The Study On The Mechanism Of LncRNA MALAT 1 As A CeRNA Of MiR-625-3p Targeting HIF-1α In Hypoxia-induced Retinopathy

Objective:Retinal disease is the leading cause of irreversible blindness,the retina is a tissue structure in the human body that relies on more oxygen,and lack of oxygen is intimately associated to the common pathological processes of many retinal diseases,such as age-related macular degeneration（AMD）,and proliferative vitreoretinopathy（PVR）.In addition,the epithelial-mesenchymal transition（EMT）of retinal pigment epithelial（RPE）cells is a remarkable pathological mechanism leading to the progression of these diseases.Lnc RNAs play a non-negligible role in many diseases through complex regulatory mechanisms,the most prominent of which is the competitive endogenous RNA（ce RNA）mechanism.Lnc RNA MALAT 1 has been shown to play a significant role in various human diseases,but little is known about its function in retinal diseases.Previous blood sequencing results from clinical specimens in our group had indicated that MALAT 1was highly elevated in hypoxic-ischemic ophthalmopathy,but miR-625-3p was significantly reduced.Therefore,this study is based on MALAT 1 as the ce RNA of miR-625-3p in retinal pigment epithelial cells,and deeply explores the mechanism of targeting HIF-1αto regulate hypoxic retina diseases,in order to provide reliable evidence for finding novel target genes for the pathogenesis,diagnosis,and therapy of hypoxia-induced retinopathy.Methods:1.To construct a hypoxia model of retinal pigment epithelial cells（ARPE-19）.ARPE-19 cells were treated with different concentrations of Co Cl₂for 24 hours,and cell viability was detected by the CCK-8 experiment.When the cell viability was about to reach 50%,the hypoxia model concentration of ARPE-19 cells was chosen for subsequent experiments,and it was named the Co Cl₂group.The cells in the control group were cultured normally and designated as the Ctrl group.Western blot was performed to identify the influence of varying concentrations of Co Cl₂on the expression of hypoxia-inducible factor-1α（HIF-1α）to verify the success of the hypoxia model.The total RNA of the two groups of cells was extracted,and the expression changes of MALAT 1,miR-625-3p,and HIF-1αwere detected by RT-q PCR.2.To determine the changes of EMT and apoptosis indexes in ARPE-19 cells in response to hypoxia.Western blot experiments were used to detect changes in the Co Cl₂-induced hypoxia group compared with Ctrl group cells in the EMT characteristic proteins E-Cadherin,Vimentin,and apoptosis-related proteins Bax,Bcl-2,c-caspase-3,and c-caspase-9.3.Detection of binding sites between MALAT 1 and miR-625-3p,as well as between miR-625-3p and HIF-1α.The Target Scan database,a software that predicted binding sites online,predicted the binding region and possible probability.The dual-luciferase reporter experiments verified their binding by co-transfecting two reporter plasmids,wild type and mutant type from MALAT 1 or HIF-1α3’-UTR,with miR-625-3p mimic and mimic NC,respectively,into ARPE-19 cells.4.The expression levels of MALAT 1 and miR-625-3p were changed to explore their biological functions in response to hypoxia.RT-q PCR was used to verify the effect of changes in one molecule on the expression of the other.Lipofection3000 was used to transfect miR-625-3p mimic,mimic NC,si-MALAT 1,and si-NC,which were used as the transfection control.ARPE-19 cell models overexpressing miR-625-3p and knocking down MALAT1 were established,and the changes of EMT and apoptosis proteins mentioned above in each group were detected by Western blot.CCK-8 was used to detect the changes in cell activity in each group at different time points,and the Ed U experiment was utilized to detect the changes in cell proliferation in each group.5.Establish the downstream target of miR-625-3p in ARPE-19 cells.The binding of miR-625-3p to HIF-1αwas confirmed using an online prediction binding site database and dual-luciferase reporter experiments.The effect of MALAT1/miR-625-3p on the protein expression of HIF-1αwas verified by cell transfection and subsequent Western blot technique.6.Finding the downstream pathway regulated by HIF-1α.The nuclear translocation effect of si-MALAT 1 on NFκB-p65 was detected by immunofluorescence staining,and