微小 RNA-16 通过靶向热变性真皮成纤维细胞中的热休克蛋白 70 抑制细胞增殖和迁移。

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

机构信息

Department of Burn Ningbo No.2 Hospital, Ningbo, China.

Department of Clinical Laboratory, Ningbo No.2 Hospital, Ningbo, China.

出版信息

Korean J Intern Med. 2019 May;34(3):634-642. doi: 10.3904/kjim.2016.315. Epub 2018 Jan 5.

DOI:10.3904/kjim.2016.315

PMID:29294597

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6506753/

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.

CONCLUSION

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.

摘要

背景/目的：本研究旨在探讨 miR-16 在热变性原代人真皮成纤维细胞中的精确作用机制和功能。

方法

从正常人皮肤样本中分离出原代人真皮成纤维细胞。在热应激下，通过实时定量聚合酶链反应（qRT-PCR）检测原代人真皮成纤维细胞中 miR-16 和热休克蛋白 70（HSP70）的水平。然后，用合成的随机阴性对照（NC）RNA（NC 组）、miR-16 模拟物、miR-16 抑制剂或 miR-16 抑制剂加针对 HSP70 的小干扰 RNA 转染热变性细胞，然后通过 qRT-PCR 检测 HSP70 的 mRNA 水平，通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）和 AlamarBlue 测定法评估细胞增殖，通过 Transwell 测定法检查细胞迁移，通过转移酶 dUTP（脱氧尿苷三磷酸）缺口末端标记（TUNEL）测定法评估细胞凋亡。此外，通过 Western blot 检测细胞凋亡相关蛋白 Bax 和 Bcl-2。

结果

与未处理的细胞相比，热应激显著降低了 miR-16 的水平并增加了 HSP70 的 mRNA 水平（p<0.05）。与 NC 组相比，过表达 miR-16 降低了 HSP70 的 mRNA 水平，抑制了细胞增殖（p<0.05 或 p<0.01）、迁移（p<0.05），并促进了细胞凋亡（p<0.001）。下调 miR-16 对热变性的原代人真皮成纤维细胞产生了相反的影响。此外，HSP70 沉默逆转了 miR16 下调对细胞增殖和迁移的影响。