Fig 1: Expression levels of RAB20 in PSCC tissues and cell lines. (A) The comprehensive CGP analysis of eight pN+ PSCC patients indicated 108 co-upregulated genes in the PCA group and LM group, of which 19 genes were more highly expressed in the LM group. The heatmap shows the expression pattern of the 19 target genes. (B) Among the 19 genes in the GSE57955 database, AIM2, MMP9 and RAB20 were the top 3 genes with a significantly high expression level in PSCC tissues (log2 tumor/control: 4.24, 3.94 and 2.01 respectively), and followed with STK17A, COL11A1, BCL2A1, MEST and WISP1 (FC: 1.86, 1.64, 1.45, 1.37 and 1.20 respectively). IFIT2, IFI44L, RGS4 and BCAT1 were downregulated in the GSE57955 database (FC: 0.68, 0.84, 0.88 and 0.95 respectively). (C,D) The RAB20 protein was overexpressed in PSCC cell lines and tumor tissues in pairs. (E) IHC staining indicated that RAB20 was highly expressed in tumors compared with the corresponding normal tissues (magnification: 200×). (F,G) The mRNA levels of RAB20 were upregulated in 78 tumor tissues compared with those in 21 normal tissues (15 pairs). GAPDH was selected as the internal reference gene, and the results are presented as the means ± SDs of three independent experiments (H) RAB20 staining scores were multiplied by the staining intensity and the staining area. The standard staining intensity score of RAB20 in the cytoplasm was 0 for no staining, 1 for weak staining, 2 for clear staining and 3 for strong staining. * p < 0.05, ** p < 0.01. FC, fold change; HaCaT, human immortalized keratinocytes; IHC, immunohistochemistry; NS, not significant. The uncropped blots are shown in Figure S5.
Fig 2: Knockdown of RAB20 inhibited the proliferation of PSCC cells in vivo and in vivo. (A) Western blotting was performed to examine the knockdown efficacy in Penl2 and 149rca RAB20-silenced cells. (B) Knockdown of RAB20 in PSCC cells inhibited cell colony formation, (C) cell proliferation, (D) cell migration in vitro, (E), and tumor growth in vivo. (F) GSEA showed that knockdown of RAB20 in Penl2 cells significantly influenced the expression of proteins in the cell cycle and proliferation. Statistics are presented as the means ± SDs of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. GSEA, gene set enrichment analysis; IHC, immunohistochemistry; NC, negative control. The uncropped blots are shown in Figure S5.
Fig 3: Knockdown of RAB20 induced G2/M cell arrest via the Chk1/cdc25c/cdc2-cyclinB1 pathways. (A) Penl2 and 149rca cells transfecting with RAB20-sh3 increased the proportion of cells in the G2 phase and inhibited cell proliferation. (B) Knockdown of RAB20 significantly reduced the expression of the G2/M checkpoint proteins cdc2 and cyclinB1 but did not influence the G1/S phase. (C,D) Knockdown of RAB20 induced cell cycle arrest at the G2/M phase via the Chk1/cdc25c/cdc2-cyclinB1 pathway rather than via the p53 pathway. (E,F) Knockdown of Chk1 dramatically attenuated G2/M cell arrest in RAB20-sh3 cells and recovered the expression of the G2/M checkpoint proteins cdc2 and cyclinB1. Flow cytometry was performed in triplicate and the results are presented as the mean ± SD. * p < 0.05, ** p < 0.01. The uncropped blots are shown in Figure S5.
Fig 4: Overexpression of RAB20 promoted and rescued the proliferative ability of PSCC cells. (A) Western blotting was performed to detect the overexpression efficiency of RAB20 in wild type or transfection with RAB20-sh3 PSCC cells. (B,C) CCK-8 proliferation assays and colony formation assays revealed that RAB20 overexpression promoted cell proliferation and colony formation in Penl2 and 149rca cells and relieved the proliferative capability of cells restrained by RAB20 knockdown. Statistics are presented as the means ± SDs of three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001. OE, overexpression. The uncropped blots are shown in Figure S5.
Fig 5: Survival analysis between RAB20 expression and clinical features in 259 PSCC patients. Kaplan–Meier survival analysis was performed to determine the RAB20 expression level in PSCC patients. (A) High RAB20 expression indicated a significantly lower CSS rate in the entire cohort, (B) pathological grade subgroup, (C) pT2-pT4 subgroup, (D) positive lymph node metastasis subgroup, (E) clinical stage III/IV subgroup, and (F) ENE subgroup of PSCC patients (p < 0.05). CSS, cancer-specific survival; ENE, extranodal extension.
Supplier Page from Abcam for Anti-Rab20 antibody