图1 头颈鳞癌组织和细胞中LASTR表达水平及其与患者生存相关性分析

Fig.1 Expression of LASTR in HNSCC tissues and cells and its correlation with survival time of HNSCC patients. A: Expression of LASTR in pan-cancer analyzed by GEPIA. B: Expression of LASTR in HNSCC tissues and normal control tissues analyzed using GEPIA. C: Expression of LASTR in laryngeal squamous cell carcinoma (LSCC) tissues and adjacent normal mucosal (ANM) tissues analyzed using transcriptome data from GEO database. D: Correlation between LASTR expression level and overall survival time of HNSCC patients analyzed using Kaplan-Meir method. E: Expression of LASTR in HNSCC cells and normal control cells determined by qPCR (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs HOK cells.

图2 敲降LASTR抑制头颈鳞癌细胞增殖和活力

Fig.2 Knockdown of LASTR inhibits proliferation and viability of HNSCC cells. A, B: TU177 and CAL-27 cells transfected with siRNAs targeting LASTR (si-LASTR) or negative control (si-NC). After 48 h of transfection, LASTR knockdown efficiency was determined by qPCR (n=3). C, D: Proliferation of TU177 and CAL-27 cells with LASTR knockdown determined by CCK8 assay (n=3). E, F: TU177 and CAL-27 cell counts under a high-content system at 24, 48 and 72 h after LASTR knockdown and seeding in 96-well plate (n=3). G, H: TU177 and CAL-27 cell viability after LASTR knockdown determined by CellTiter-Glo® Luminescent Cell Viability Assay. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs si-NC group.

图3 敲降LASTR抑制头颈鳞癌细胞迁移和侵袭能力

Fig.3 Knockdown of LASTR inhibits migration and invasion of HNSCC cells. A, B: TU177 and CAL-27 cells transfected with siRNAs targeting LASTR (si-LASTR) or negative control (si-NC). After 24 h of transfection, the cells were seeded into Transwell insert for 48 h and examined for migration (A) and invasion (B) capacity using Transwell assay (Scale bar=50 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01 vs si-NC group.

图4 头颈鳞癌细胞LASTR靶基因预测及验证

Fig.4 Prediction and validation of the target genes of LASTR in HNSCC cells. A: Venn diagram of predicted LASTR target genes and genes that upregulated in HNSCC tissues. B: KEGG pathway analysis of LASTR target genes. C: GO analysis of biological process involving LASTR target genes. D, E: TU177 and CAL-27 cells transfected with siRNAs targeting LASTR (si-LASTR) and negative control (si-NC) respectively. After 48 hours of transfection, the expression of BCAM mRNA was determined by qPCR (n=3). F: Western blotting for detecting expression of BCAM in HNSCC cells with LASTR knockdown. Data are presented as Mean±SD. **P<0.01, ***P<0.001 vs si-NC.

图5 LASTR调控靶基因BCAM表达的机制研究

Fig. 5 Mechanistic study of regulation of BCAM expression by LASTR. A: RNA pulldown assay using Biotin-labeled LASTR probes in TU177 cells and qPCR analysis of enrichment of the indicated miRNAs. B, C: Expression of miR-4476 in TU177 and CAL-27 cells with LASTR overexpression or knockdown determined by qPCR analysis. D: Luciferase reporter assay of TU177 cells cotransfected with miR-4476 mimics and luciferase reporter plasmid containing wild-type (WT) and miR-4476 binding site mutated (Mut) BCAM 3' UTR. E: Expression of BCAM in TU177 and CAL-27 cells transfected with miR-4476 mimics or NC mimics detected by qPCR. F: Expression of BCAM protein in TU177 and CAL-27 cells transfected with miR-4476 mimics or NC mimics detected by Western blotting. G: Expression of BCAM protein in TU177 and CAL-27 cells transfected with miR-4476 mimics or co-transfected with miR-4476 mimics and LASTR overexpressing plasmid (LASTR-OE) detected by Western blotting. Error bars represent SD of 3 independent experiments. *P<0.05, **P<0.01 vs NC/control/si-NC/NC mimics group.

图6 BCAM在头颈鳞癌中的表达水平及生物学功能

Fig.6 Expression of BCAM and its biological functions in HNSCC. A: Expression of BCAM mRNA in HNSCC tissues and normal control tissues determined by GEPIA analysis. B: Expression of BCAM in laryngeal squamous cell carcinoma (LSCC) tissues and adjacent normal mucosal (ANM) tissues analyzed using transcriptome data from GEO database. C: Correlation between BCAM expression level and overall survival time of HNSCC patient analyzed using by Kaplan-Meir method. D, E: BCAM mRNA expression detected by qPCR in TU177 and CAL-27 cells transfected with siRNAs targeting BCAM (si-BCAM) or negative control (n=3). F: Western blotting for detecting BCAM expression in si-BCAM transfected HNSCC cells. G, H: CCK-8 assay of assessing proliferation of TU177 and CAL-27 cells 24 h after transfection with si-BCAM or si-NC (n=3). I: Transwell assay of migration capacity of HNSCC cells with BCAM knockdown (n=3). J: Invasion capacity of HNSCC cells with BCAM knockdown determined by Transwell assays. Scale bar=50 μm. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs si-NC.

图7 LASTR通过上调BCAM表达水平促进头颈鳞癌细胞恶性表型

Fig. 7 LASTR promotes malignant phenotypes of HNSCC cells via upregulating BCAM expression. TU177 and CAL-27 cells were transfected with si-LASTR, BCAM overexpression plasmid (BCAM-OE), or co-transfected with si-LASTR and BCAM-OE, respectively. A, B: After 48 h transfection, the expression of BCAM mRNA was determined by qPCR (n=3). C: Western blotting for detecting expression of BCAM protein. D, E: CCK8 assay for assessing proliferation of the transfected cells (n=3). F: Migration capacity of the transfected cells determined by Transwell assay (n=3). G: Invasion capacity of the transfected HNSCC cells determined by Transwell assay. Scale bar=50 μm. Data are presented as Mean±SD. *P<0.05, **P<0.01, ***P<0.001.