Korean J Intern Med.  2019 May;34(3):634-642. 10.3904/kjim.2016.315.

MicroRNA-16 inhibits cell proliferation and migration by targeting heat shock protein 70 in heat-denatured dermal fibroblasts

• Zhang C
• Dai J
• Fan Y
• He X
• Wei R

• KMID: 2448356
• DOI: http://doi.org/10.3904/kjim.2016.315

Abstract

BACKGROUND/AIMS
This study aimed to investigate the precise mechanism and function of miR-16 in heat-denatured primary human dermal fibroblasts.
METHODS
Primary human dermal fibroblasts were separated from normal human skin samples. Under heat stress, the levels of miR-16 and heat shock protein 70 (HSP70) were detected in primary human dermal fibroblasts by quantitative real-time polymerase chain reaction (qRT-PCR). Next, heat-denatured cells were transfected with synthetic scrambled negative control (NC) RNA (NC group), miR-16 mimics, miR-16 inhibitor or miR-16 inhibitor accompanied by small interfering RNA targeting HSP70, then the mRNA level of HSP70 was detected by qRT-PCR, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and AlamarBlue assay, cell migration was examined by Transwell assay and cell apoptosis was assessed by transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) assay. In addition, cell apoptosis-related proteins, Bax and Bcl-2, were detected by Western blotting.
RESULTS
Heat stress significantly reduced miR-16 level and increased the mRNA level of HSP70 compared with untreated cells (p < 0.05). Overexpressed miR-16 reduced the mRNA level of HSP70, suppressed cell proliferation (p < 0.05 or p < 0.01), migration (p < 0.05), and promoted cell apoptosis (p < 0.001) compared with the NC group. Down-regulated miR-16 exerted an opposite effect on primary human dermal fibroblasts with heat-denaturation. Furthermore, effects of miR16 down-regulation on cell proliferation and migration were reversed by HSP70 silence.
CONCLUSIONS
MiR-16 might have an inhibitory effect on cell proliferation and migration in heat-denatured human dermal fibroblasts, and HSP70 might be associated with the cell proliferation and migration as a target gene of miR-16.

Keyword

MIRN16 microRNA; Wound healing; HSP70; Cell proliferation; Cell movement

MeSH Terms

Apoptosis
Blotting, Western
Cell Migration Assays
Cell Movement
Cell Proliferation*
Down-Regulation
Fibroblasts*
Heat-Shock Proteins*
Hot Temperature*
HSP70 Heat-Shock Proteins*
Humans
Real-Time Polymerase Chain Reaction
RNA
RNA, Messenger
RNA, Small Interfering
Skin
Transferases
Wound Healing
HSP70 Heat-Shock Proteins
Heat-Shock Proteins
RNA
RNA, Messenger
RNA, Small Interfering
Transferases