Exploring the potential mechanism of artesunate in intervening with glioma cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology

Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. In this research, we aim to explore the regulatory mechanism of artesunate acting on glioma through constructing the interaction network of therapeutic effects-related genes of artesunate based on transcriptome sequencing data of glioma cells, and then verified by apoptosis staining and molecular docking at cellular level. In vitro cell activity and proliferation assay of two human glioma cell lines indicated that artesunate exerted more obvious inhibitory effects on the cell activity and proliferation of U87 cells than that of U251 cells. It could significantly promote apoptosis in U87 cells (P<0.01), while not in U251 cells (P>0.05), detected by using Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained, respectively, by transcriptome sequencing and differential data analysis using the lysis buffer of U87 and U251 cells before and after artesunate treatment. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which ATF4-DDIT3-PARP1 signaling axis, that was related with cell apoptosis, was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that artesunate may induce the apoptosis and inhibited cell proliferation of glioma cells through regulating the abnormal alterations of corresponding proteins in ATF4-DDIT3-PARP1 signaling axis, which provided the novel scientific basis for its potential application in treating glioma. Overall design: Before and after ART treatment for 24 hours, U87 and U251 glioma cell lysates were centrifuged at 3000 rpm for 15 minutes, and then total RNA was extracted according to the routine procedure. After the quality inspection is qualified, the double stranded cDNA is further purified and reverse transcribed, which is used as a template for PCR amplification to construct a cDNA library. High throughput sequencing was carried out on the illumina NovaSeq 6000 platform and entrusted to Beijing Zhimeiyinuo Biotechnology Co., Ltd. (Beijing, China). Use fastp software to clean and filter the original offline data to obtain high-quality sequences (Clean Reads); Use Hisat2 2.1.0 software to compare Clean Reads with the reference gene (the reference genome version is GRCh 38.91); Stringtie 1.3.3b software was used to compare the results and quantify the transcriptome expression; TMM (trimmed mean of M values) algorithm was used for normalization, and finally FPKM (Fragments PerKilobase Million) value of each gene was calculated, and edgeR 3.30.3 software package was used for differential analysis of gene expression between samples/groups.