Comprehensive Bioinformatics Analysis of Lipopolysaccharide-Induced Altered Autophagy in Acute Lung Injury and Construction of Underlying Competing Endogenous RNA Regulatory Mechanism

Acute lung injury (ALI) is a fatal syndrome frequently induced by lipopolysaccharide (LPS) released from the bacterial cell wall. LPS could also trigger autophagy of lung bronchial epithelial cell to relieve the inflammation, while the overwhelming LPS would impair the balance of autophagy consequently inducing serious lung injury.

Our research indicated that the hsa-miR-663b-related ceRNA network may contribute to the key regulatory mechanism in LPS-induced changes of autophagy and ALI.

We observed the autophagy variation of 16HBE, human bronchial epithelial cell, under exposure to different concentrations of LPS through western blot, immunofluorescence staining, and electron microscopy. Eight strands of 16HBE were divided into two groups upon 1000 ng/ml LPS stimulation or not, which were sent to be sequenced at whole transcriptome. Subsequently, we analyzed the sequencing data in functional enrichment, pathway analysis, and candidate gene selection and constructed a hsa-miR-663b-related competing endogenous RNA (ceRNA) network.

We set a series of concentrations of LPS to stimulate 16HBE and observed the variation of autophagy in related protein expression and autophagosome count. We found that the effective concentration of LPS was 1000 ng/ml at 12 hours of exposure and sequenced the 1000 ng/ml LPS-stimulated 16HBE. As a result, a total of 750 differentially expressed genes (DEGs), 449 differentially expressed lncRNAs (DElncRNAs), 76 differentially expressed circRNAs (DEcircRNAs), and 127 differentially expressed miRNAs (DEmiRNAs) were identified. We constructed the protein-protein interaction (PPI) network to visualize the interaction between DEGs and located 36 genes to comprehend the core discrepancy between LPS-stimulated 16HBE and the negative control group. In combined analysis of differentially expressed RNAs (DERNAs), we analyzed all the targeted relationships of ceRNA in DERNAs and figured hsa-miR-663b as a central mediator in the ceRNA network to play when LPS induced the variation of autophagy in 16HBE.