LINC01638 promotes epithelial-to-mesenchymal transition in endometriosis epithelial cells by upregulating RHOB via HDAC1 suppression
Endometriosis is a common disease in women characterized by the growth of endometrial-like lesions at ectopic sites, associated with chronic pelvic pain and infertility. LINC01638 is a long non-coding RNA (lncRNA) that in several cancers has been implicated in the regulation of proliferation and epithelial-to-mesenchymal transition (EMT). Given that these processes are also important in endometriosis, we investigated the role of LINC01638 in this disease. We found that LINC01638 is upregulated in the epithelial layer of endometriosis lesions, and that knockdown of the gene in 12Z epithelial endometriosis cell line led to reduced proliferation, adhesion, migration and invasion. The reduction in proliferation was associated with increased p21 and p27 expression and G1 phase arrest of 12Z cells, showing that LINC01638 acts as an important epigenetic regulator of endometriosis epithelial cells cell cycle progression. Further, transcriptome analysis of LINC01638 control and knockdown cells revealed that a number of transcription factors associated with EMT are downregulated in the knockdown, including FOXC2, SNAI2, TWIST1 and ZEB1, along with the mesenchymal marker VIM. These genetic and phenotypic changes in 12Z knockdown cells were accompanied by a significant reduction in expression of cytoskeleton regulatory gene RHOB and an upregulation of HDAC1 expression. Chromatin immunoprecipitation analysis and HDAC1 inhibitory treatment in LINC01638 control and knockdown cells indicated that LINC01638 regulates RHOB expression via HDAC1 mediated regulation of its promoter acetylation. In vivo, RHOB is upregulated in the epithelial layer of endometriosis lesions compared to control and endometriosis patient eutopic endometrium, supporting a role in the disease. Together these results indicate that LINC01638 is an epigenetic regulator of pathogenesis of endometriosis promoting proliferation and EMT of endometriosis lesions. Overall design: To investigate the role of LINC01638 in endometriosis we performed an siRNA knockdown experiment in the 12Z endometriosis epithelial cell line followed by transcriptomic analysis 72 hours post knockdown. We performed 50bp paired-end stranded total RNA-seq (Ribo-Zero depletion) with a NovaSeq SP Illumina machine on 3 control siRNA and 3 LINC01638 knockdown samples.