Splicing Factor Derived CircZNF652 Contributes To Goblet Cell Metaplasia By Targeting MiR-452-5p/JAK2 In Asthma

Bronchial asthma(asthma)is a common heterogeneous chronic airway inflammatory disease,which seriously threatens people’s health.The World Health Organization estimates that asthma affects about 180,000 people worldwide each year.Among them,excessive mucus secretion leads to blockage of the airway is the main cause of critical illness and death.The synthesis and secretion of airway mucus is dependent on airway mucins,mainly mucin 5AC(MUC5AC)and MUC5 B,especially MUC5 AC.Circular RNAs(circ RNAs)are important non-coding regulatory RNAs,which are first discovered in viruses.They are often expressed at low levels and are characterized by the cell and tissue specificity.Covalently closed circ RNAs are often produced by reverse splicing of pre-m RNA(pre-m RNA)in the exons of the gene(part of introns)under the action of shearing factors.A large number of circ RNAs flanking introns are rich in Alu Repetitive sequences promote the production of circ RNAs.The occurrence of reverse splicing mainly depends on intronic complementary sequence(ICS)and RNA binding protein(RBP).Studies have found that circ RNAs play an important role in many diseases,but the biological mechanism of circ RNAs in the goblet cell metaplasia of asthma remains mostly unknown.Objective:1.Explore whether circZNF652 is involved in the regulation of the expression of airway mucin MUC5 AC in asthma;2.Explore the regulatory effect and mechanism of circZNF652 in the expression of airway mucin MUC5 AC in asthma;3.To clarify the key regulatory effect and mechanism of the splicing factor ESRP1 on the production of circZNF652.Methods:1.To collect BALF from asthma and FBA patients,and performed RNA-seq to screen for differentially expressed circZNF652,and then detected the expression level and location of circZNF652 in the OVA induced mouse models and BALF from asthma and FBA patients by immunofluorescence.2.CircZNF652 knockdown compound were intratracheally administered to OVA induced mouse model,and then we tested the pulmonary inflammatory cell infiltration,goblet cell metaplasia and mucous protein MUC5 AC expression.3.To collect BALF from asthma and FBA patients,and performed RNA-seq to screen differentially expressed miRNAs,and then screened out miRNAs that interacted with circZNF652 through databases,RNA pull-down,dual luciferase reporter genes etc.,and then detected the expression level and location of miRNAs in the OVA induced mouse models and BALF from asthma and FBA patients by immunofluorescence.4.Screened out target genes that interacted with miRNAs through RNA-seq,databases,dual luciferase reporter genes etc,and further verified them at the RNA and protein levels.5.CircZNF652 knockdown compound,miRNAs antagonist,and a mixture of the two were intratracheally administered to OVA induced mouse model to perform rescue experiments,and then we tested the infiltration of pulmonary inflammatory cell,goblet cell metaplasia and mucous protein MUC5 AC expression.6.Constructed wild-type and mutant plasmids of circZNF652 flanking intron regions that interacted with splicing factor,and knock-down plasmids of splicing factor ESRP1,and observed their effects on the formation of circ RNAs and downstream target genes.Results:1.We found that circZNF652 was significantly high expression in asthma patients by sequencing,and further immunofluorescence staining found that circZNF652 was mainly highly expressed in airway epithelial cells.At the same time,the content of circZNF652 in the BALF of asthma patients was significantly higher than that in the FBA.Immunofluorescence staining showed that circZNF652 was mainly expressed in carat cells with CC10 positive.2.CircZNF652 knockdown compound were intratracheally administered to OVA induced mouse model,and it could dose-dependently inhibit the goblet cell metaplasia and MUC5 AC expression.3.By collecting the BALF from asthma and FBA patients for sequencing and database comparison analysis,we screened out miR-452-5p that is low expressed in asthma patients and interacted with circZNF652.Then we performed a pull-down assay with a biotinylated circZNF652 probe and showed that miR-452-5p were more enriched in RNA pull-down by the circZNF652 probe.After the mimic of miR-452-5p was transfected in 16 HBE cells,the activity of MUC5 AC luciferase reporter gene was significantly reduced.The wild-type and mutant plasmids of circZNF652,which was interacted with miR-452-5p was constructed,and the co-transformation of the wild-type plasmid and miR-452-5p significantly reduced the activity of the luc-circZNF652-WT.In 16 HBE cells showed that circZNF652 and miR-452-5p had obvious co-localization in the cytoplasm by immunofluorescence.And further immunofluorescence staining found that miR-452-5p was mainly expressed in airway epithelial cells.At the same time,the content of miR-452-5p in the BALF from asthma patients was significantly lower than that in the FBA.Immunofluorescence staining showed that miR-452-5p was mainly expressed in carat cells with CC10 positive.4.The circZNF652 knockdown plasmid was transfected into the cells and RNA-seq was X performed,and combined with database comparison analysis,we selected the target gene of JAK2.The wild-type and mutant plasmids of JAK2,which was interacted with miR-452-5p was constructed,and the co-transformation of the wild-type plasmid and miR-452-5p significantly reduced the activity of the luc-JAK2-WT.After transfection with circZNF652 knockdown plasmid,the levels of JAK2 m RNA and protein were decreased.While after transfection with miR-452-5p inhibitor,the levels of JAK2 m RNA and protein were increased.5.CircZNF652 knockdown compound,miRNAs antagonist,and a mixture of the two were intratracheally administered to OVA induced mouse model,and they could partially rescue the pulmonary inflammatory cell infiltration,goblet cell metaplasia and mucous protein MUC5 AC expression caused by circZNF652 knockdown.6.Through database comparison and analysis,we found that the flanking intron region of circZNF652 contains the binding sequence of the splice factor ESRP1,and constructed wild type and mutant plasmids of these binding sequences for RIP experiments,and found that ESRP1 can interact with these sequences of wild type instead of mutant.After constructing the ESRP1 knockdown plasmid,it was further proved that it could inhibit the formation of circZNF652 and the expression of downstream target genes JAK2 and STAT6.Conclusions:1.The circZNF652 was high expression in airway epithelial cells in mouse asthma models and asthma patients.2.CircZNF652 acted as a sponge for miRNA-452-5p to release its inhibitory effect on the target gene JAK2,thereby activating the JAK/STAT6 signaling pathway,and promoting the metaplasia of goblet cells.3.The splicing factor ESRP1 interacted with the sequence of the flanking intron region of circZNF652 to promote the cyclization of circZNF652,thereby promoting the metaplasia of goblet cells.