School of Basic Medical Sciences, Lanzhou University, Lanzhou, China.
Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, China.
J Cell Mol Med. 2023 Apr;27(7):991-1005. doi: 10.1111/jcmm.17716. Epub 2023 Mar 13.
Cyclin D1 (CCND1), a mediator of cell cycle control, has a G870A polymorphism which results in the formation of two splicing variants: full-length CCND1 (CCND1a) and C-terminally truncated CCND1 species (CCND1b). However, the role of CCND1a and CCND1b variants in cancer chemoresistance remains unknown. Therefore, this study aimed to explore the molecular mechanism of alternative splicing of CCND1 in breast cancer (BC) chemoresistance. To address the contribution of G870A polymorphism to the production of CCND1 variants in BC chemoresistance, we sequenced the G870A polymorphism and analysed the expressions of CCND1a and CCND1b in MCF-7 and MCF-7/ADM cells. In comparison with MCF-7 cells, MCF-7/ADM cells with the A allele could enhance alternative splicing with the increase of SC-35, upregulate the ratio of CCND1b/a at both mRNA and protein levels, and activate the CDK4/CyclinD1-pRB-E2F1 pathway. Furthermore, CCND1b expression and the downstream signalling pathway were analysed through Western blotting and cell cycle in MCF-7/ADM cells with knockdown of CCND1b. Knockdown of CCND1b downregulated the ratio of CCND1b/a, demoted cell proliferation, decelerated cell cycle progression, inhibited the CDK4/CyclinD1-pRB-E2F1 pathway and thereby decreased the chemoresistance of MCF-7/ADM cells. Finally, CCND1 G870A polymorphism, the alternative splicing of CCDN1 was detected through Sequenom Mass ARRAY platform, Sanger sequencing, semi-quantitative RT-PCR, Western blotting and immunohistochemistry in clinical BC specimens. The increase of the ratio of CCND1b/a caused by G870A polymorphism was involved in BC chemoresistance. Thus, these findings revealed that CCND1b/a ratio caused by the polymorphism is involved in BC chemoresistance via CDK4/CyclinD1-pRB-E2F1 pathway.
细胞周期蛋白 D1（CCND1）是细胞周期调控的介质，存在 G870A 多态性，导致两种剪接变体的形成：全长 CCND1（CCND1a）和 C 端截断的 CCND1 物种（CCND1b）。然而，CCND1a 和 CCND1b 变体在癌症化疗耐药中的作用尚不清楚。因此，本研究旨在探讨乳腺癌（BC）化疗耐药中 CCND1 可变剪接的分子机制。为了研究 G870A 多态性对 BC 化疗耐药中 CCND1 变体产生的贡献，我们对 G870A 多态性进行了测序，并分析了 MCF-7 和 MCF-7/ADM 细胞中 CCND1a 和 CCND1b 的表达。与 MCF-7 细胞相比，携带 A 等位基因的 MCF-7/ADM 细胞可通过增加 SC-35 增强可变剪接，上调 mRNA 和蛋白水平的 CCND1b/a 比值，并激活 CDK4/CyclinD1-pRB-E2F1 通路。此外，通过 Western blot 和 MCF-7/ADM 细胞中 CCND1b 的敲低分析 CCND1b 表达和下游信号通路。CCND1b 的敲低下调了 CCND1b/a 的比值，抑制了细胞增殖，减缓了细胞周期进程，抑制了 CDK4/CyclinD1-pRB-E2F1 通路，从而降低了 MCF-7/ADM 细胞的化疗耐药性。最后，通过 Sequenom Mass ARRAY 平台、Sanger 测序、半定量 RT-PCR、Western blot 和免疫组织化学检测临床 BC 标本中的 CCND1 G870A 多态性、CCND1 的可变剪接。G870A 多态性引起的 CCND1b/a 比值增加参与了 BC 化疗耐药。因此，这些发现表明，多态性引起的 CCND1b/a 比值通过 CDK4/CyclinD1-pRB-E2F1 通路参与 BC 化疗耐药。
