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.

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“…Este factor juega un papel determinante en la regulación transcripcional de las MMPs al desencadenar el aumento de expresión de dichos genes para las enzimas MMPs-1, -3, -9 lo que resulta en la degradación del colágeno y la elastina. 8,20,22,32,39,[43][44][45][46][47][48][49][50] Otro de los efectos que provoca la activación de AP-1 por la RUV es la alteración de la vía de señalización del factor de crecimiento transformante beta (TGF-β), una de las principales citocinas profibróticas, al disminuir la síntesis del receptor II de TGF-β (TβRII). La menor disponibilidad de este receptor conduce a la unión reducida de TGF-β a la superficie de los fibroblastos, lo que provoca la disminución de la sensibilidad de las células al TGF-β y la inhibición de la fosforilación de SMAD 2 y 3.…”

“…Es útil vía intravenosa en pacientes neonatos en cuidados intensivos con riesgo de invasión sistémica (catéter venoso central), 28 además, con menor índice de recaídas en estudios realizados en adultos. 47 Como parte de nuestra experiencia, en caso de falta de respuesta puede agregarse tratamiento queratolítico en gel o loción, ya sea urea, retinoide 25 o ácido salicílico.…”