Table 1.
The hub genes’ primer sequences were used for RT-qPCR assay.
Fig 1.
The potential targets and mechanism analysis of AS/BJO-NEs against oral squamous cell carcinoma (OSCC).
(A) Drug-target-disease network diagram of AS/BJO-NEs and OSCC. (B) Venn diagram showing the triple intersection of the targets of the two components of AS/BJO-NEs and OSCC targets. (C) Visualization of PPI network diagram of intersection targets. (D) Visualization of the KEGG enrichment results(15 results are selected for display). (E) Visualization of the GO enrichment results(15 results are selected for display).
Fig 2.
Single-gene bioinformatics analysis of CDK1.
(A) Box plot of the differential expression of CDK1 between tumors and non-tumors in pan-cancer analysis. (B) CDK1 and MTFR2 exhibit a strong positive correlation (R = 0.8, P < 0.001). (C-D) Visualization of CDK1 protein expression across different tumor stages in HNSCC. (E) Differential expression of CDK1 between OSCC samples and normal controls in TCGA-OSCC transcriptome data. (F) Differential expression of CDK1 between OSCC samples and normal controls in GSE37991 transcriptome data. (G) ROC curve analysis of CDK1 based on TCGA-OSCC transcriptome data. (H) ROC curve analysis of CDK1 based on GSE37991 transcriptome data. (I) Overall survival prognosis analysis of CDK1, P < 0.05. (J) Disease-free interval survival analysis of CDK1, P < 0.05. (K) Disease-Specific Survival analysis of CDK1, P < 0.05. (L) Progression-Free Interval analysis of CDK1, P < 0.05. (M-N) Based on the TCGA-OSCC dataset and its clinical data (survival data, age, sex, etc.), univariate (M) and multivariate Coxanalyses (N) were performed on CDK1, and the expression of CDK1 was still significantly negatively correlated with the poor prognosis of the patients.
Fig 3.
(A-C) Molecular docking complex of AS-IV and CDK1. (D-F) Molecular docking complex of luteolin and CDK1.(G) RMSD of ligand. (H) RMSF of chain A that binding with ligand. (I) Hydrogen bonds generated between ligand and protein. (J) RG of total axe of protein. (K) RG of total and around axes of protein. (L) 2D Gibbs free energy landscape of chain A in complex. (M) 3D Gibbs free energy landscape of chain A in complex.
Fig 4.
AS/BJO-NEs inhibited the proliferation, migration and invasion of OSCC cells.
(A-B) CCK −8 assay was used to detect the effects of AS/BJO-NEs at different concentrations on the viability of SCC9 and CAL27 cells. (C-D) Clone formation assay was employed to examine the influence of AS/BJO-NEs on the proliferation ability of SCC9 and CAL27 cells. (E-G) Wound healing assay was conducted to investigate the impact of AS/BJO-NEs on the migration ability of SCC9 and CAL27 cells. (H-I) Transwell assay was utilized to evaluate the influence of AS/BJO-NEs on the invasion ability of SCC9 and CAL27 cells. n = 3, Scale bar: 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001,and ****p < 0.0001.
Fig 5.
Western blot and RT-qPCR experiments were conducted to detect the expression levels of related molecules.
(A-C) Western blot assessment of the CDK1,MTFR2 and EMT markers (E-cadherin,N-cadherin)protein levels in SCC9 and CAL27 cells intervened with AS/BJO-NEs(0,2,4 μg/ml) for 24h. (D-E) RT-qPCR assessment of the mRNA level of the CDK1,MTFR2 and EMT markers(E-cadherin,N-cadherin) in SCC9 and CAL27 cells intervened with AS/BJO-NEs (0,2,4 μg/ml) for 24h. n = 3,*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 6.
RT-qPCR and Western blot assays validate the knockdown and overexpression efficiency of CDK1.
(A) RT-qPCR assays were utilized to examine the CDK1 mRNA expression after knockdown. (B) RT-qPCR assays were utilized to examine the CDK1 mRNA expression subsequent to overexpression. (C-D) Western blotting experiments were carried out to evaluate the CDK1 protein expression after knockdown. (E-F) Western blotting experiments were carried out to evaluate the CDK1 protein expression following overexpression. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 7.
The Effects of CDK1 Knockdown and Overexpression on the Proliferation, Migration and Invasion of CAL27 Cells.
(A-B) The clone formation assay was utilized to investigate the impact of CDK1 knockdown on the proliferation of CAL27 cells. (C-D) The clone formation assay was utilized to investigate the impact of CDK1 overexpression on the proliferation of CAL27 cells. (E-F) Wound healing assay was applied to assess the influence of CDK1 Knockdown on the migration of CAL27 cells. (G-H) Wound healing assay was applied to assess the influence of CDK1 overexpression on the migration of CAL27 cells. (I-J) Transwell assay was utilized to explore the influence of CDK1 Knockdown on the invasion of CAL27 cells. (K-L) Transwell assay was carried out to explore the impact of CDK1 overexpression on the invasion of CAL27 cells. n = 3,Scale bar: 100μm, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 8.
Western blot and RT-qPCR experiments were conducted to detect the expression levels of related molecules.
(A-C) Western blot assessment of the CDK1,MTFR2,E-cadherin,and N-cadherin protein levels. (D-E) RT-qPCR assessment of the CDK1,MTFR2,E-cadherin,and N-cadherin mRNA levels. n = 3,*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig 9.
AS/BJO-NEs inhibited the growth of CAL27 xenograft tumors in nude mice.
(A) Representative photographs of the tumors from nude mice. (B) The weights of tumors from the control groups, Blank-NEs treated groups, AS/BJO-NEs treated groups, and Cisplatin treated groups. (C) The growth curves of tumors derived from CAL27 xenografts in mice after normal saline, Blank-NEs, AS/BJO-NEs, and Cisplatin treatment. (D) The expression of CDK1 and MTFR2 were detected by immunohistochemical staining and the expression of CDK1 and MTFR2 were decreased after AS/BJO-NEs and Cisplatin treatment. Scale bar: 100μm, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 10.
Mechanism of AS/BJO-NEs on OSCC.
AS/BJO-NEs downregulate CDK1, and then reduce the expression of MTFR2, thereby inhibiting the proliferation, migration, invasion as well as the EMT process of OSCC cells.