Supervisors info:
Παντελής Χατζής, Ερευνητής Β', ΕΚΕΒΕ Φλεμινγκ
Παναγιώτα Καφασλα, Ερευνήτρια Γ, ΕΚΕΒΕ Φλεμινγκ
Δέσποινα Σανουδου, Επίκουρη Καθηγήτρια Ιατρική Σχολής, ΕΚΠΑ
Summary:
The canonical WNT pathway plays a pivotal role in stem cell maintenance and affects the expression profile of target genes that are implicated in development, cell proliferation and selfrenewal of the intestinal epithelium. Constitutive and deviating activation of the pathway, caused by prominent mutations in its main components, such as APC, AXIN and β-catenin, is among the primary factors leading to the onset of Colorectal Cancer. Thus far, the mechanisms involved in WNT-dependent regulation of the target genes, due to their apparent complexity, remain elusive. Nonetheless, solid evidence indicates that a plethora of targets and regulatory elements of the effector β-catenin/TCF4 transcriptional complex reside in non-coding regions, reporting the existence of long non-coding RNAs that mediate transcriptional responses. Our research, surprisingly, has identified lnc-IGSF9, a lncRNA whose expression is down-regulated in colorectal cancer patients and also negatively regulated by the β-catenin/TCF4 canonical pathway. Overexpression of lnc-IGSF9 in CRC cell lines increases the expression of genes that control cell adhesion, ultimately promoting cell differentiation. Amongst the up-regulated genes, we distinguished IGSF9, a neighbouring gene that resides in the vicinity of lnc-IGSF9, as a putative mediator of lnc-IGSF9 function. IGSF9 is a cell adhesion protein, whose reduced expression strongly correlates with Colorectal Cancer manifestation. Further functional experiments demonstrate that lnc-IGSF9 and IGSF9 follow a similar expression pattern, with lnc-IGSF9 affecting directly IGSF9 expression but not vice versa. Analysis of ChIP-seq experiments against β-catenin in CRC cell lines revealed two strong binding sites in the locus of interest, particularly in the promoter and in a putative enhancer of lncIGSF9. Excision of these two loci and subsequent expression analysis of the clones showed that the absence of the putative enhancer does not impact lnc-IGSF9 expression, which indicates that its importance in the mechanism may be minor. On the other hand, excision of the promoter resulted in hardly no expression of lnc-IGSF9 as expected, however little impact on IGSF9 expression was observed attributed to its already low expression in CRC cell lines. In parallel, we investigated the role of CDX2 transcription factor, a protein that was identified as interactor of lnc-IGSF9 in RNA pull-down experiments. Re-analysis of publicly available ChIP-seq data in LS174T cells demonstrated the global binding pattern of CDX2. Specifically, CDX2 strongly binds to both the promoter and the putative intronic enhancer region of lnc-IGSF9 implying its role on the regulation of lnc-IGSF9 expression. Follow-up experiments clearly show that in-trans overexpression of CDX2, yields the upregulation of lnc-IGSF9 and IGSF9 as well, proposing a regulatory mechanism between those three components which may be subverted during carcinogenesis. In conclusion, further investigation should be done in order to delineate the exact mechanism of action of lnc-IGSF9 by thorough examination of their putative mediators CDX2 and IGSF9 as well.
Keywords:
LncRNAs, Colorectal cancer, Intestinal homeostasis, Long non coding RNAs, WNT pathway, Β-catenin, TCF4,
