| Objective Non-small cell lung cancer(NSCLC)is one of the highly prevalent malignancies in clinical practice.Triptolide(TP)has strong broad-spectrum antitumor activity,and although the mechanism of TP inhibition of NSCLC through induction of apoptosis has been widely reported,the related inhibition mechanism involving ribosomal proteins needs to be further investigated.This study intends to initially elucidate the molecular mechanisms involved in TP inhibition of NSCLC,and provide clues and scientific basis for the development of TPbased therapeutic strategies for NSCLC.Methods(1)The R language package was used to screen the transcriptomic differential mRNAs in the dataset after TP treatment of A549 cells.and then these differential mRNAs were combined with the proteomic differential proteins of TP acting on A549 cells screened by the project team in the pre-project phase,and the PPI network between the differential mRNAs and proteins as well as the hotspot proteins were jointly integrated and analyzed using the STRING database.(2)The TIMER database and GSCA database were used to analyze the differential expression of Ribosomal Protein L4(RPL4)in lung cancer,and the K-M database was used to plot the K-M curve of RPL4 in Lung Adenocarcinoma(LUAD).Western blot was used to verify the differential expression of RPL4 protein in cell lines and the effect of TP on RPL4 protein,and Q-PCR was used to detect the effect of TP on RPL4 mRNA.(3)Cell proliferation,apoptosis,cycle and invasion were detected by CCK-8,Flow Cytometry and Transwell,respectively.Western blot and CO-IP were used to detect the expression and interaction of pathway proteins.(4)BALB/cAnNCr-nu/nu nude mice were xenografted and grouped for TP treatment,and tumor sections were stained using TUNEL assay and IHC to detect the effects of TP administration on apoptosis of xenograft cells as well as RPL4 expression.Results(1)Transcriptomic and proteomic integration analysis revealed that the down-regulated RPL4 protein had more pairs of relationships of action in the differential protein PPI network of TP acting on NSCLC cells and was among the top 10 hub proteins.(2)RPL4 was highly expressed in LUAD and Lung Squamous Carcinoma(LUSC)tissues and correlated with poor prognosis;RPL4 was highly expressed in NSCLC cells;TP inhibited the expression of RPL4 in NSCLC cells.(3)Interfering with RPL4 combined with TP treatment enhanced the downregulation of RPL4 protein;interfering with RPL4 or TP treatment alone inhibited the proliferation and invasion of NSCLC cells,induced apoptosis,and blocked the cell cycle at G1 phase,respectively;interfering with RPL4 combined with TP administration,compared with TP administration alone,interfering with RPL4 enhanced the inhibition of proliferation and invasion of NSCLC cells by TP and enhanced apoptosis induction and cycle blocking effects of TP on NSCLC cells;downregulation of RPL4 activated the MDM2-P53 pathway and regulated the expression of apoptosis,invasion and cycle-related protein PARP1/Snail/cyclin Dl.(4)Animal experiments confirmed that TP inhibited NSCLC tumor size,induced apoptosis in NSCLC cells and decreased RPL4 expression in vivo.Conclusion(1)RPL4 was highly expressed in tumor tissues and cells.(2)Down-regulation of RPL4 inhibited the proliferation and invasion of NSCLC cells and induced apoptosis and cycle arrest in NSCLC cells.(3)Down-regulation of RPL4 enhanced the inhibitory effects of TP on NSCLC cell proliferation and invasion,and enhanced apoptosis induction and cycle arrest.(4)Down-regulation of RPL4 enhanced the inhibitory effect of TP on NSCLC cells by activating the MDM-P53 pathway and regulating PARP1/Snail/cyclin D1 protein expression. |
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