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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。 -
关键词:
- 套细胞淋巴瘤 /
- 滤泡树突状细胞 /
- miR-548m /
- 细胞周期蛋白依赖性激酶6 /
- 集落形成
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. -
Key words:
- mantle cell lymphoma /
- FDCs /
- miR-548m /
- CDK6 /
- colony formation
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图 1 miR-548m与CDK6-3'UTR的结合位点，以及野生型和突变型CDK6-3'UTR的结构
Figure 1. MiR-548m binding site at the CDK6-3'UTR and the construct of wild and mutational CDK6-3'UTR
图 2 Western blot实验检测Jeko-1细胞与/不与HK细胞共培养CDK6的表达水平。Suspension，即悬浮细胞，与HK细胞无粘附；HK-adhesion，与HK细胞直接粘附的细胞，*P＜0.05
Figure 2. Western blot was used to test the expression of CDK6 after Jeko-1 cells adhered to FDCs. Suspension, cells in suspension; HK-adhesion, cells after adhesion to HK cells, *P＜0.0
图 3 Western blot实验检测转染pre-miR-548m或anti-miR-548m后CDK6表达水平的变化，*P＜0.0
Figure 3. Western blot was used to test the expression level of CDK6 after miR-548m overexpression or inhibition, *P＜0.05
图 4 pGL3-CDK6-WT和pre-miR-548m共转染HEK293细胞以后荧光素酶相对活性变化，*P＜0.05
Figure 4. Relative activity of luciferase in HEK293 cells after being cotransfected with pre-miR-548m and pGL3-CDK6-WT, *P＜0.05
A. RT-qPCR was used to test the expression of miR-548m after being transfected with pre-miR-548m in HEK293 cells; B. Luciferase report assay confirmed that miR-548m directly targeted 3'-UTR of CDK6. Ctrl, pGL3-control; WT, pGL3-CDK6-WT; MUT, pGL3-CDK6-MUT
图 5 miR-548m和CDK6对HK细胞介导的HBL-2细胞的集落形成能力的影响
Figure 5. miR-548m and CDK6 affected the HK cell-mediated colony forming activity in HBL-2 cells
A. Overexpression of miR-548m suppressed the colony formation of HBL-2 cell with or wothout HK cell adhesion; B. Knockdown of CDK6 suppressed the colony formation of HBL-2 cell with or wothout HK cell adhesion
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