Function Of MiR-664 In UVB-Induced Apoptosis And Autophagy In HaCaT Cells

BackgroundMiRNA is an endogenous micro ncRNA.Studies have shown that it is involved in the regulation of a variety of physiological and pathological processes and is closely related to human health and various diseases.Autophagy and apoptosis are two basic biological mechanisms that may interact or antagonize They play important roles in mammalian development,cellular homeostasis,and tumorigenesis.They exist simultaneously in the cell and share a common upstream signal and stimulus,so they can be combined or mutually exclusive.MiRNAs can regulate autophagy by regulating autophagy proteins,and can also act as upstream of pro-apoptotic and anti-apoptotic proteins to affect apoptosis.CircRNA is ncRNA like miRNA,but circRNA is a long endogenous ncRNA molecule with a closed continuous loop structure linked by covalent bonds.CircRNA can have a significant impact on cellular regulatory networks and epigenetics through targeted binding to mirna.However,the role of CircRNA and miR-664 in UVB-induced skin damage has not been studied in depth.This study aims to investigate the relationship between CircRNA and miR-664 and their effects on UVB-induced skin damage,which is induced by UVB.Skin damage provides a theoretical basis for clinical treatment.Methods(1)UVB irradiation treatment:HaCaT cells were irradiated with UVB of 30 mJ/cm 2 intensity,and cells were cultured for corresponding period of time after cell culture incubator for corresponding detection.(2)Tansient transfection:The small interfering RNA of miR-664 was synthesized.The overexpression vector of SMAD4 was transiently transfected into HaCaT cells by liposome,and the cell model of miR-664 down-regulated or SMAD4 up-regulated was constructed.(3)transient transfection combined with UVB treatment:liposome transfection for 24h for UVB irradiation treatment.(4)The apoptosis rate and cell cycle distribution of HaCaT cells after irradiation,miR-664 or up-regulated by SMAD4 were detected by flow cytometry.(5)The mRNA levels of the target genes(miR-664,SMAD4 and circFBXW8)in the treated HaCaT cells were detected by qPCR technique.(6)Western blot was used to detect the expression of target proteins(SMAD4,LC3,BECN1,Bcl2 and GAPDH,etc.).(7)Using the bioinformatics website to predict the target gene of miR-664,the targeting relationship between circRNA and miR-664 and the analysis of cyclin sequencing results.(8)Fluorescein Reporting Experiments Verify that SMAD4 is the target gene for miR-664.(9)Amplification of cNDA and gDNA using convergent primers and divergent primers,and circFBXW8 loopability by horizontal gel electrophoresis and Sanger sequencing.10)After construction of the cDNA library of circRNA,the library was quantified by Agilent 2100 and quantified by qPCR and sequenced using an Illumina sequencer.(11)The overexpression vector of circFBXW8 was constructed,and the cells were infected with lentivirus to establish a stable strain of circFBXW8 overexpressing HaCaT cells,and the overexpression efficiency was detected by qPCR.(12)AGO2-RIP verified the targeting relationship between circFBXW8 and miR-664.Res?lts(1)Apoptosis increased in HaCaT cells after UVB irradiation,autophagy increased,S phase arrest,and miR-664,LC3 was up-regulated and Bcl2 was down-regulated.(2)The expression of SMAD4 in HaCaT cells was decreased after UVB irradiation,and negatively correlated with miR-664.Fluorescein reporter assay confirmed that SMAD4 is the target gene of miR-664.(3)Illumina sequencing revealed differential expression of 103 circRNAs,15 of which had a targeting relationship with miR-664.(4)qPCR confirmed that circFBXW8 showed down-regulation of expression and negative correlation with miR-664 after UVB irradiation.(5)After overexpression of circFBXW8,the expression of LC3,BECN1,Bcl2 were up-regulated.The expression of Bax was down-regulated.Therefore apoptosis was suppressed,indicating that UVB-induced apoptosis and autophagy of HaCaT cells were inhibited.(6)AGO2-RIP experiments confirmed that circFBXW8 targets and regulates miR-664 to regulate the apoptosis and autophagy of SMAD4 in HaCaT cells.ConclusionCircFBXW8,via miR-664 regulation of SMAD4 is involved in UVB-induced apoptosis and autophagy in HaCaT cells.