Up-regulation of the MicroRNA miR-30c Induced by High Mobility Group Box 1 in A549 Cells Used as an Alveolar Epithelial Model.

Abstract

Background: MicroRNAs (miRNAs) have been reported to be involved in multiple diseases, including chronic obstructive pulmonary disease (COPD), a progressive disease in which alveolar apoptosis may play a role. We hypothesized that miRNAs are associated with the response to injury. induced by high mobility group box 1 (HMGB1), a cytokine crucial for the development of COPD, and studied the potential link between HMGB1 and miRNAs.

Materials and methods: A549 cells were stimulated with recombinant HMGB1. RNA and protein were extracted and culture supernatants were collected. Molecules downstream of HMGB1 signaling were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Expression levels of miRNA were analyzed by quantitative RT-PCR. Cellular injury was evaluated by western blotting of relevant proteins. Apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL).

Results: HMGB1 treatment of A549 cells resulted in the up-regulation of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 mRNAs and over expression of matrix metalloprotease (MMP)-7 protein in the supernatant. The miRNA miR-30c was also up-regulated in response to HMGB1 treatment. Cellular injury and apoptosis were observed following HMGB1 treatment, as demonstrated by the oyerexpression of cyclin A2 (CCNA2) and phosphatase and tensin homolog (PTEN) proteins and-b'y decreased levels of pro-caspase-7 protein. The TUNEL assay showed that A549 cells with HMGB1 stimulation underwent apoptosis.

Conclusions: Up-regulation of miR-30c and apoptosis of A549 cells were observed following HMGB1 stimulation. Our model demonstrates the potential for utilization of HMGB1 and miR-30c in further studies of alveolar apoptosis in COPD.