MiR-145-5p Targets KLFs To Regulate The Expression Of Inflammatory Factors In Human Corneal Fibroblasts

Keratoconus is a kind of corneal dilation disease,which is one of the common blinding diseases in clinic.Cornea is a typical load-bearing tissue.Under the action of intraocular pressure,the cornea mainly bears tensile and shear loads.Keratoconus local tissue thinning,structural damage,material properties decreased,resulting in the redistribution of corneal internal stress.A variety of inflammatory factors are over expressed in corneal tissues,cells and tears of keratoconus patients.As an important gene expression regulator,miRNAs are closely related to the physiological and pathological process of cornea.MiRNAs also interact with transcription factors KLFs to regulate the expression of inflammatory mediators.KLFs family is one of the important transcription factors that regulate the expression of inflammatory mediators.It is unclear whether miRNAs and KLFs are involved in the regulation of inflammatory factors expression in cornea under mechanical stimulation.The aim of this study was to investigate the effects of mechanical stimulation on the expression of inflammatory factors,KLFs and matrix remodeling related genes in cultured human corneal fibroblasts;The effects of KLF4 and KLF5 on the expression of inflammatory factors and matrix remodeling related genes in corneal fibroblasts were studied by siRNA;At the same time,miRNAs that can target KLF4 and KLF5 at the same time and have negative correlation with their expression were screened to explore whether miRNAs regulate the expression of inflammatory factors in human corneal fibroblasts through KLFs,and then participate in the regulation of corneal stromal remodeling related proteins.The main work of this paper is as follows:(1)Normal corneal fibroblasts cultured in vitro were subjected to periodic mechanical stretching for 0 h,3 h,6 h and 12 h.The gene expressions of transcription factors KLFs,interleukin(ILS),matrix metalloproteinases(MMPs),LOX and Collagen in cells were detected by real-time quantitative PCR.The results showed that the expression of transcription factors(KLF4,KLF5),inflammatory factors(IL-1β,IL-6,IL-8,ICAM-1),matrix metalloproteinases(MMP-1,MMP-2,MMP-9,MMP-13,and MMP-14)in human corneal fibroblasts were significantly increased under the stimulation of cyclic mechanical stretch.The expression of lysyl oxidase-like proteins(LOX-1,LOX-2,LOX-4)and Collagen(COL1A2 and COL3A1)were significantly down-regulated.It is suggested that mechanical stretching can induce the expression of transcription factors KLFs,inflammatory factors and matrix remodeling related genes in human corneal fibroblasts.(2)SiRNA interference fragments of KLF4 and KLF5 genes were constructed and transfected into human corneal fibroblasts,respectively.The effects of KLF4 and KLF5 on the expression of inflammatory factors and matrix remodeling related genes in human corneal fibroblasts were detected by real-time fluorescence quantitative PCR.The results showed that the expression of IL-6,IL-8,MMP-1,MMP-2,MMP-9,MMP-13 and MMP-14 in human corneal fibroblasts were decreased after the expression of KLF4 and KLF5 was interfered with.The COL1A2,COL3A1,LOX-1,LOX-2 and LOX-4 expressions were significantly increased.These results suggest that KLF4 and KLF5 may regulate the expression of inflammatory factors(except IL-1β)in human corneal fibroblasts,and then participate in the regulation of matrix remodeling related proteins.(3)The miRNAs(miR-145-5p,miR-5195-3p)that simultaneously target KLF4 and KLF5 were predicted by miRBase and Target Scan websites.The expressions of KLF4,KLF5 and the above miRNAs in human corneal fibroblasts cultured in vitro were detected by quantitative real-time PCR.The results showed that the expression trend of miR-145-5p was opposite to that of KLF4 and KLF5,and the expression trend was most obvious at 3 h.Therefore,miR-145-5p was selected for subsequent experimental studies.(4)The simulants and inhibitors of miR-145-5p were transfected into human corneal fibroblasts in vitro,and the expressions of KLFs,inflammatory factors and stromal remodeling related genes in the transfected corneal fibroblasts were detected.The results showed that miR-145-5p mimics could significantly down-regulate the expression of transcription factors KLF4 and KLF5,and decrease the expression of inflammatory factors IL-6,IL-8 and matrix metalloproteinases MMP-1,MMP-2,MMP-9,MMP-13 and MMP-14.COL1A2 COL3A1,LOX-1 and LOX-2 were increased.It is suggested that miR-145-5p decreased under mechanical stretching stimulation,causing increased KLF4 and KLF5 expression,and promoting the expression of inflammatory factors(except IL-1β)and matrix remodeling related proteins(except LOX-4).This may provide a potential target for the pathogenesis and treatment of keratoconus.