Long Noncoding RNA MIATNB Regulates Hypeosmotic Stress-induced Corneal Epithelial Cell Injury By Inhibiting Autophagy In Dry Eye Disease

Objective: To investigate the regulatory effects of long non-coding RNA(lnc RNA)myocardial infarction associated transcription neighbor(MIATNB)on autophagy and apoptosis in human corneal epithelial cell(HCEC).Methods: The immortalized human corneal epithelial cell line was used in vitro.1.Different concentrations of Na Cl solution were used to establish dry eye model.After HCECs were cultured in 70,90,100,110,120 m M Na Cl for 24 h,CCK8 was used to detect the proliferation ability of HCECs.The m RNA expressions of LC3 B,ATG16L,BECN1,ATG1,ATG7,ATG13,ATG5 and ATG10 were detected by RT-q PCR,and protein expressions of LC3 B and P62 were detected by Western blot.2.The experimental groups were:(1)Normal group: culture medium without sodium chloride for 24 h.(2)Hyperosmotic group: hyperosmotic medium was used for 24 h culture.(3)si NC group:24h was transfected with small interfering RNA si NC.(4)si MIATNB group: small interfering RNA si MIATNB transfection treatment for 24 h.(5)si NC combined with hyperosmotic group: 24 h after transfection with small interfering RNA si NC,24 h after hyperosmotic treatment.(6)si MIATNB combined with hyperosmolar group: 24 h after transfection with small interfering RNA si MIATNB,24 h after hyperosmotic treatment.(7)Chloroquine(CQ)group: Cells were treated with 50μm chloroquine for 24 h.(8)Chloroquine combined hyperosmotic group: cells treated with hyperosmotic for 24 h were treated with chloroquine for 24 h.(9)Chloroquine combined with si NC group: 24 h after transfection with small interfering RNA si NC,24 h after treatment with chloroquine.(10)Chloroquine combined with si MIATNB group: After transfection with small interfering RNA si MIATNB for 24 h,chloroquine was treated for 24 h.The expressions of caspase 3,BCL2 and BAX were detected by flow cytometry and western blot.Autophagosomes were detected by transfection with plasmid m RFP-GFP-LC3,and autophago-related proteins and m RNA expressions were detected by Western blot and RT-q PCR.Transwell was used to detect cell migration and CCK8 was used to detect cell proliferation.Results: Autophagy and apoptosis of human corneal epithelial cells(HCECs)were activated under hyperosmotic conditions: The m RNA expression levels of LC3 B,Beclin1(BECN1),ATG5,ATG7,ATG10,ATG13,ATG16 L and ATG101 increased and the protein expression levels of LC3 B increased after treatment with sodium chloride at 70,90,100,110 and 120 m M for 24 h.The expression level of P62 protein decreased.At the concentration of 90 m M,more red particles were found in plasmid transfection.Hyperosmotic activation of apoptosis and inhibition of HCEC migration: at 70,90,100,110,and 120 m M sodium chloride treatment for 24 h,CCK8 detection showed decreased cell activity,decreased ratio of BCL2/BAX protein expression,and increased caspase3 and cleaved caspase3 protein expression.Transwell showed decreased cell migration at70,90 and 120 m M Na Cl concentrations.Inhibition of autophagy can promote apoptosis of HCEC: After treatment with 90 m M sodium chloride for 24 h,the expression ratio of BCL2/BAX protein decreased with the addition of chloroquine,and the number of apoptotic cells increased by flow cytometry.Hyperosmotic can up-regulate MIATNB expression,and knockdown of MIATNB can inhibit autophagosome degradation and promote apoptosis: After hyperosmolar treatment of 70,90,100,110 and 120 m M,PCR detected the increased expression of MIATNB.After the gene expression was knocked down by si-RNA,the protein expression of LC3 B increased,and the number of green spots increased after plasmid transfection,indicating that the number of autophagosomes increased.The results of co-treatment with chloroquine and si-RNA showed that the degradation of autophagosome was inhibited after MIATNB knockdown.After MIATNB knockdown,BCL2/BAX protein expression ratio decreased,flow cytometry showed more apoptotic cells,Transwell experiment showed that after knockdown,cell migration ability decreased.Under hyperosmotic conditions,downregulation of MIATNB also inhibited the degradation of autophagic lysosomes and promoted apoptosis: m RNA expressions of ATG5,BECN1 and ATG101 decreased after MIATNB knockout under hyperosmotic conditions.Meanwhile,only LC3 B protein expression was slightly decreased under these conditions.Using m RFP-GFP-LC3,it was found that autophagolysosome cleavage decreased.The expression ratio of BCL2/BAX protein decreased,and the expression of caspase 3 increased.Conclusion: MIATNB plays an important role in the pathogenesis of dry eye and acts as a bridge between autophagy and apoptosis.It can potentially provide a new therapeutic target for the pathogenesis and treatment of DED.