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. 2025 Jul 1;26(1):37.
doi: 10.1186/s12868-025-00956-6.

Shikonin inhibits epithelial-mesenchymal transition in glioblastoma cells by upregulating p53 and promoting miR-361-5p level to suppress ZEB1 expression

Fengying Zhang #  1   2 , Zhiyi Liu #  3 , Yingbin Wang  4 , Lin Zuo  2 , Sicong Xu  2 , Yin Liu  2 , Hao Liang  2 , Yixue Xue  5   6
Affiliations

Shikonin inhibits epithelial-mesenchymal transition in glioblastoma cells by upregulating p53 and promoting miR-361-5p level to suppress ZEB1 expression

Fengying Zhang et al. BMC Neurosci. .

Abstract

Objective: Shikonin, an active compound from the rhizome of Lithospermum erythrorhizon, exerts anti-tumor effects in various cancers, including glioblastoma multiforme (GBM). This study explored the mechanism of Shikonin for inhibiting the migration and invasion of GBM cells, providing a rationale for developing novel glioma therapies.

Methods: The effects of Shikonin on GBM cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) were detected by CCK-8, scratch wound-healing, Transwell, and Western blot assays. The effect of Shikonin on miR-361-5p expression in GBM cells was examined by RT-qPCR and the effect of miR-361-5p inhibitor transfection on proliferation, migration, invasion, and EMT in Shikonin-treated GBM cells was examined. Shikonin's target genes were identified and validated using dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay, focusing on its induction of miR-361-5p expression. The downstream target genes of miR-361-5p were also identified and validated under Shikonin action. A GBM cell nude mouse xenograft tumor was established to confirm the regulatory role of Shikonin.

Results: Shikonin inhibited cell proliferation, migration, invasion, and EMT and upregulated miR-361-5p expression in GBM cells. Shikonin upregulated the glioma-associated protein p53, which promoted miR-361-5p transcription. miR-361-5p inhibited ZEB1 expression. Therefore, Shikonin inhibited GBM cell proliferation, migration, invasion, and EMT via p53/ miR-361-5p/ ZEB1 axis in vitro and in vivo.

Conclusion: Shikonin suppresses glioma cell proliferation, migration, invasion, and EMT by inhibiting ZEB1 expression through the p53/miR-361-5p axis.

