MCL细胞与滤泡树突状细胞(FDCs)共培养后miR-548m和CDK6的变化。以生物信息学软件预测miR-548m的靶点, Western Blot检测MCL细胞系分别转染pre-miR-548m和anti-miR-548后细胞周期蛋白依赖激酶6(CDK6)的变化。荧光素酶报告基因实验验证CDK6是否为miR-548m的直接作用靶点。MCL细胞系与/不与FDCs共培养, 过表达miR-548m或者敲低CDK6后MCL的集落形成能力。结果 FDCs与MCL的粘附作用可以下调miR-548m并上调CDK6。生物信息学软件预测显示CDK6的3'UTR是miR-548m的潜在靶点, 且过表达miR-548m能够减少CDK6的表达, 抑制miR-548m表达能够增加CDK6。荧光素酶报告基因实验证实CDK6的3'UTR是miR-548m的一个直接作用靶点。集落形成实验显示过表达miR-548m或者敲低CDK6后, 细胞的集落形成能力明显减弱。结论 FDCs通过抑制MCL中miR-548m表达上调CDK6, 增强MCL的集落形成能力。Abstract:Objective To study the role of FDCs-miR-548m-CDK6 axis on clonogenicity in mantle cell lymphoma.Methods RT-qPCR and Western blot were used respectively to test the expression of miR-548m and CDK6. Bioinformatics assay was applied to predict the targets of miR-548m, and Western Blot was used to test the expression level of CDK6 after miR-548m overexpression or inhibition. Luciferase report assay was performed to test whether CDK6 was a direct target of miR-548m. Colony forming assay was used to test the colony forming activity in MCL after overexpression of miR-548m or knockdown of CDK6.Results Cell adhesion to FDCs induced downregulation of miR-548m and CDK6 expression in MCL. Bioinformatics assay revealed that miR-548m could target the 3'-UTR of CDK6 and that a negative correlation exists between the level of miR-548m and the CDK6 expression. Luciferase report assay confirmed that miR-548m directly targeted 3'-UTR of CDK6. Colony forming assay showed that overexpression of miR-548m or knockdown of CDK6 significantly suppressed MCL colony formation.Conclusion This study reveals that FDC-enhanced mantle cell lymphoma clonogenicity is mediated by the miR-548m/CDK6 axis.
下载: