Abstract
Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.
Highlights
MiRNAs are small non-coding RNAs consisting of 19–25 nucleotides[1]
Lung cancer is a devastating disease, with more than 1.6 million of lung cancer-related deaths recorded per year world-wide[9], and approximately 85% of the cases attributed to non-small cell lung cancer (NSCLC)[10]
We report that the combinatorial treatment of A549 cells with the two miRs induces strong downregulation of CDK1, 4 and 6, and causes strong cell cycle arrest, accompanied with apoptotic and cytotoxic activity, and caspase 3/7 activation
Summary
MiRNAs (miRs) are small non-coding RNAs consisting of 19–25 nucleotides[1]. These unique molecules regulate at least 30% of all human gene expressions, either by translational repression or target messenger RNA destabilization. For gene regulation to take place, miRs require base-pair complementarity between the targeted messenger RNA (mRNA) and the seed region of the miR, with their activity relying on the cell’s natural RNA interference mechanism[2,3]. Org)[19], we determined that miR-143 and miR-506 have base pair complementarity with the CDK1 and CDK4/6 mRNAs, respectively (Fig. 1), demonstrating a potential to combinatorially regulate the cell cycle on different stages. We determined that the combinatorial treatment significantly inhibited tube formation in an in vitro angiogenesis model, endowing the proposed treatment with multifaceted activity against the tumor cells and the tumor microenvironment
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