Methods

Expression of miR-26a-5p/-26b-5p and their target genes were evaluated in 69 BC clinical BC specimens and 23 normal bladder epithelia (NBE) by real-time PCR. We performed gain-of-function studies (cell proliferation, migration and invasion) by using miR-26a/b transfectants in BC cell lines (T24 and BOY). Putative target genes were listed by in-silico study using the gene expression omnibus (GEO) and TargetScan. We performed loss-of-function studies by using si-RNA transfectants to evaluate the functional role of the target gene. Luciferase reporter analyses were employed to validate direct binding between the target gene and miR-26a/b in BC cells. Overall survival (OS) of the BC patients was evaluated by the Kaplan-Meier analysis.

Results

The expression levels of miR-26a-5p/-26b-5p in clinical BCs were significantly downregulated compared to that in NBE (p < 0.0001 and p = 0.0006, respectively). miR-26a-5p/-26b-5p transfectants significantly suppressed cell migration and invasion, suggesting these miRNAs act as tumor-suppressors. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was identified as a direct regulation of miR-26a-5p/-26b-5p by luciferase reporter assay. Kaplan-Meier analysis revealed that the patients with higher PLOD2 expression showed lower overall survival probabilities than those with lower expression (p = 0.0153). Loss-of-function study showed that cell migration and invasion were significantly inhibited in si-PLOD2 transfectans.

Conclusions

PLOD2 was directly regulated by tumor-suppressive miR-26a-5p/-26b-5p, and might be good prognostic markers for survival of BC patients. Recent studies showed that PLOD2 function as a collagen cross-linking enzyme associate with extracellular matrix (ECM) stiffness. Aberrant expression of PLOD2 by regulation of these miRNAs might cause extracellular matrix (ECM) disruption and promoting metastasis.

Funding

none