To analyze the effects and molecular mechanism of Noggin (NOG) genes in bleomycin (BLM)-induced A549 cell senescence model of idiopathic pulmonary fibrosis (IPF) based on the bioinformatics.

IPF datasets including GSE28042 and GSE135893 were downloaded from GEO database.In GSE28042, R software was applied to analyze the differential expression analysis of senescence related genes, genes related to prognosis were screened out by univariate Cox regression, Random Forests and Least Absolute Shrinkage and Selection Operator-logistic regression (LASSO-LR) were applied to screen out key genes, and Kaplan-Meier method was applied to draw the survival curve of patients with high and low NOG expressions.Uniform manifold approximation and projection algorithm (UMAP) was used to implement the data dimension reduction of single-cell sequencing, and the differential expression of NOG in type Ⅱ alveolar epithelial cells in GSE135893 databases was analyzed.Twelve C57BL/6 mice aged at 6-8 weeks were divided into the control group and experimental group with six mice in each group by a random number table, the mice in the experimental group were injected with BLM in totracheas of mice to build the pulmonary fibrosis model, and the expression level of NOG protein and mRNA was detected by extracting lung tissues of mice by means of immunohistochemical staining and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). A549 cells were divided into the blank control group, BLM group (5 mg/L), negative control group (BLM in 5 mg/L+ empty carrier lentivirus) and NOG over-expression group (BLM in 5 mg/L+ NOG over-expression lentivirus). The expressions of senescence related proteins P16, P21, Wnt/beta-catenin signaling pathway related proteins and Ⅰα-collagen and α-smooth muscle actin were detected by Western blotting in groups of cells, and SA-β-Gal staining was used to detect the proportion of senescent cells in groups of cells.

Ninety five differentially expressed senescence related genes were screened out from GSE28042 dataset, 35 genes were up-regulated and 60 genes were down-regulated.The hub senescence related genes NOG were screened out by combining univariate Cox regression, LASSO regression and random forest algorithm.IPF patients with high NOG expression had longer overall survival duration than IPF patients with low NOG expression (P<0.05). Single cell sequencing showed that the expression quantity of NOG in type Ⅱ alveolar epithelial cells of IPF patients was lower than that in normal group (P<0.05). RT-qPCR and immunohistochemical staining showed that NOG was low expressed in BLM-induced lung fibrosis in mouse.NOG protein expression in A549 cells of BLM group was lower than that of blank control group and NOG over-expression group (all P<0.05). The expression level of senescence related proteins, Wnt/β-catenin signaling pathway related protein, Ⅰα-collagen and α smooth muscle actin were higher in the BLM group than the control group and NOG over-expression group (all P<0.05). SA-β-Gal staining results showed that the proportion of senescence cells in the BLM group were significantly higher than those in the control group and the NOG over-expression group (all P<0.05).

NOG is lowly expressed in bleomycin-induced pulmonary fibrosis tissues of mice.Over-expression of NOG attenuates bleomycin-induced senescence in A549 cells through the Wnt/β-catenin signaling pathway, thereby inhibiting the progression of pulmonary fibrosis.

Pulmonary fibrosis; Aging; Nod signaling adaptor proteins; Noggin; Computational biology