Abstract
GADD45A (growth arrest and DNA damage inducible alpha), a stress response gene induced by genotoxic and nongenotoxic stresses, is implicated in various key processes, including the control of cell cycle checkpoints and DNA repair. The expression of GADD45A is directly regulated by numerous transcription factors, with p53 being the most representative. Moreover, post-transcriptional regulation also plays a role in GADD45A expression. However, little is known about the regulatory effects of microRNAs (miRNAs) on GADD45A expression. As a potential tumour suppressor, miR-138 has pleiotropic biological functions in various cancers. We have previously reported p53-mediated activation of miR-138 in human non-small-cell lung cancer (NSCLC) cells. In this study, we found that miR-138 specifically targeted AGO2, which affects the stability and maturation of miR-130b. Decreased expression of miR-130b promoted the expression of GADD45A and resulted in the G2/M phase arrest and proliferation inhibition in human NSCLC cells. Our results suggested that p53 could alternatively upregulate GADD45A in human NSCLC cells through a post-transcriptional pathway in which miR-138 is involved.
Highlights
As a transcription regulator, p53 plays a prominent role in cellular responses to stress signals, such as DNA damage, oncogene activation, and hypoxia
The columns represent the mean normalized relative luciferase activity (RLU) from three independent experiments, with 95% confidence intervals. *P < 0.05 vs. NC by rank-sum test. (e) AGO2 mRNA and miR-138 were quantified using real-time PCR, and AGO2 protein expression was detected by western blotting in H460 and H1299 cells. *P < 0.05 and **P < 0.01 vs. NC by ranksum test
Full-length blots are in Supplementary information. (f) AGO2 mRNA was quantified using real-time PCR, and the expression of AGO2 protein was analyzed by western blotting in H1299 cells transfected with miR-138, anti-miR-138, AGO2 siRNA, and negative control. *P < 0.05 vs. NC by LSD test
Summary
It has recently been reported that Ago[2], known as eIF2C2, is important both for maintaining miRNA abundance through its involvement in miRNA processing[32,33,34] and enhancement of miRNA stability[28, 35], and for enhancing miRNA function via assembly of RISC (RNA-induced silencing complex)[36, 37]. We demonstrated that AGO2 is capable of slicing pre-miR-130b into an Ac-pre-miRNA precursor, followed by Dicer-mediated cleavage of intermediates to produce mature miRNAs. miR-130b is a 3′-end miRNA, and we demonstrated that pre-miR130b is sliced at the passenger strand from the 5′ end. The results suggested the pivotal impacts of AGO2 on the abundance of miR-130b and unveiled variant mechanisms of AGO2-mediated processing. Our work re-confirmed the p53-mediated regulation of GADD45A by demonstrating a miRNA-directed post-transcriptional pathway. This study provides direct evidence that there is an alternative microRNA-mediated pathway in p53 regulation of GADD45A in human NSCLC cells, in addition to canonical transcriptional regulation. Our results demonstrated the complexity of the miRNA-protein mutual interaction network in the regulation of gene expression in human cells
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