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Mild overexpression of a novel transcription factor Grainy head like-2 (Grhl2) elicits hybrid phenotype in human astrocytoma cells

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Title Mild overexpression of a novel transcription factor Grainy head like-2 (Grhl2) elicits hybrid phenotype in human astrocytoma cells
 
Creator Prasannaraj, Govindaraj
Prabhakaran, Vasudevan
 
Subject Boyden chamber assay
Brain cancer
Cell migration
Chemotherapy
Embryonic development
Epithelial markers
Invasion
Mesenchymal markers
Scratch assay
Temozolomide
Tumor progression/suppression
 
Description 386-396
The Grainy head like-2 (Grhl2) transcription factor plays a major role in embryonic and cancer development. The role of
Grhl2 has been intensively studied in various cancers but not for brain cancer. Hence, in this study, we provide a preliminary
understanding on the role of Grhl2 that regulate the transition of astrocytoma cells. The human A172 astrocytoma cell line, a
mesenchymal cell characterized by mild overexpression of Grhl2 transcription factor, was used in this study. At first, the
Grhl2 stably overexpressing A172 clones into three types i.e., Grhl2+ (mild), Grhl2++ (moderate) and Grhl2+++ (high)
based on mRNA and protein expression levels of Grhl2 were characterized. Phenotypic characteristics of vector and Grhl2+
cells were observed using phase contrast microscopy. Quantitative PCR (qPCR), Western blot and immunofluorescence
were used to detect the level of mesenchymal markers (N-cadherin/vimentin) and also epithelial markers (E-cadherin/ β-
catenin) in vector and Grhl2+ cells. The migration and invasion characteristics of vector and Grhl2+ cells were determined
by scratch assay and Boyden chamber assay. Further, the Grhl2+ cells were characterized to determine the effect of
temozolomide chemotherapy drug which were widely used in treating brain cancer. As expected, in phase contrast image,
we observed the mesenchymal characteristic of A172 cells becomes hybrid phenotype i.e., mixture of mesenchymal
(spindle-like fibroblast morphology) and epithelial (cobblestone like appearance) cells upon Grhl2 mild expression (Grhl2+)
when compared to vector cells. Further, we found that there was a significant upregulation of E-cadherin at both mRNA and
protein levels in Grhl2+ cells when compared to vector cells. There was a significant upregulation of β-catenin, N-cadherin
and vimentin at mRNA levels, but there was no significant upregulation at the protein levels in Grhl2+ cells compared to the
vector cells. The migration and invasion were diminished in Grhl2+ cells when compared to the vector control cells. We
observed that the Grhl2+ were sensitive to the temozolomide compared to the vector cells. This infers that the Grhl2+ cells
were unable to attain complete transition of mesenchymal to epithelial state, and hence we categorized the Grhl2+ cells as
hybrid phenotype. The results provide a better understanding of the largely unknown function of Grhl2 in human
astrocytoma cells as tumor progression or suppression.
 
Date 2022-06-03T06:51:30Z
2022-06-03T06:51:30Z
2022-06
 
Type Article
 
Identifier 0975-1009 (Online); 0019-5189 (Print)
http://nopr.niscpr.res.in/handle/123456789/59847
 
Language en
 
Publisher NIScPR-CSIR, India
 
Source IJEB Vol.60(06) [June 2022]