Identification of Autophagy-Related Targets of Berberine against Non-Small Cell Lung Cancer and Their Correlation with Immune Cell Infiltration By Combining Network Pharmacology, Molecular Docking, and Experimental Verification.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is the most common lung cancer type with high incidence. This study aimed to reveal the anti-NSCLC mechanisms of berberine and identify novel therapeutic targets.

METHODS: Berberine-related targets were acquired from SuperPred, SwissTargetPrediction, and GeneCards. NSCLC-re-lated targets were collected from GeneCards and DisGeNET. Differentially expressed genes (DEGs) were identified GEO database, UCSC Xena, and limma. GO and KEGG analyses were performed using clusterProfiler. Autophagy-related genes and transcriptional factors were collected from HADb and KnockTF, respectively. STRING and Cytoscape were used for PPI network analysis. Immune cell infiltration in NSCLC was assessed using CIBERSORT, and its correlation with autophagy-related targets was evaluated. Molecular docking was conducted using PyMOL and AutoDock. qRT-PCR and CCK-8 assay was used for in vitro verification.

RESULTS: Thirty intersecting targets of berberine-related targets, NSCLC-related targets, and DEGs were obtained. GO and KEGG analyses revealed that the intersecting targets were mainly implicated in oxidative stress, focal adhesion, and cell-substrate junction, as well as AGE-RAGE, relaxin, FoxO, and estrogen signaling pathways. Significantly, CAPN1, IKBKB, and SIRT2 were identified as the foremost autophagy-related targets, and 21 corresponding transcriptional factors were obtained. PPI network analysis showed that CAPN1, IKBKB, and SIRT2 interacted with 50 other genes. Fifty immune cell types, such as B cells naive, T cells CD8, T cells CD4 naive, T cells follicular helper, and monocytes, were implicated in NSCLC pathogenesis, and CAPN1, IKBKB, and SIRT2 were related to immune cells. Molecular docking revealed the favorable binding activity of berberine with CAPN1, IKBKB, and SIRT2. In vitro assays showed lower CAPN1, IKBKB, and SIRT2 expression in NSCLC cells than that in normal cells. Notably, berberine inhibited the viability and elevated CAPN1, IKBKB, and SIRT2 expression in NSCLC cells.

CONCLUSIONS: Berberine might treat NSCLC mainly by targeting CAPN1, IKBKB, and SIRT2.