Wang Yu, Liu Shuwei
Research Center for Sectional and Imaging Anatomy Cheeloo College of Medicine, Shandong University, Jinan, China.
School of Basic Medical Sciences, Shandong University, Jinan, China.
Front Cell Dev Biol. 2021 Apr 1;9:643515. doi: 10.3389/fcell.2021.643515. eCollection 2021.
This study was to assess the specific impacts and mechanism of lncRNA GHET1 in the development of triple-negative breast cancer (TNBC).
The lncRNA GHET1 expression in TNBC tissues and adjacent healthy tissues was detected by qRT-PCR, and its expression was then measured at the cellular level, including TNBC cells and human normal breast epithelial cell line MCF10A. On the completion of transfection of negative shRNA or lncRNA GHET1 shRNA, the TNBC cells, HCC1937 and MDA-MB-468, were then cultured in a normoxia or hypoxia environment, respectively. 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, and transwell assay were applicable to the determination of cell proliferation, cell viability, and invasion in each group, respectively. Reagent kits were used for testing glucose consumption and lactate production levels. HCC1937 cells with knockdown or overexpression of lncRNA GHET1 were injected into the nude mice, followed by the examination of resulting tumor volume and weight. The distribution and expression of Hippo/YAP signaling pathway-related proteins were probed using western blotting.
Highly expressed lncRNA GHET1 in TNBC tissues and cells and induction of lncRNA GHET1 by hypoxia were proved. Knockdown of lncRNA GHET1 significantly reduced proliferation, viability, and invasion of TNBC cells, and decreased glucose consumption and lactate production levels under the hypoxia condition. Furthermore, lncRNA GHET1 knockdown decreased HIF-1α expression in hypoxia and significantly inhibited tumor development . Knockdown of lncRNA GHET1 increased the phosphorylation levels of LATS1 and Yes-associated protein (YAP) to retain YAP within the cytoplasm, while the overexpression of lncRNA GHET1 or hypoxia promoted nuclear translocation of YAP and TNBC development.
LncRNA GHET1 expression can be induced by hypoxia, which leads to excessive activation of the Hippo/YAP signaling pathway, thus promoting TNBC progression.
本研究旨在评估长链非编码RNA（lncRNA）GHET1在三阴性乳腺癌（TNBC）发生发展中的具体影响及机制。
采用qRT-PCR检测TNBC组织及癌旁正常组织中lncRNA GHET1的表达，并在细胞水平进行检测，包括TNBC细胞和人正常乳腺上皮细胞系MCF10A。在转染阴性对照短发夹RNA（shRNA）或lncRNA GHET1 shRNA后，分别在常氧或缺氧环境下培养TNBC细胞HCC1937和MDA-MB-468。分别采用5-乙炔基-2'-脱氧尿苷（EdU）检测法、集落形成检测法和Transwell检测法测定各组细胞的增殖、活力及侵袭能力。使用试剂盒检测葡萄糖消耗和乳酸生成水平。将lncRNA GHET1敲低或过表达的HCC1937细胞接种到裸鼠体内，随后检测形成肿瘤的体积和重量。采用蛋白质免疫印迹法检测Hippo/YAP信号通路相关蛋白的分布及表达。
证实lncRNA GHET1在TNBC组织和细胞中高表达，且缺氧可诱导lncRNA GHET1表达。敲低lncRNA GHET1可显著降低TNBC细胞的增殖、活力及侵袭能力，并降低缺氧条件下的葡萄糖消耗和乳酸生成水平。此外，敲低lncRNA GHET1可降低缺氧时缺氧诱导因子-1α（HIF-1α）的表达，并显著抑制肿瘤生长。敲低lncRNA GHET1可增加大肿瘤抑制因子1（LATS1）和Yes相关蛋白（YAP）的磷酸化水平，使YAP滞留于细胞质中，而lncRNA GHET1过表达或缺氧则促进YAP核转位及TNBC进展。
缺氧可诱导lncRNA GHET1表达，导致Hippo/YAP信号通路过度激活，从而促进TNBC进展。
