Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells

Abstract

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasisand is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.According to the newest projections published by the American Cancer Society, despite significant overall reductions in colorectal cancer (CRC) incidence and mortality, there is a need for further efforts to advance therapies on the early stage of cancer and metastasis development 1 . To escape from the primary tumour site and to form metastatic lesions, epithelial cancer cells must acquire a more migratory phenotype to overcome several anatomical barriers. The process of conversion of the epithelial cell phenotype towards a more mesenchymal phenotype (EMT process) is considered to be an initial and critical for metastasis. Although there are many concepts of how EMT is modified in cancer, (reviewed by Gurzu et al. 2 ), the mechanism of EMT is still incompletely elucidated and there are many conflicting results published. During cancer EMT, there is a dynamic modulation of the interplay between transcription factors, gene expression and miRNAs (microRNAs /small non-coding RNAs) 3 . Thus, the understanding of the molecular interactions during the phenotype switch towards more mesenchymal and invasive cells has become important for future therapeutic strategies.We previously performed a global transcriptomic and phenotypic characteristic of the HT29 colorectal adenocarcinoma cell line affected by the transcription factor Snail (Snai1), a core regulator of the early stages of the epithelial phenotype conversion that initiates metastasis. We have shown that upregulation of Snail in HT29 cells results in an incomplete phenotype conversion, up to the intermediate epithelial state 4 . We and others have found that enhanced Snail expression is associated with a more aggressive phenotype, poorer clinical outc...

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail

“…Our previous studies showed that Snail, an early regulator of EMT, affects human HT29 CRC cells transcriptome and miRNA profile and changes HT29 cells phenotype to a promigratory one [9,10]. In this paper, we described Snail effect on mouse CRC line MC38 in order to characterize it before moving to in vivo mouse models of colon cancer.…”

“…EV fractions were obtained after sequential centrifugations and ultracentrifugation for 1.5 h at 100,000× g and followed by washing in PBS and another ultracentrifugation. The final EV pellets were resuspended in PBS and stored at −80 • C until use ( [10] and modified from [34]). We submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV190098) [35].…”

“…In numerous studies, metastatic cancer cells were found to express the Snail protein, a zinc-finger transcription factor that represses the transcription of E-cadherin and triggers EMT [ 7 , 8 ]. The impact of Snail on the characteristics of CRC cells undergoing EMT has been investigated by our group in various aspects for several years [ 9 , 10 , 11 ].…”

Angioregulatory microRNAs in Colorectal Cancer

“…MiR-194 is involved in EMT induction and invasion in CRC cell lines [135]; nevertheless, its downregulation in CRC tissue compared to adjacent non-cancerous tissue and its association with inhibition of cell proliferation via regulation of the MAP4K4/c-Jun/MDM2 signaling pathway indicate tumor suppressive features of miR-194 [136]. It has been reported that the miR-194 promoter has a binding site for Snail which leads to miR-194 downregulation and THBS1 upregulation in snail-mediated EMT in HT-29 cells [137]. THBS1 (thrombospondin 1) is an intrinsic inhibitor of angiogenesis which also has a suppressive effect on CRC proliferation and migration.…”

Functional mechanisms of miR‐192 family in cancer

“…58 It was found that overexpression of SNAIL, a key transcription factor in early EMT, in HT29 CRC cells repressed the expression of miR-192 and miR-194. It was suggestedthat the presence of SNAIL alters the complex mRNA/miRNA interactions in the early steps of metastasis and impacts the content of extracellular vesicles (EVs), which are small membrane vesicles, released from HT29 cells 59.…”