The Regulatory Mechanism Of PiR-hsa-229734 On Corneal Neovascularization After Alkali Burn

Objective Corneal alkali burn is a relatively serious chemical injury,and the saponification of alkali can easily induce the formation of a large number of corneal neovascularization(CNV),which can seriously lead to the formation of corneal blindness.It has been reported that PIWI interacting RNA(piRNAs)may be involved in the regulation of corneal injury and act as important regulators in the angiogenesis process of some disease,while the relationship between piRNAs and corneal neovascularization is rarely explored.The purpose of this study is to explore the regulatory effect and related mechanism of piR-hsa-229734 on corneal neovascularization induced by alkali burn in vivo and in vitro.Methods High-throughput sequencing technology was used to obtain differentially expressed piRNAs in corneal alkali burn and normal corneal tissue;Human umbilical vein endothelial cells(HUVECs)which were treated by basic fibroblast growth factor(b FGF)to simulate alkali burn environment,the differentially expressed piR-hsa-229734 was screened by quantitative real-time fluorescence PCR(qRT-PCR),and its piRNA structure was verified by RTL-P experiments.piR-hsa-229734 agomir and antagomir were designed to verifyit the regulation in the proliferation,migration and tube formation of HUVECs.The protein binding of piR-hsa-229734 was obtained by RNA pull down assay,and the binding relationship between piR-hsa-229734 and target protein was verified by liquid chromatography tandem mass spectrometry.Western blot(WB)was used to detect the regulatory effect of piR-hsa-229734 on downstream target proteins at the protein level.Finally,HUVECs pre-transfected with piR-hsa-229734 agomir were mixed with Matrigel and implanted in nude mice to observe whether piR-hsa-229734 could regulate angiogenesis in vivo.Results(1)The results of qRT-PCR aanlysis showed that the expression of piR-hsa-229734 in corneal tissues with obvious angiogenesis was significantly lower than that in normal corneal tissues.After treated with 20ng/ml b FGF,the expression of piR-hsa-229734 in HUVECs was significantly down-regulated(P<0.05).(2)The cell function test showed that agomir could significantly promote the expression of piR-has-229734,thereby inhibiting the proliferation,migration and tube formation of HUVECs.However,the result of knockdown of piR-hsa-229734 by antagomir was the opposite(P<0.05).(3)The results of Western blot showed that overexpression of piR-hsa-229734 could down-regulate the expression of vascular endothelial growth factor A(VEGFA)in human corneal epithelial cells,while,the effect of knockdown of piR-hsa-229734 showed the opposite(P<0.05).(4)RNA pull down experiments confirmed that piR-hsa-229734 can bind to vimentin in HUVECs,and overexpression of piR-hsa-229734 can down-regulate the expression of vimentin at the protein level.The level promoted the expression of vimentin at protein level(P<0.05).(5)The experiment in vivo showed that the color of Matrigel plug pre-transfected with piR-hsa-229734 agomir was significantly lighter than the control group,and the results of paraffin sections of emboli using HE staining showed that the number of new capillaries was also less than the control group,indicating that piR-hsa-229734 could inhibit the formation of neovascularization in animals to a certain extent.It was confirmed that piR-hsa-229734 has the potential to inhibit corneal neovascularization.Conclusion The expression of piR-hsa-229734 in alkali-burned cornea with neovascularization and b FGF-induced HUVECs was significantly down-regulated,while overexpression of piR-hsa-229734 could inhibit the tube-forming ability of HUVECs in vivo and in vitro.At the same time,Western blot experiments found that it can inhibit the expression of VEGFA,a gene closely related to neovascularization in human corneal epithelial cells,and can also bind and inhibit the protein VIM involved in angiogenesis in HUVECs.It is proved that piR-hsa-229734 can inhibit the development of neovascularization after corneal alkali burn,and it may inhibit angiogenesis through negative regulation of VEGFA and VIM.