Dataset Information

Let-7c miRNA Inhibits the Proliferation and Migration of Heat-Denatured Dermal Fibroblasts Through Down-Regulating HSP70.

ABSTRACT: Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured fibroblasts and that inhibiting let-7c expression leads to the increased proliferation and migration of dermal fibroblasts, whereas the overexpression of let-7c exerts an opposite effect. Further investigation has identified heat shock protein 70 as a direct target of let-7c and has demonstrated that the expression of HSP70 in fibroblasts is negatively correlated with let-7c levels. Moreover, down-regulation of let-7c expression is accompanied by up-regulation of Bcl-2 expression and down-regulation of Bax expression, both of which are the downstream genes of HSP70. Notably, the knockdown of HSP70 by HSP70 siRNA apparently abrogates the stimulatory effect of let-7c inhibitor on heat-denatured fibroblasts proliferation and migration. Overall, we have identified let-7c as a key regulator that inhibits fibroblasts proliferation and migration during wound healing.

SUBMITTER: Jiang T

PROVIDER: S-EPMC4844942 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Let-7c miRNA Inhibits the Proliferation and Migration of Heat-Denatured Dermal Fibroblasts Through Down-Regulating HSP70.

Jiang Tao T Wang Xingang X Wu Weiwei W Zhang Fan F Wu Shifeng S

Molecules and cells 20160229 4

Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured fibroblasts and that inhibiting let-7c expression leads to the increased proliferation and migration of dermal fibroblasts, whereas the overexpression of let-7c exerts an opposite effect. Further inv ...[more]

PMID: 26923191

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Project description:Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti‑inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis‑adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to measure miR‑16‑1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col‑I), collagen III (Col‑III) and α‑smooth muscle actin (α‑SMA) proteins. Agomir‑16‑1 and antagomir‑16‑1 were used to up and downregulate the expression of miR‑16‑1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5‑bromo‑2‑deoxyrudidine assays. Cell apoptosis was determined by caspase‑3 activity and TUNEL assays. The results demonstrated that the levels of Col‑I, Col‑III and α‑SMA were all significantly upregulated in SAT compared with NT. miR‑16‑1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR‑16‑1/HSP70 pathway. The results demonstrated that the elevated expression of miR‑16‑1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR‑16‑1 reversed the effect of TPL on the miR‑16‑1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR‑16‑1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)‑associated protein expression of fibroblasts from SAT. Finally, downregulation of miR‑16‑1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir‑16‑1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR‑16‑1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.

Project description:BackgroundThe urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein.MethodsWe performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay.ResultsHSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P?<?0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR.ConclusionsThese findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.

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Let-7c miRNA Inhibits the Proliferation and Migration of Heat-Denatured Dermal Fibroblasts Through Down-Regulating HSP70.

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Let-7c miRNA Inhibits the Proliferation and Migration of Heat-Denatured Dermal Fibroblasts Through Down-Regulating HSP70.

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