Background
WNT and TGFβ signaling pathways play critical regulatory roles in cardiomyocyte fate determination and differentiation. MiRNAs are also known to regulate different biological processes and signaling pathways. Here, we intended to find candidate miRNAs that are involved in cardiac differentiation through regulation of WNT and TGFβ signaling pathways.
Methods
Bioinformatics analysis suggested
hsa-miR-335-3p
and
hsa-miR-335-5p
as regulators of cardiac differentiation. Then, RT-qPCR, dual luciferase, TOP/FOP flash, and western blot analyses were done to confirm the hypothesis.
Results
Human embryonic stem cells (hESCs) were differentiated into beating cardiomyocytes, and these miRNAs showed significant expression during the differentiation process. Gain and loss of function of
miR-335-3p
and
miR-335-5p
resulted in
BRACHYURY
,
GATA4
, and
NKX2-5
(cardiac differentiation markers) expression alteration during the course of hESC cardiac differentiation. The overexpression of
miR-335-3p
and
miR-335-5p
also led to upregulation of
CNX43
and
TNNT2
expression, respectively. Our results suggest that this might be mediated through enhancement of WNT and TGFβ signaling pathways.
Conclusion
Overall, we show that
miR-335-3p/5p
upregulates cardiac mesoderm (
BRACHYURY
) and cardiac progenitor cell (
GATA4
and
NKX2-5
) markers, which are potentially mediated through activation of WNT and TGFβ signaling pathways. Our findings suggest
miR-335-3p/5p
to be considered as a regulator of the cardiac differentiation process.
Electronic supplementary material
The online version of this article (10.1186/s13287-019-1249-2) contains supplementary material, which is available to authorized users.
Crizotinib is an anticancer tyrosine kinase inhibitor that is approved for use as a first-line treatment for some non-small-cell lung cancers. L1196M is the most frequently observed mutation in NSCLC patients. This mutation, known as the gatekeeper mutation in the ALK kinase domain, confers resistance to crizotinib by sterically blocking the binding of the drug. However, the molecular mechanism of crizotinib resistance caused by the L1196M mutation is still unclear. Molecular dynamics simulation was therefore utilized in this study to investigate the mechanism by which the L1196M mutation may affect crizotinib resistance. Our results suggest that larger fluctuations in some important regions of the mutant complex compared to the wild-type complex may contribute to the resistance of the mutant complex to crizotinib. Also, mutation-induced alterations to the secondary structure of the complex as well as unstable hydrogen-bonding patterns in the A-loop and P-loop regions decrease the total binding energy of the complex. This study therefore provides a molecular explanation for the resistance to crizotinib caused by the L1196M mutation, which could aid the design of more efficient and selective drugs.
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