Page 83 - Annual report 2021-22

Annual Report 2021-22 |

Malabika Datta

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The roles of long non-coding RNAs in the diabetic liver were studied in mechanistic detail with a special
emphasis on the implications in fatty liver and gluconeogenesis. The main objectives were to study
the cellular consequences of ncRNA-mictochondria associated membrane (MAM) correlation and
effects on hepatic metabolism and explore MAM proteins as targets of altered ncRNAs in the liver
during diabetes. Altered lipid metabolism is a significant phenotype associated with the liver during
diabetes. Since levels of H19 and Vdac1 are altered in the liver during diabetes, it was important to
explore if interactions between H19 and Vdac1 might influence lipid levels. While Vdac1 levels were
increased, the levels of Vdac2 and Vdac3 were analyzed by qRT-PCR and only mRNA levels are Vdac2
were increased with no change in Vdac3 levels in the livers of db/db mice. Also, levels of H19 that are
downregulated in the livers of diabetic mice, promoted an increase in the levels of Vdac1, while
exploring potential transcription factors binding on the Vdac1 promoter using online available tools,
it was found that around 59 transcription factors had binding sites on the Vdac1 promoter. Among
these, around nine transcription factors were found to be predicted interactors with H19 as obtained
from a lncRNA interactome database. This suggests that by interacting with these transcription
factors, H19 might influence the cellular levels of Vdac1 and subsequent cellular processes.

Regulation of Vdac1 by H19 significantly increased in the interaction between the ER and mitochondria
as demonstrated by proximity ligation assay where there was significant increase in the number of
dots representing ER-mitochondria contacts. Together with this, H19 inhibition increased
mitochondria calcium levels, ROS generation and decreased ATP content. Interestingly, H19 inhibition
increased the transcript levels of gluconeogenic genes suggesting an increase in hepatic glucose
output. However, there was no change in the hepatic lipid content during H19 inhibition, although
some lipid metabolism genes showed modest increases. In-vivo injection of H19 siRNA increased
Vdac1 and gluconeogenic genes' levels within the liver, all suggesting that H19 inhibition alone is
sufficient enough to increase hepatic gluconeogenesis. Overexpression of Vdac1 alone was sufficient
to increase hepatic gluconeogenic gene expression. Also, H19 inhibition induced ER stress and JNK
phosphorylation without altering the activation of the other MAPKs. H19 inhibition increased Ser
phosphorylation of IRS1 suggesting its effect on the insulin signalling cascade.

In a previous study, this group had demonstrated altered levels of miRNAs in the livers of diabetic
db/db mice. To study if these altered miRNAs might regulate hepatic MAM proteins, they extracted
the predicted targets to the altered miRNAs and filtered MAM proteins from among the predicted
targets. miR-378a-3p and miR-93-5p were prioritized for validation as these were found to target more
than one MAM protein and therefore, expected to significantly affect the MAM complex. Hepa 1-6
cells were transfected with the scramble or miR-93-5p (25 and 50 nM) and after 48 h, the levels of its
target, FACL-4 were quantified by Western Blot analysis. There was a significant decrease in the
endogenous levels of the target, FACL4 following transfection, indicating that FACL-4 might be a
potential target of miR-93-5p to be investigated.

In an attempt to re-engineer epidermal cells and evaluate matrices for regeneration in diabetic
wounds, Malabika Datta's group has performed in vitro assays for wound closure. Transfection of