Mechanism Of LncRNA MALAT1 Affecting Apoptosis Through Transcriptional Regulation Of CD80

Objective: Neonatal respiratory distress syndrome(NRDS)is a common disease in neonatal intensive care units.Apoptosis is involved in the mechanism of NRDS lung injury.CD80 gene can be expressed in non-bone marrow-derived cells after external stimulation,and participates in the mechanism of pathogenisis.The long non-coding RNA(lnc RNA)metastasis-associated lung adenocarcinoma transcript(MALAT1)is associated with inflammation induced by lipopolysaccharide(LPS)and is involved in gene transcriptional regulation.In this study,we intend to determine whether lnc RNA MALAT1 is involved in the regulation of CD80 and lung cell apoptosis and its underlying mechanisms,laying the foundation for NRDS mechanism research and precision therapy.Methods: Real-time quantitative PCR was used to detect the expression of CD80 m RNA and MALAT1 RNA in peripheral blood mononuclear cells in newborns with NRDS and LPS-induced cell lines;knockdown of MALAT1 to observe the changes of CD80 m RNA,protein expression and transcriptional activity.(2)Bioinformatics prediction,site-directed base mutation and chromatin immunoprecipitation to find potential transcription factors for CD80 promoter after LPS induction.RNA-binding protein immunoprecipitation was used to verify whether MALAT1 binds to CD80 potential transcription factor;knockdown of MALAT1 to detect the effect of MALAT1 on potential transcription factors from m RNA,protein expression level,and molecular activity.The effect of MALAT1 on the ability of potential transcription factors to bind to the CD80 promoter was examined again by chromatin immunoprecipitation experiments.The subcellular distribution of MALAT1 regulating CD80 was localized by cellular immunofluorescence and nuclear separation techniques.(3)CCK-8 and flow cytometry were used to detect the effect of CD80 and MALAT1 on cell proliferation and apoptosis.The cytoskeleton was observed by cytoskeleton staining.The changes of key protein molecules in apoptosissignal pathway were observed by Western blotting.Results:(1)CD80 expression was increased in newborns with NRDS exposed to prenatal infection and in low-dose LPS-induced A549 cells;MALAT1 negatively regulated CD80 expression and transcriptional activity.(2)There were NF-?B binding sites upstream of CD80,and NF-?B can bind to CD80 after LPS induction.CD80 activity was significantly decreased after mutation of NF-?B binding sites,and CD80 expression level was significantly decreased after knockdown of NF-?B active subunit p65.After LPS stimulation,MALAT1 and NF-?B binded in the nucleus;after knocking down MALAT1,NF-?B activity and the binding ability to CD80 promoter were enhanced,but the expression level of NF-?B is not significantly changed.It was confirmed that MALAT1 exerts the above-mentioned subcellular localization in the nucleus via cellular immunofluorescence and nuclear isolation RNA quantification.(3)CCK-8 and flow apoptosis assay showed that CD80 overexpression can lead to apoptosis,inhibit cell proliferation,cytoskeleton destruction,and decrease the content of cytoskeleton;the expression and the activity of PARP and caspase-3 were increased.Knockdown of MALAT1 also promoted apoptosis while increasing CD80 expression,consistent with CD80-mediated apoptosis.Conclusion: CD80 expression was elevated in newborns with NRDS exposed to prenatal infection and in low-dose LPS-induced A549 cells;MALAT1 negatively regulated CD80 expression and transcriptional activity.NF-?B is a CD80-dependent transcription factor after LPS stimulation;MALAT1 can interfere with NF-?B activity and affect NF-?B binding to CD80 promoter by binding to NF-?B;MALAT1exerts the above-mentioned subcellular localization in the nucleus.Overexpression of CD80 promotes apoptosis,and MALAT1 is involved in CD80-mediated apoptosis by affecting the expression of CD80.