Department of Emergency Surgery, Sichuan Academy of Medical Sciences & Sichaun Provincial People's Hospital, Chengdu, Sichuan, China.
J Cell Mol Med. 2019 Jun;23(6):3808-3823. doi: 10.1111/jcmm.14071. Epub 2019 Apr 17.
We aimed to explore the mechanism of the KCNQ1OT1/miR-760/PPP1R1B axis acting to regulate methotrexate (MTX) resistance of colorectal cancer (CRC). Differentially expressed mRNAs and lncRNAs in MTX-sensitive CRC cell lines and MTX-resistant cell lines were determined through microarray analysis. Application of bioinformatics analysis was aimed to uncover the relationships among the lncRNAs/miRNAs/mRNAs, and to demonstrate the effects of cAMP signalling pathway in MTX-resistant CRC. The expression level of RNA and proteins was, respectively, detected using qRT-PCR and Western blot assays, whereas the dual-luciferase reporter gene assay was implemented to verify the targeted relationship. The influence of the lncRNA/miRNA/mRNA axis on biological functions of MTX-resistant cells and on the growth of tumours determined through both vitro and vivo experiments. LncRNA KCNQ1OT1 and PPP1R1B mRNA were overexpressed in MTX-resistant CRC tumour cells. KCNQ1OT1 functioned as a sponge of miR-760, which targeted PPP1R1B. Knockdown of KCNQ1OT1 enhanced chemosensitivity towards MTX through the sponging of miR-760. MiR-760 expressed at low levels targeted PPP1R1B in the activated cAMP signalling pathway under MTX treatment. Knockdown of KCNQ1OT1 dampened the proliferation of MTX-resistant (HT29/MTX) cells by regulating the miR-760/PPP1R1B axis, which also induced cell cycle arrest together with apoptosis. KCNQ1OT1 regulated the expression of PPP1R1B and the downstream genes CREB and CBP in the cAMP signalling pathway. MTX showed a suppressive function on CRC progression. KCNQ1OT1 enhanced the MTX resistance of CRC cells by regulating miR-760-mediated PPP1R1B expression via the cAMP signalling pathway.
我们旨在探索 KCNQ1OT1/miR-760/PPP1R1B 轴调控结直肠癌细胞（CRC）中甲氨蝶呤（MTX）耐药的机制。通过微阵列分析确定 MTX 敏感 CRC 细胞系和 MTX 耐药细胞系中差异表达的 mRNAs 和 lncRNAs。应用生物信息学分析旨在揭示 lncRNA/miRNA/mRNA 之间的关系，并证明 cAMP 信号通路在 MTX 耐药 CRC 中的作用。使用 qRT-PCR 和 Western blot 检测 RNA 和蛋白质的表达水平，而通过双荧光素酶报告基因检测来验证靶向关系。通过体外和体内实验确定 lncRNA/miRNA/mRNA 轴对 MTX 耐药细胞的生物学功能和肿瘤生长的影响。lncRNA KCNQ1OT1 和 PPP1R1B mRNA 在 MTX 耐药 CRC 肿瘤细胞中过度表达。KCNQ1OT1 作为 miR-760 的海绵体，靶向 PPP1R1B。敲低 KCNQ1OT1 通过海绵 miR-760 增强了对 MTX 的化学敏感性。在 MTX 处理下，激活的 cAMP 信号通路中 miR-760 表达水平低，靶向 PPP1R1B。敲低 KCNQ1OT1 通过调节 miR-760/PPP1R1B 轴，抑制 MTX 耐药（HT29/MTX）细胞的增殖，同时诱导细胞周期停滞和凋亡。KCNQ1OT1 调节 cAMP 信号通路中 PPP1R1B 及其下游基因 CREB 和 CBP 的表达。MTX 对 CRC 进展具有抑制作用。KCNQ1OT1 通过调节 miR-760 介导的 PPP1R1B 表达，通过 cAMP 信号通路增强 CRC 细胞对 MTX 的耐药性。
