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Fig. 5 | Molecular Cancer

Fig. 5

From: CircMRPS35 suppresses gastric cancer progression via recruiting KAT7 to govern histone modification

Fig. 5

CircMRPS35 Inhibits Gastric Cancer Progression by Upregulating the Transcriptional Activity of FOXO1 and FOXO3a. a RNA-seq and pathway analysis of circMRPS35-mediated mRNA expression profiles. b-c qRT-PCR assay for FOXO1, FOXO3a, FOXO4 and FOXO6 mRNA after overexpression or knockdown of circMRPS35. SGC7901 cells were transfected with circMRPS35 overexpression plasmid b, while MGC803 cells were transfected with siRNAs targeting circMRPS35 and corresponding control for 24 h c. d Western blot for FOXO1, FOXO3a, FOXO4 and FOXO6 after overexpression or knockdown of circMRPS35. Cells were treated as in b, c and harvested 48 h later. e-f qRT-PCR assay for FOXO1 target genes p21 and p27, and FOXO3a target genes Twist1 and E-cadherin. Cells were treated as in b, c and harvested 48 h later. g p21 and p27 mRNA expression after FOXO1 knockdown in circMRPS35-overexpressed SGC7901 cells. SGC7901 cells were transfected with circMRPS35 overexpression plasmid and control plasmid, together with siRNA targeting FOXO1 and siNC for 48 h. h Flow cytometry in SGC7901 cells. Cells were treated as in g. i CCK-8 assay in SGC7901 cells. Cells were treated as in g, six hours after transfection, cells were resuspended and seeded in 96-well plates, and CCK-8 assays were performed on the indicated days. j Twist1 and E-cadherin mRNA expression after FOXO3a knockdown in circMRPS35-overexpressed SGC7901 cells. SGC7901 cells were transfected with circMRPS35 overexpression plasmid and control plasmid, together with siRNA targeting FOXO3a and siNC for 48 h. k Cell invasion assay was performed in the above cells as in j and the number of invasion cells was calculated. Scale bar, 200 μm. l FISH assay of circMRPS35 in both SGC7901 and MGC803 cells. Nuclei were stained with DAPI. Scale bar, 10 μm. m qRT-PCR analysis for circMRPS35 expression and Western blot for Lamin B1, GAPDH. The nuclear and cytoplasmic fractions of MGC803 cells were isolated, followed by qRT-PCR and Western blot assays. Lamin B1 as a nuclear control, and GAPDH as a cytoplasmic control. n The luciferase activity of FOXO1 and FOXO3a promoters after circMRPS35 overexpression. SGC7901 cells in 24-well plates were transfected with circMRPS35 plasmids or control, together with pGL3-basic-FOXO1 (pGL3-FOXO1) or pGL3-basic-FOXO3a (pGL3-FOXO3a) plasmid. The luciferase activity was measured 24 h later. o The luciferase activity of FOXO1 and FOXO3a reporters after circMRPS35 knockdown. MGC803 cells in 24-well plates were transfected with circMRPS35 siRNA, together with the luciferase reporters. The luciferase activity was measured 24 h later. *P < 0.05, **P < 0.01, ***P < 0.001

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