Fig 1: RASSF10 impaired cell adhesion through MMP2 via FAK signaling. (a) Eight genes modulated by RASSF10 in HCC migration or invasion were found by cDNA adhesion array, which were further confirmed by qRT-PCR. White bars indicate the result of cDNA adhesion array, and black bars represent qRT-PCR data (the value of 2−ΔΔCT) in QGY 7703 transfected with pcDNA3.1/RASSF10 or empty vector (pcDNA3.1). (b) Western blot confirmed the association between RASSF10 and MMP2 or TIMP2 by over-expression or knock-down assay. (c) Regulatory effect of RASSF10 on FAK and MAPKs. Over-expression of RASSF10 has suppressed the accumulation of total or phosphorylation FAK and p38 MAPK; consistently, down-regulation of RASSF10 by SiRNA/RASSF10 in Huh 7 cells induced the activity of FAK, as well as p38 MAPK. The expression of ERK1/2 or JNK1/2 was independent on the level of RASSF10. (d) Regulatory effect of FAK on MMP2. Depletion of FAK suppressed the expression of MMP2. (e) Rescued assay for MMP2 in stable cell lines (QGY7703/RASSF10) was evidenced by western blot. (f) The change of cell invasion property was re-evaluated following MMP2 rescued assay. More invaded cells were observed with the restored expression of MMP2 in QGY7703/RASSF10. Invaded cells were stained with cell stain solution, counted by microscope in five random high power fields. Data are mean±s.d. The asterisk indicates statistical significance (*P<0.05, **P<0.01).
Fig 2: Effect of RASSF10 on ESCC cell metastasis. (A-C) Representative images of the migration capability of treated ECA109 and TE-10 cells as evaluated by the wound-healing assay. *P<0.05 vs. RASFF10, **P<0.01 vs. RASFF10 or sh-RASFF10. (D-F) Representative images of the invasive capability of the treated ECA109 and TE-10 cells as evaluated by the Matrigel invasion assay. **P<0.01 vs. RASFF10 or vs. sh-RASFF10. RASSF10, Ras-association domain family 10; ESCC, esophageal squamous cell carcinoma.
Fig 3: Effect of RASSF10 on ESCC cell proliferation. (A) Expression of RASSF10 in ESCC cell lines and the Het-1a cell line. *P<0.05, **P<0.01 vs. Het-1a cell line. (B and C) Western blot analysis of RASSF10 expression in RASSF10-overexpressing ECA109 and TE-10 cells and RASSF10-silenced ECA109 cells. (D-F) Cell proliferation was evaluated for 24, 48, 72, 96 and 120 h by CCK-8 assay. ***P<0.001 vs. RASFF10 or sh-RASFF10. (G-I) Representative images of the colony formation induced by RASSF10-overexpressing ECA109 and TE-10 cells and RASSF10-silenced ECA109 cells. **P<0.01 vs. RASFF10; ***P<0.001 vs. sh-RASFF10. RASSF10, Ras-association domain family 10; ESCC, esophageal squamous cell carcinoma.
Fig 4: RASSF10 inhibits the Wnt/β-catenin signaling pathway. (A) The results of TOPflash/FOPflash indicated that the Wnt/β-catenin signaling pathway was inhibited by overexpression of RASSF10 while knockdown of RASSF10 activated the Wnt/β-catenin signaling pathway. *P<0.05, **P<0.01. (B) The protein expression of β-catenin in the treated ESCC ECA109 and TE-10 cells was evaluated by western blot analysis. (C) The Wnt/β-catenin inhibitor IWR-1 suppressed the protein levels of β-catenin nuclear accumulation. (D and E) The Wnt/β-catenin inhibitor IWR-1 reversed the effect of RASSF10 knockdown on cell growth and cell invasion as determined using the CCK-8 and Matrigel invasion assays, respectively. **P<0.01 vs. sh-RASFF10. RASSF10, Ras-association domain family 10; ESCC, esophageal squamous cell carcinoma.
Fig 5: RASSF10 expression is downregulated in human ccRCC specimens and negatively associated with the malignant features and targeted drug resistance of ccRCC. (a), The mRNA expression of RASSF10 in ccRCC specimens (n = 479) and normal renal tissues (n = 72) was analyzed using the TCGA database. The z-score of RASSF10 was determined to compare the expression differences (***p < .001; Wilcoxon test). (b), The mRNA expression of RASSF10 was detected in ccRCC specimens and the matched paracancerous tissues (n = 62) from the TCGA database. The z-score of RASSF10 was calculated to compare the expression differences. (c), The mRNA expression of RASSF10 was analyzed by real-time PCR in paired ccRCC samples and adjacent renal tissues (n = 55; 45/55) (***p < .001; Wilcoxon test). (d), The protein expression of RASSF10 in the matched ccRCC specimens and paracancerous tissues was determined by Western blot assays (n = 20; 17/20) (**p < .01; Wilcoxon test). (e), The IHC assay was performed in ccRCC to detect RASSF10 expression (n = 375; the different scale bars have been shown on the images). Representative images of hematoxylin and eosin (H&E) and IHC staining are presented. The H-score was analyzed to compare the expression of RASSF10 in the matched ccRCC specimens with that in normal renal tissues, which is shown in the statistical chart (***p < .001; Wilcoxon test). (f), The mRNA expression of RASSF10 was determined by real-time PCR in normal renal cell lines (HK-2) and ccRCC cell lines (OS-RC-2, 769-P, 786-O, and ACHN). (g–h), Representative H&E and IHC staining and statistical charts for RASSF10 in different comparative groups ((g), TNM stage; h, WHO/ISUP grading) are presented (the different scale bars have been shown on the images). (i–j), Real-time PCR assays were performed to detect RASSF10 expression in sunitinib- and pazopanib-treated and -resistant ccRCC cell lines (786-O–SR and 786-O–PR) compared with that in naïve 786-O cells. All p values are defined as *p < .05, **p < .01 and ***p < .001, and the values are presented as the means ± SD.
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