Dataset Information

Construction and evaluation of an efficient C-Jun siRNA to downregulate matrix metalloproteinase in human keratinocytes and fibroblasts under UV exposure.

ABSTRACT:

Background

C-Jun and EGFR have not been explored as targets via the mechanism of RNA silencing. Hence, this study designed an efficient C-Jun-h-825 small interfering RNA (siRNA) and investigated its effect on matrix metalloproteinase (MMP) and collagen expression in human keratinocytes exposed to UV radiation.

Methods

Five C-Jun siRNAs were designed and screened for their ability to downregulate C-Jun expression in human fibroblasts. These constructs were used to study changes in skin cancer-related protein expression. HaCaT cells were grouped into 5-carboxyfluorescien (FAM-labeled) C-Jun-h-825 siRNA + 2 hr prior irradiation; mock transfected + 2 hr prior irradiation; normal control; irradiation only for 2 hr; and Blank. Twenty-four hours posttransfection, mRNA and protein levels of MMP-I, MMP-III, collagen-I and collagen-III were determined using standard RT-PCR and ELISA kits.

Results

FAM-labeled C-Jun siRNA showed 80%-90% transfection efficiency. There was a significant increase in MMP-I and MMP-III and decrease in Col-I and III mRNA levels when the cells were exposed to UV irradiation without siRNA transfection compared to blank (p < .05). This effect was reversed upon transfection with C-Jun-h-825 (p < .01).

Conclusion

Thus, C-Jun-h-825 siRNA might help restore skin collagen by decreasing MMP expression in cells exposed to UVA. Constructs and vectors designed herein have the potential to be translated into a treatment for photoaging induced skin cancer.

SUBMITTER: Xiao H

PROVIDER: S-EPMC6978249 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

Construction and evaluation of an efficient C-Jun siRNA to downregulate matrix metalloproteinase in human keratinocytes and fibroblasts under UV exposure.

Xiao Hong H Yang Ruinian R Yang Fang F Zhao Yanan Y Liu Yin Y

Molecular genetics & genomic medicine 20191114 1

<h4>Background</h4>C-Jun and EGFR have not been explored as targets via the mechanism of RNA silencing. Hence, this study designed an efficient C-Jun-h-825 small interfering RNA (siRNA) and investigated its effect on matrix metalloproteinase (MMP) and collagen expression in human keratinocytes exposed to UV radiation.<h4>Methods</h4>Five C-Jun siRNAs were designed and screened for their ability to downregulate C-Jun expression in human fibroblasts. These constructs were used to study changes in ...[more]

PMID: 31729200

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Project description:BACKGROUND:RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. METHODS:Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. RESULTS:rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. CONCLUSIONS:Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

Dataset Information

Construction and evaluation of an efficient C-Jun siRNA to downregulate matrix metalloproteinase in human keratinocytes and fibroblasts under UV exposure.

Background

Methods

Results

Conclusion

Publications

Construction and evaluation of an efficient C-Jun siRNA to downregulate matrix metalloproteinase in human keratinocytes and fibroblasts under UV exposure.

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