Abstract 3759: Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells
Authors: Shrestha Archana , Lahooti Behnaz , Madadi Mahboubeh , Mikelis Constantinos M. , Mattheolabakis George 
Published: April 2023

Abstract
Lung cancer (LC) is among the leading causes of death from malignancies worldwide. miRNAs (miRs), small noncoding RNAs capable of regulating gene expression through the cell’s RNAi mechanism, have emerged as promising molecules for cancer treatment. In our previous work, we identified two miRs, miR-143 and miR-506, whose transient transfection induced apoptosis, cell cycle inhibition, and cyclin-dependent kinase (CDK) 1, 4, and 6 downregulations. Here, we developed stable deregulations of the miRs’ expressions in A549 cells, individually or in combination, to evaluate their effect on LC cell behavior. To establish homogeneous populations of deregulated cells, A549 cells with stable up and downregulated miR-143 and miR-506, alone or in combination, were sorted according to marker expression using fluorescence-activated cell sorting (FACS). miR deregulations were confirmed with quantitative real-time polymerase chain reaction (qRT -PCR) analysis, and the cells were analyzed for changes in cell proliferation through cell cycle analysis using flow cytometry and cell doubling time using Quantitative Phase Imaging (QPI). With the same method, morphometric characteristics were quantified. Finally, we evaluated the motility of the cells using a scratch assay and determined the gene expression of CDKs. Taqman qPCR analysis indicated a &gt;5-fold increase of the respective miR’s basal expression due to the stable transfections. The cell cycle analysis indicated a complex behavior for the individual miRs, while, in contrast, the combined upregulation of the two miRs indicated a G2 inhibition, representing a well-defined behavior in comparison to both the control and downregulation groups. Furthermore, the combined miR upregulation increased the cell doubling time compared to the control and respective downregulations. Similarly, using QPI microscopy, we observed that the miR combination upregulation had higher cellular sphericity, perimeter, and area compared to the control group. The wound healing assay did not indicate statistically significant differences among the different groups, with miR-506 upregulation demonstrating the slowest closure rate, followed by miR -143. In contrast, the differences between the miR-combination and control group were small, although the anti-miR-combination indicated an accelerated closure. In conclusion, the combined upregulation of both miRs inhibited cell proliferation through a G2 arrest, while the deregulation of the individual miRs demonstrated a complex behavior. Thus, the miR-combination’s consistent, promising behavior would indicate potential benefits using the two miRs against LC that merits further evaluation.
Citation Format: Archana Shrestha, Behnaz Lahooti, Mahboubeh Madadi, Constantinos M. Mikelis, George Mattheolabakis. Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3759.