Role of microRNA-4516 involved autophagy associated with exposure to fine particulate matter

Abstract

Metals are vital toxic components of fine particulate matter (PM2.5). Cellular responses to exposure to PM2.5 or PM metal components remain unknown. Post-transcriptional profiling and subsequent cell- and individual-based assays implied that the metal ion-binding miR-4516/RPL37/autophagy pathway could play a critical role in cellular responses to PM2.5 and PM metal stresses. miR-4516 was up-regulated in A549 cells exposed to PM2.5 and in the serum of individuals living in a city with moderate air pollution. The expression levels of the miR-4516 target genes, namely, RPL37 and UBA52, were involved in ribosome function and inhibited by exposure to PM2.5 and PM metal components. Autophagy in A549 cells was induced by PM2.5 exposure as a response to decreased RPL37 expression. Moreover, enhanced miR-4516 expression was positively correlated with the augmentation of the internal burden of aluminum and lead in individuals living in a city with moderate air pollution. Hereby, the miR-4516/RPL37/autophagy pathway may represent a novel mechanism that mediates responses to PM metal components.

Keywords: PM2.5; autophagy; metal; microRNA; ribosome.

Figure 1. Modulation of miR-4516 expression in A549 cells and individual serum

(A) The heatmap showed the induced and suppressed miRNAs of samples exposed to 500 μg/ml PM_2.5 relative to matched controls in terms of the fold-change values as 2.0, P = 0.05. (B) Enhanced expression of miR-4516 in A549 cells treated with PM_2.5 in a dose-dependent manner. *P < 0.05, compared with control. (C) miR-4516 levels were significantly increased in the serum of residents living in a moderately polluted city. *P < 0.05, compared with city with clean air.

Figure 2. Functional annotations of miR-4516 targets

A total of 25 targets of miR-4516 were determined by target prediction coupled with proteomics data. (A) Enriched GO terms of the 25 targets are shown by bar plot. Enrichment score of each term represents the term involved gene numbers. (B) UBA52 and RPL37 were enriched in the KEGG term “ribosome”. (C) The interaction between RPL37 and UBA52 was revealed by PPI analysis and confirmed by CO-IP assays. (D) The potential biding site of miR-5416 to RPL37 3′-UTR was predicted by the Targetscan online tool. The corresponding mutated sites of RPL3 3′-UTR were labeled in green. (E) The luciferase reporter assay showed that miR-4516 mimics the significantly decreased luciferase activity in 3′-UTR of RPL37. *P < 0.05, compared with the other three groups. (F) The expression of RPL37 in A549 cells was significantly decreased by treatment with 100 μg/ml PM_2.5 for 24 h and increased after miR-4516 inhibitor transfection for 48 h. Co-treatment of PM_2.5 and the miR-4516 inhibitor rescued RPL37 expression to control levels. (G) Treatment with 100 μg/ml PM_2.5 significantly decreased UBA52 expression in A549 cells. *P < 0.05, compared with untreated control, ^#P < 0.05, compared with control within each group.

Figure 3. Expression of miR-4516 target genes as inhibited by PM_2.5 and PM metals

Proportion of different metal components in PM_2.5 reference material. (B) Proportion of different metal components in PM_2.5sjz. mRNA expression levels of RPL37 and UBA52 were modulated by different doses of (C) Al, (D) Pb, and (E) Cu metal particles. mRNA expression levels of RPL37 and UBA52 were slightly modulated by (F) Fe or (G) Mn metal particles. (H) mRNA expression levels of RPL37 and UBA52 were inhibited by the Al, Pb, and Cu metal mix and PM_2.5. (I) Immunoblot analysis demonstrated the down-regulation of RPL37 and UBA52 protein expression induced by PM_2.5, as well as the Al and Pb metal particles. *P < 0.05, compared with control, ^#P < 0.05, compared with 500 μg/mL PM_2.5 treatment group.

Figure 4. PM_2.5 treatment induced autophagy in A549 cells as regulated by miR-4516 and its target gene

(A) A549 cells were treated with control medium for 24 h, no autophagosomes were observed in the cytoplasm. A549 cells were treated with (B) 100 μg/mL or (C) 500 μg/mL PM_2.5 for 24 h. Typical autophagosomes with double membranes and cellular contents were observed in the cytoplasm, as shown by the black arrow. N, nucleus, CY, cytoplasm. Insets are magnified below each image highlighted with black box). Black arrows show typical autophagosome and black arrows show intracellular PM. (D) Increased number of autophagic vacuoles were observed in A549 cells. Autophagic vacuoles were showed by white arrows. (E) Expression levels of LC3B-I and LC3B-II were increased in A549 cells after treatment with 100 μg/ml PM_2.5 for 24 h. Co-treatment of PM_2.5 and the miR-4516 inhibitor could reverse the increased expression of LC3B-I and LC3B-II. (F) Expression levels of RPL37 and UBA52 were inhibited after RPL37 siRNA transfection in A549 cells. Meanwhile, increased expression of LC-3B-I and -II were also observed. (G) S stage arrest was induced by PM_2.5 exposure for 24 h. (H) The proportion of apoptosis in A549 cells was significantly increased after PM_2.5 treatment for 24 h.

Figure 5. Scatter plot of miR-4516 and metal levels in serum

(A) Expression of miR-4516 was negatively associated with the Al burden in serum of individuals from a moderately polluted city. (B) Expression of miR-4516 was negatively relative to the Pb burden in serum of individuals from a moderately polluted city. (C) Expression of miR-4516 was slightly affected by Cu levels in serum samples.

Figure 6. Alterations of miR-4516 expression and the metal burden between baseline and post-exposure levels

(A) Relative expression level of miR-4516 and (B) Al burden were significantly increased after 3 months in individual sera. *P < 0.05, compared with baseline level. (C) Pb and (D) Cu burdens were not significantly modulated after 3 months. Increment of miR-4516 expression was positively associated with the increment of (E) the Al burden and (F) Pb burden. (G) No association between the increment of miR-4516 and that of the Cu burden was found.