miRNAs Potentially Involved in Post Lung Transplant-Obliterative Bronchiolitis: The Role of miR-21-5p

Abstract

Epigenetic changes, including miRNAs deregulation, have been suggested to play a significant role in development of obliterative bronchiolitis (OB) in transplanted lungs. Many studies have tried to identify ideal candidate miRNAs and the downstream pathways implicated in the bronchiolar fibro-obliterative process. Several candidate miRNAs, previously indicated as possibly being associated with OB, were analyzed by combining the quantitative real time-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) of lung tissues of OB affected patients. Disease and OB-lesion-specific expression of miR-21-5p was confirmed and by computational analysis we were able to identify the network of genes most probably associated miR-21-5p in the context of OB fibrogenesis. Among all potentially associated genes, STAT3 had a very high probability score. Immunohistochemistry showed that STAT3/miR-21-5p were co-over expressed in OB lesions, thus, suggesting miR-21-5p could regulate STAT3 expression. However, miR-21-5p inhibition in cultures of bronchiolitis obliterans syndrome (BOS) derived myofibroblasts did not significantly affect STAT3 mRNA and protein expression levels. This study demonstrates the specificity of miR-21-5p over-expression in OB lesions and contributes to existing knowledge on the miR-21-5p downstream pathway. Activation of STAT3 is associated with miR-21-5p upregulation, however, STAT-3 network activation is most likely complex and miR-21-5p is not the sole regulator of STAT3.

Keywords: bronchiolitis obliterans; chronic lung allograft dysfunction; in situ hybridization; miR-21-5p; microRNA.

Figure 1

Tissue localization of miRNAs in normal lung (miR-21-5p (a); miR-146b-5p (c); miR-34a-5p (e); miR-15a-5p (g); miR-145-5p (i); let-7d-5p (k)) and BOS samples (miR-21-5p (b); miR-146b-5p (d); miR-34a-5p (f); miR-15a-5p (h); miR-145-5p (j); let-7d-5p (l)) by ISH.

Figure 2

Quantitative analysis of miRNAs expression. qRT-PCR miRNAs expression levels in BOS samples, mir-21 (a), mir-146 (b), mir34(c), mir15 (d), mir145 (e), let7 (f), represented as fold changes relative to RNU6.

Figure 3

miR-21-5p regulatory network. Interactions between miR-21-5p and genes that appeared to be associated with BOS in at least 3 different databases (Mirtarbase, Targetscan, Miranda, Pictar4, and Oictar5). miR-21-5p-related genes (green boxes) encoding for proteins that interact with BOS-related genes listed in DisGeNet database (light blue boxes).

Figure 4

Tissue localization of miR-21-5p and STAT3 in OB lesions. Photomicrographs representative of the co-localization of miR-21-5p (a) and STAT3 (b) in fibroblast of OB lesions and in the adjacent tissue. High magnification pictures show details of STAT3 expression in (c) bronchial epithelium (arrow) and OB fibroblast (arrowhead), and in (d) alveolar pneumocytes (arrow), endothelia (arrowhead), and macrophages (asterisk).

Figure 5

qRT-PCR analysis of miR-21-5p/STAT3 mRNA expression levels: (a) in A549, mean ± standard deviations of replicates; (b) in Lung fibroblasts (LFs) derived from BOS patients, mean ± standard deviations of all cell lines; (c) in LFs derived from patients without chronic lung allograft dysfunction (CLAD), mean ± standard deviations of replicates. *** p < 0.001.

Figure 6

Western blot of STAT3 protein expression levels in LFs treated with anti-miR-21. Quantification of immunoblots using anti-STAT3 of LFs from BAL of CLAD patients treated with anti-miR-21 after 48h. Control cells (CTR); negative control treated cells (NC).