Keywords: GBM; Shikonin; miR-361-5p.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The animal experiments reported in the manuscript were approved by the Animal Experimentation Ethics Committee of China Medical University, China. All experiments were performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ) for the reporting of animal experiments. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Shikonin inhibits proliferation, migration, and invasion of GBM cells. Cell proliferation, migration, and invasion of U87 cells treated with different doses of Shikonin were detected using (A) CCK-8, (B) scratch wound-healing, and (C) Transwell assays. The expression of EMT-related proteins in U87 cells was detected using (D) Western blot. *, vs. Control group, p < 0.05
Fig. 2
Fig. 2
Shikonin inhibits malignant progression of GBM cells through miR-361-5p upregulation. Expression of miR-361-5p in U87 cells treated with different doses of Shikonin detected using (A) RT-qPCR. Following transfection of miR-361-5p inhibitor into U87 cells treated with 7 µM Shikonin, the expression of miR-361-5p was detected using (B) RT-qPCR. Cell proliferation, migration, and invasion were detected using (C) CCK-8, (D) scratch wound-healing, and (E) Transwell assays, respectively. The expression of EMT-related proteins in U87 cells was detected using (F) Western blot. *, vs. Control group, p < 0.05; #, vs. Shikonin group, p < 0.05; $, vs. Shikonin + inhibitor NC group, p < 0.05
Fig. 3
Fig. 3
Screening for target genes associated with GBM after Shikonin treatment. (A) The chemical structure of Shikonin was analyzed by PubChem. (B) GBM-related genes were identified among the target genes of Shikonin using Venn analysis. (C) Protein interaction network. (D) Hub genes were identified by Cytoscape
Fig. 4
Fig. 4
Shikonin upregulates miR-361-5p expression through promoting p53 expression. Expression of p53 mRNA and protein in U87 cells treated with different doses of Shikonin were detected using (A) RT-qPCR and (B) Western blot, respectively. Following transfection of p53 siRNA into U87 cells treated with 7 µM Shikonin, miR-361-5p expression was detected using (C) RT-qPCR, and p53 protein expression was detected using (D) Western blot. *, vs. Control group, p < 0.05; #, vs. Shikonin group, p < 0.05; $, vs. Shikonin + si-NC group, p < 0.05. (E) The potential p53 binding site on the promoter of miR-361-5p was predicted using JASPAR. miR-361-5p’s status as a p53 transcriptional target was confirmed using (F) dual luciferase reporter gene assays (*, vs. Vector group, p < 0.05) and (G) ChIP-qPCR (*, vs. Control group, p < 0.05)
Fig. 5
Fig. 5
miR-361-5p targets and inhibits the expression of ZEB1. (A) The GBM-related genes were identified among the downstream target genes of miR-361-5p using Venn analysis. (B) EMT-related genes were identified among GBM-related miR-361-5p target genes using Venn analysis. (C) The expression of ZEB1 in GBM tissues was analyzed by GEPIA. Red indicates tumor tissue; gray indicates normal tissue. *, p < 0.05. (D) The predicted binding site of miR-361-5p and ZEB1 mRNA. (E) miR-361-5p binding to ZEB1 mRNA was confirmed by a dual luciferase reporter gene assay. *, vs. mimics NC group, p < 0.05. (F) miR-361-5p binding to ZEB1 mRNA was confirmed by the AGO2-RIP assay. *, vs. IgG group, p < 0.05. Following transfection of miR-361-5p mimics and inhibitor into U87 cells, expression of miR-361-5p, ZEB1 mRNA, and ZEB1 protein was detected by (G) RT-qPCR and (H) Western blot. *, vs. mimics NC group, p < 0.05; #, vs. inhibitor NC group, p < 0.05
Fig. 6
Fig. 6
Shikonin inhibits the proliferation, migration, and invasion of GBM cells through p53/miR-361-5p/ZEB1 axis. 7 µM Shikonin-treated U87 cells were transfected with p53 siRNA and miR-361-5p mimics. (A) Expression of miR-361-5p, p53, and ZEB1 mRNA of U87 cells was detected using RT-qPCR. (B) Expression of ZEB1 protein of U87 cells was detected using Western blot. (C) Cell proliferation of U87 cells was detected using CCK-8 assay. (D) Migration of U87 cells was detected using scratch wound-healing assay. (E) Invasion of U87 cells was detected using Transwell assay. (F) The expression of EMT-related proteins in U87 cells was detected using Western blot. 1, Control group; 2, Shikonin group; 3, Shikonin + si-NC group; 4, Shikonin + si-p53 group; 5, Shikonin + si-p53 + mimics NC group; 6, Shikonin + si-p53 + miR-361-5p mimics group. *, vs. Control group, p < 0.05; #, vs. Shikonin group, p < 0.05; $, vs. Shikonin + si-NC group, p < 0.05; &, vs. Shikonin + si-p53 group, p < 0.05; @, vs. Shikonin + si-p53 + mimics NC group, p < 0.05
Fig. 7
Fig. 7
Shikonin inhibits GBM cell tumor growth and EMT through ZEB1 downregulation in vivo. (A) Tumor volume and weight. (B) Expression of miR-361-5p in tumor was detected using RT-qPCR. (C) Expression of p53 and ZEB1 proteins in tumor was detected using Western blot. (D) Expression of EMT-related proteins protein in tumor was detected using Western blot. *, vs. Control group, p < 0.05; #, vs. Shikonin group, p < 0.05; $, vs. Shikonin + ov-NC group, p < 0.05
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