the expression of nuclear proteins and cytoplasmic proteins in each group of NFκB-p65 was detected by Western blot.By transfecting sh HIF-1αinto ARPE-19 cells to verify the effect of HIF-1αon the NFκB/Snail pathway,and by co-transfecting si-MALAT 1 with HIF-1αoverexpression plasmids into ARPE-19cells to assess the effect of MALAT 1/miR-625-3p/HIF-1αon this pathway.Western blot was able to detect protein levels in each group of p-p65,p65,p-IκB,IκB,and Snail.Results:1.The results of CCK-8 showed that when the concentration of cobalt chloride was 600μmol/l,the cell survival rate was 48.7%.A hypoxia model of ARPE-19 cells was constructed at this concentration,and the protein expression of HIF-1αwas highest at all concentrations.The RT-q PCR results showed that MALAT 1 was significantly up-regulated,miR-625-3p was greatly down-regulated,and HIF-1αwas significantly up-regulated in the Co Cl₂group.The results of Western blot revealed significant decreases in E-Cadherin and Bcl-2,and significant increases in Vimentin,Bax,c-caspase-3,and c-caspase-9,proving that the hypoxia model was successfully induced and EMT and cell apoptosis took place in the model.2.After overexpression of miR-625-3p,Western blot results revealed that E-Cadherin and Bcl-2 were increased to variable levels in hypoxia,while Vimentin,Bax,c-caspase-3,and c-caspase-9 were reduced to variable levels,indicating that the overexpression of miR-625-3p had a certain inhibitory effect on apoptosis and EMT in ARPE-19 cells caused by hypoxia.3.RT-q PCR results revealed that the expression of MALAT 1 was dramatically decreased when miR-625-3p was overexpressed,while the expression of miR-625-3p was significantly increased when MALAT 1 was knocked down,indicating a competitive relationship between the two.The Target Scan database predicted the existence of binding sites,and the dual-luciferase reporter assays demonstrated that miR-625-3p significantly decreased luciferase activity,suggesting that miR-625-3p could bind to MALAT 1.Results from CCK-8,Ed U,and Western blot showed that miR-625-3p inhibitor could partially offset the apoptosis,cell proliferation,and EMT behavior caused by knocking down MALAT 1.This suggested that MALAT 1 and miR-625-3p constituted a ce RNA,which was crucial for the apoptosis and EMT of ARPE-19 cells caused by hypoxia.4.The Target Scan online database prediction and dual-luciferase reporter experiments demonstrated that there was a binding relationship between miR-625-3p and HIF-1α.Western blot results showed that the reduction in HIF-1αexpression caused by knocking down MALAT 1 could be partially offset by miR-625-3p inhibitor,indicating that HIF-1αwas the downstream target of the MALAT 1/miR-625-3p axis.5.Immunofluorescence and Western blot results showed that the transfection of si-MALAT 1 led to a significant decrease in NF-κB p65 expression in the nucleus under hypoxia conditions,indicating that si-MALAT1 inhibited the nuclear translocation of NF-κB p65 subunits.Western blot results revealed that hypoxia conditions significantly enhanced the expression of p-p65/p65,p-IκB/IκB,and Snail proteins while knocking down HIF-1αand MALAT 1 could partially reverse this trend.Interestingly,overexpression of HIF-1αcould reverse the effects of MALAT 1 knockdown on the expression of p-p65/p65,p-IκB/IκB,and Snail proteins,indicating that the NFκB/Snail signaling pathway was involved in the process of regulating hypoxia retinal diseases by the MALAT 1/miR-625-3p/HIF-1αaxis.Conclusion:This study showed that MALAT 1 may play an important role in hypoxia retinal diseases.It may adsorb miR-625-3p,constitute the ce RNA,and participate in the regulation of apoptosis,proliferation,and EMT in cobalt chloride-induced retinal hypoxia models.It is anticipated that MALAT 1 will one day be used as a therapeutic target for hypoxia retinopathy.Further studies had shown that HIF-1αwas the downstream target of MALAT 1/miR-625-3p,which together constituted the MALAT 1/miR-625-3p/HIF-1αaxis.The axis influenced the occurrence and development of hypoxia retinal diseases by regulating the NFκB/Snail signaling pathway.In conclusion,the discovery of MALAT1/miR-625-3p/HIF-1αaxis provided new possibilities for further research on the pathogenesis and therapy of hypoxia-induced retinopathy.