A Preliminary Study Of The Receptor-Interacting Protein 1 In Ultraviolet B-induced Damage Of Dermal Fibroblasts

Background:Receptor interacting protein (RIP) is an important signal transduction molecule. Recently, three independent international studies have found that RIP is an important crossroad of cell death and survival and RIP plays a key role in apoptosis, cell survival and programmed cell necrosis. RIP1 is one of the most important proteins in RIPs family. Our previous proteomics study firstly discovered that RIP1 was significantly increased by ultraviolet B in the human dermal fibroblasts which was also confirmed by RT-PCR and Western Blot. The function of RIP1 in ultraviolet B-irradiated fibroblasts was not mentioned in previous literature.Objective:(1) To establish RIP silence fibroblasts by RNA interference and find out the duration of RIP silence.(2) To find out the function of RIP in apoptosis, reactive oxygen species and matrix metalloproteinases in ultraviolet B-irradiated fibroblasts.(3) To understand the UVB-induced damage mechanism of fibroblasts and provide experimental evidence and theoretical basis for the treatment and prevention of photodermatosesMethods:(1) The dermis separated from foreskin was cut into pieces to disperse the cells and obtain single-cell suspension to obtain the primary human fibroblasts. PCR and cell Immunofluorescence were used to identify the primary fibroblasts.(2) Cell viability affected by different doses of UVB was measured by the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-diphenytetrazoliumromide (MTT) assay.(3) Quantitative real-time PCR was performed to select reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts.(4) RNA interference was introduced to establish RIP silence NIH3T3 fibroblasts, and the RNA interference was evaluated by RT-qPCR and Western Blot. (5) RT-qPCR and Western Blot were used to determine the mRNA and protein levels of MMP-1 and MMP-3 in ultraviolet B-irradiated RIP1 silence NIH3T3 cell.(6) Through Hoechst staining, the morphological changes of nuclear in cells irradiated by UVB were observed in RIP1 silence NIH3T3 fibroblasts.(7) Flow cytometry was performed to detect the cell apoptosis rate of RIP1 silence NIH3T3 fibroblasts irradiated by UVB.(8) Flow cytometry and colorimetry was used to detect the activity of Caspase-8 and Caspase-3 in RIP1 silence NIH3T3 fibroblasts irradiated by UVB.(9) Fluorescence microscope and flow cytometry was used to detect the ROS in RIP1 silence NIH3T3 fibroblasts irradiated by UVB.Results:(1) Highly pure human dermal fibroblasts were successfully obtained from the foreskin samples.(2) Cell viability of human skin fibroblasts was lowered to different extents at 24h after 20-100mJ/cm2UVB radiation. When cells were treated with 20mJ/cm2 UVB, cell viability decreased at 48h after treatment.(3) ACTB and TUBB1 are suitable reference genes in human skin fibroblasts irradiated by UVB, whereas VIM and TUBA1A are not and should therefore be excluded as reference genes in any gene expression studies involving UVB-irradiated human skin fibroblasts.(4) RIP1 silence in NIH3T3 fibroblasts was conformed and the duration of RNA interference was more than 96h.(5) The mRNA level of MMP-1 and MMP-3 were raised in RIP1 silence NIH3T3 cell after 24h post-irradiated by UVB and the protein of MMP-1 was also raised at the same time.(6) After 24h exposed to UVB, RIP1 silence NIH3T3 cell appeared apoptotic morphology, and cell apoptosis rate was enhanced while the activity of Caspase-8 and Caspse-3 were significantly increased.(7) ROS was inhibited by UVB after 12h in RIP1 silence NIH3T3 cell.Conclusions:(1) The utility of the selected reference gene(s) must be confirmed by each research group for each particular experimental setup. (2) RIP1 can inhibit the expression of MMP in ultraviolet B-irradiated fibroblasts by some signal transduction.(3) RIP1 may inhibit the apoptosis of fibroblasts induced by UVB through some signal transduction.(4) RIPI may be one of the important molecules in signal transduction of ROS generation induced by UVB in fibroblasts.