Abstract
Non-small cell lung cancer (NSCLC) represents 85% of all lung cancers and can be difficult to treat. Despite significant progress in targeted cancer therapies, some NSCLC patients develop resistance to treatment, resulting in variations in treatment effectiveness, highlighting the need for more comprehensive treatment options.
MicroRNAs (miRNAs) are small non-coding RNA molecules endogenous to the cell which contribute to regulation of cellular pathways, such as growth and proliferation and apoptosis pathways. Some miRNAs are dysregulated in NSCLC and therefore pose targets for new therapies to take advantage of. Current compounds targeting specific miRNAs in cancer cells do so by introducing a mimic of a down-regulated miRNA or inhibiting an up-regulated miRNA, thereby returning miRNA levels to that of a non-cancerous cell.
A novel dual-action silencing construct (DASC) targeting miRNA-34a (down-regulated) and miRNA-21 (up-regulated) in NSCLC has been created. This DASC combines a miR-34a mimic with a miR-21 inhibitor. To confirm the DASC is changing miRNA levels, RT-qPCR on NSCLC cell lines (PC-9 and H358) were performed which confirmed upregulation of miR-34a and downregulation of miR-21. Next western blots were performed to investigate levels of miR-34a (BCL-2) and miR-21 (SOX2, PDCD4) targets, the results of which are inconclusive and require further analysis at this time.
To test the efficacy of this construct on lung cancer cells, transfections of cancerous PC-9 and H358 and non-cancerous BEAS-2B cell lines were performed for growth inhibition assays using SYBR Green stain. All cell lines were treated with miRNA-34a mimic, miR-21 inhibitor, a combination of the mimic and inhibitor, the DASC, negative control and positive control. In both PC-9 and H358 cells, it was found that the DASC showed a similar decrease in cell growth to the combination of the miR-34a mimic and miR-21 inhibitor. A lesser effect on growth was observed in the BEAS-2B cells.
Overall, these results show that the DASC effectively inhibits NSCLC cell growth, and RT-qPCR and western blots provide insight into how these changes in growth may be occurring.