High Throughput RNA Sequencing Of Airway Epithelial Cells Induced By Cationic Polypeptides

Background and Objective: Major basic protein(MBP)is a kind of toxic protein released by eosinophil cells in the pathophysiology of asthma.It can damage the airway epithelial cells and further lead to a severe of asthma.Poly-L-Arginine is a kind of cationic poly-peptides and is widely used to mimic the biological effects of MBP in vitro.However,the underlying mechanism of epithelial cells destroyed by MBP remains unclear.The objective of this research is to explore the effects of Poly-L-Arginine on the airway epithelial cells NCI-H292 by analyzing the differential expressed genes(DEGs)on the basis of the transcriptome sequencing.Materials and Methods: Airway epithelial cells NCI-H292 were divided into the two groups,one is the group of Control(C)and another is PLA(P).Each group has three biological repeats.The samples were sequenced by Illumina Hiseq 4000.Differential expressed genes(DEGs)were selected by the foldchanges(29)1.5 and p-value ? 0.05.DEGs were then analyzed by GO(gene ontology)reports and KEGG(kyoto encyclopedia of genes and genomes)pathways to further exploring its related functions and the signaling pathways they involved in.Besides,qRTPCR and Western Blot were used to validate the DEGs.Results: Compared with the control,there are 230 DEGs in the group treated by PLA,of which 86 DEGs were upregulated and 144 DEGs were downregulated.GO report analyzes the function of DEGs by biological process(BP),molecule function(MF)as well as cellular component(CP)respectively.According to BP terms,upregulated DEGs are mainly related to the biosynthesis and regulation of cholesterol;downregulated DEGs are mainly related to the cell adhesion.MF terms show that upregulated genes are primarily related to the combination and activation of some proteins;downregulated DEGs are mainly related to the combination of some ions' proteins.Besides,CP terms indicate that upregulated DEGs are mainly related to the formation of the membranes,while downregulated DEGs are mainly enriched in the composition of the basal membrane.KEGG have analyzed significantly differential expressed pathways that involved in DEGs.It shows that upregulated DEGs are mainly involved in the steroid biosynthetic pathways,while downregulated pathways are mainly enriched in the ECM-receptor interaction pathways.Next,we chose 6 candidate genes for validation by qRT-PCR,and GAPDH was used as an internal reference.Among these,3 up-regulated DEGs SQLE,HMGCS1,ACAT2 were all involved in cholesterol biosynthesis and enriched in metabolic pathway.While three down-regulated genes COL4A3,DAG1 and LAMB2 were all enriched in extracellular matrix(ECM)organization and implicated in the cytoskeleton organization and cell adhesion.The results are consistent with the RNA-Seq.In addition,we chose HMGCS1 for further validation by western blot.The result was in consistent with the RNA-Seq and qRT-PCR.Furthermore,we found that PLA promotes expression of HMGCS1 by activating mTOR,while inhibition of mTOR by rapamycin could blocked the expression of HMGCS1.Conclusion: Our research has revealed that the effects of cationic polypeptides on airway epithelial cells are mainly related to the expression of genes involve in the process of composition,metabolism and function.This research has provided insights of the role of natural cationic protein on airway epithelial cells in the pathophysiology of asthma.