The Role Of MiRNA-22-3p In The Inflammatory Activation Of RPE Cells Under Oxidative Stress

Background and AimsAge-related macular degeneration(AMD)is the leading cause of central vision loss and blindness in western developed countries.During the past decades,with the development of the national economy and improvement of people’s life quality,which exacerbates the population aging,AMD has become the main course of serious vision impairment in China.Based on the differential retinal pathological features,AMD can be divided into dry-AMD which characterizes by fundus atrophy and wet-AMD which characterizes by choroidal neovascularization.Of note,fundus atrophy in late stage of dry-AMD arising from death of retinal pigment epithelia(RPE)and photoreceptors,and the exudation and hemorrhage arising from choroidal neovascularization both cause serous vision impairment.Herein,it is the hot topic of great significance to deepen the investigation of mechanism behind the AMD pathology and to explore novel targets for future AMD intervention.However,the exact etiology of AMD still remains unclear.Typically,a series of genetic and environmental factors collectively activate the occurrence of this retinopathy,aging and oxidative stress exacerbate the progression,and chronic inflammation leads to an inevitable pathological change.Actually,those factors play their roles in a cross-talk instead of independent manner.However,the exact etiology of AMD still remains unclear.Growing evidence has also suggested inflammation and immune response may play important and interacting roles in AMD.In this study,a new oxidative stress model was constructed to explore a new mechanism for the inflammatory activation in Retinal Pigment Epithelial cells(RPE).The changes of microRNA in RPE cells of oxidative stress were detected by unbiased sequencing,and the role of miRNA22-3p in the activation of NLRP3 inflammasome was further verified.It is known that NLRP3 plays a vital role in the inflammatory response and is more prone to inflammation in oxidative stress.And this is also an important process for the pathogenesis of AMD.It is meaningful to clarify the activation mechanism of NLRP3 signaling pathway in the RPE cells during oxidative stress for the prevention and early diagnosis of AMD.And it also has important value for developing targeted new drugs.Methods1.Construction of oxidative stress model for RPE cell and evaluation of its effectiveness.The ARPE-19 cell line was divided into 11 groups,one group as negative control,two groups treated only with LPS(10μg/mL,50μg /mL)for 24 hours,four groups treated only with rotenone(0.5μM,1μM,2.5μM,5μM)for 24 hours,and the other four groups were pretreated with LPS for 4 hours,and then treated with different concentrations of rotenone(Rot)for 24 hours.Cells were cultured in normal cell culture conditions.The proliferation activity of each cell was detected by CCK-8 method.Subsequently,the condition that did not affect cell proliferation was chosen for model establishment.The fluorescence signal intensity of ROS probe was detected by flow cytometry.2.Detection of inflammation in RPE cells under oxidative stress The RPE cell model of oxidative stress was established in the first step.The mRNA and protein levels of NLRP3 and its associated protein caspase-1,IL-1βwere detected both in the experimental group and the control group by real-time quantitative PCR(qRT-PCR)and Western Blot.3.The mechanism of inflammasome activation in the RPE under oxidative stress We take the miRNA sequencing in RiboBio to screen the candidates which might regulate the activation of NLRP3 in the RPE cells under oxidative stress.And the results were verified by qRT-PCR.Furthermore,the effect of microRNA on the activation of NLRP3 inflammasome in RPE cells under oxidative stress was verified by rescue experiments.Results1.The oxidative stress damage model of RPE cells treated with lipopolysaccharide (LPS)and rotenone(Rot)was successfully constructed.The intracellular ROS level of RPE cell under oxidative stress was significantly up-regulated without affecting the normal proliferation of cells(P<0.05).2.Using the last oxidative stress model treated with lipopolysaccharide and rotenone,we observed that the mRNA and protein expression level of NLRP3 and its related protein caspase-1 and IL-1β in RPE cells of the experimental group were significantly higher than the control group(P<0.05).3.Screening by unbiased miRNA sequencing,we found that miRNA22-3p in RPE cells under oxidative stress was significantly lower than that in the control group (P<0.05).The rescue experiment further confirmed that activation of NLRP3 was dependent on miRNA22-3p,and the difference was significant(P<0.05).Conclusions1.Lipopolysaccharide(LPS)and rotenone(rot)can induce oxidative stress damage in RPE cells and activate NLRP3 inflammatory signaling pathway.2.MiRNA22-3p is a negative regulator of NLRP3,which could upregulate NRLP3 transcription level in RPE cells,thereby activating downstream inflammatory response.This reveals a new mechanism of inflammatory activation in the RPE cells under oxidative stress,and it provides a new idea for the diagnosis and treatment of AMD disease.