Yonsei Med J.  2015 Nov;56(6):1619-1626. 10.3349/ymj.2015.56.6.1619.

Hsp70 Knockdown by siRNA Decreased Collagen Production in Keloid Fibroblasts

• Shin JU
• Lee WJ
• Tran TN
• Jung I
• Lee JH

• KMID: 2345891
• DOI: http://doi.org/10.3349/ymj.2015.56.6.1619

Abstract

PURPOSE
There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts.
MATERIALS AND METHODS
We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay.
RESULTS
The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts.
CONCLUSION
Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.

Keyword

Heat shock proteins; keloids; Hsp70

MeSH Terms

Adolescent
Adult
Blotting, Western
Collagen/*drug effects/metabolism
Female
Fibroblasts/metabolism
Gene Expression Regulation
HSP70 Heat-Shock Proteins/genetics/metabolism/*pharmacology
Humans
Keloid/*drug therapy/genetics/metabolism
Male
RNA, Messenger/*genetics
RNA, Small Interfering/*genetics
Real-Time Polymerase Chain Reaction
Transfection
Up-Regulation
Collagen
HSP70 Heat-Shock Proteins
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 Immunohistochemical staining of keloids and adjacent normal dermal tissues. Abnormal dense collagen fibers were extending over clinical keloid margin (dotted line). The expression of Hsp70 and TGF-β1 were also increased along with extending collagen fibers (×100, scale bar: 1 mm). Each experiment was performed in triplicate; representative data are shown here. Hsp70, heat shock protein 70; TGF-β1, transforming growth factor-beta1.

• 

  Fig. 2 The expression of Hsp70 was down-regulated by Hsp70 siRNA transfection. (A) MTT viability of keloid fibroblasts after siRNA transfection. Results are shown as mean±SD. Experiment was performed in triplicate. (B) Hsp70 mRNA levels determined by RT-PCR; results are shown as mean±SD. Experiment was performed in two sets of triplicate. (C) Detection of the Hsp70 protein in keloid fibroblasts by western blot analysis 24 h and 48 h after transfection (*p<0.05). Each experiment was performed in triplicate; representative data are shown here. Hsp70, heat shock protein 70; siRNA, small interfering RNA; MTT, tetrazolium-based colorimetric assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, real-time polymerase chain reaction.

• 

  Fig. 3 Collagen mRNA and protein levels in keloid fibroblasts decreased significantly, corresponding to the down-regulation of Hsp70. (A) Expression of collagen-I and III mRNAs after Hsp70 siRNA transfection. Each experiment was performed in two sets of triplicate. (B) Histogram of the extracellular secreted-collagen content of keloid fibroblast cultures by Sircol collagen assay 24 h and 48 h after transfection; results are shown as mean±SD (*p<0.05). Each experiment was performed in triplicate. Hsp70, heat shock protein 70; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 4 Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) 24 h and 48 h after transfection. (A) Expression of MMP and TIMP mRNAs after Hsp70 siRNA transfection: MMP-14, TIMP-1, and TIMP-2 mRNAs were significantly suppressed; however, MMP-2 was not; results are shown as mean±SD. Each experiment was performed in two sets of triplicate. (B) Western-blot analysis of MMP-2, MMP-14, TIMP-1, and TIMP-2 protein levels (*p<0.05). Each experiment was performed in triplicate. Hsp70, heat shock protein 70; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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