Page 95 - Annual report 2021-22

Annual Report 2021-22 |

Sonam Dhamija

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Sonam Dhamija is interested in understanding the role of lncRNAs and lncRNA-derived peptides in
EMT and cancer cell migration. Identification of lncRNAs with a role in EMT and cell migration followed
by analysis of molecular mechanism of functional lncRNAs are the immediate aims. Functional analysis
of small ORF (smORF) harboring lncRNAs & their encoded micropeptides (miPs) as well as
identification of non-coding variants of protein-coding genes and their biological function are long
term goals. A TGF-beta induced murine EMT model was used to identify the candidate
lncRNAs/mRNAs and human orthologs of these candidates were identified based on sequence and/or
expression from syntenic loci. Expression of these candidates was compared with the publicly
available human lncRNA expression datasets to select more targets relevant to EMT and migration.
Primers were designed to quantify the candidates in the human EMT model being established. Cell
lines including A549 (lung cancer), U-87 MG and LN-229 (glioma), MCF10A and HMLE (breast) were
used for the establishment of TGFbeta-induced EMT model. Morphological effects confirm that all
models respond to TGFbeta and the RNA/protein samples were isolated and processed to monitor
lncRNA and EMT-marker expression. Both A549 and U-87 MG showed an enhanced expression of
mesenchymal markers CDH2 and ACTA2 and only A549 cells had reduced epithelial marker CDH1 RNA
expression. Top ten candidate genes were quantified in these cells and based on significant differential
expression, four of them are being continued for further analysis. The other cell types analyzed require
more standardization in marker expression at different time-points. One of the target genes is
C15orf48. TCGA data analysis shows that it is significantly upregulated in lung cancer compared to the
normal and its high expression correlates with poor patient survival. TGFbeta time kinetics shows
upregulation of C15orf48 concomitant with the morphological (and EMT marker expression) changes
in A549 cells and a return to basal levels upon induction of mesenchymal-to-epithelial transition. To
analyse the signaling pathways involved, cells treated with a panel of inhibitors and TGFbeta indicate
PKC and/or IKK activation is important for TGFbeta-induced upregulation of C15orf48. A second
candidate gene GATA6-AS1 which was found to be downregulated by TGF-beta in EMT model and in
lung cancer patient samples in TCGA analysis show that it is enriched in immunoprecipitation of EZH2
(polycomb repressor complex 2 component). C15orf48 is a bifunctional transcript that gives rise to a
MIR147B and encodes for a micropeptide. To investigate and distinguish the role of MIR147b and
C15orf48 micropeptide, lentiviral expression vectors with insert that expresses either MIR147b or miP
or both were created. EMT markers, morphology and migration will be analyzed after generating
stable lentivirally transduced cell lines.

A small molecule inhibitor screen in A549 cell has revealed the signalling pathways involved in the
modulation of lncRNA expression. This will be further verified by siRNA approaches to identify the
networks regulating lncRNA expression during EMT. CRISPR guide RNAs have been designed and
cloned for selected candidates keeping loss of-function cell line generation as the next target in the
plan. Polysome analysis and in vitro translation experiments will be used to segregate targets into
genuine lncRNAs and micropeptide encoders. Custom antibodies will be generated to investigate the
biological functions of micropeptides with potential roles in EMT and cancer. RNA-seq in
lncRNA/smORF loss-of-function and/or over expressing cells will be used to understand its genome