Fig 2: YY1 is an essential target of GRSF1, and feedback promoted GRSF1 expression. A GRSF1 and YY1 expression in HCC cells cotransfected with sh-GRSF1 and ov-YY1 vectors. B-E YY1 overexpression rescued the tumor-inhibiting effect caused by GRSF1 knockdown on HCC cell proliferation (B), cell apoptosis (C), colony formation (D) and migration (E) ability. The invaded cells in Transwell assays were quantified by counting the cells in 10 random fields (magnification, 200×). F GRSF1 expression levels in HCC cells were decreased following inhibition of YY1. G GRSF1 expression in HCC cells was increased following overexpression of YY1. H ChIP analysis of YY1-HA tagged fusion vector levels at the GRSF1 promoter in MHCC-97H and Hep3B cells. ChIP results were analyzed via qRT–PCR using GRSF1 promoter-specific primers and are expressed as the percentage of the input. Rpb1 was used as a positive control for GRSF1 ChIP. IgG was used as a negative control. Values are the mean± SEM (n = 3). *p < 0.05, **p < 0.01

Fig 3: ARGLU1 enhanced the transcriptional activity of AP1 and YY1 on MLH3, MSH2, MSH3 and MSH6. (a) Venn diagram of ARGLU1 IP-MS result and transcription factors of 4 MMR genes (MLH3, MSH2, MSH3 and MSH6) predicted by JASPAR. (b) Co-IP result of ARGLU1 with YY1 and SP1. (c) ChIP-qPCR results of YY1 and SP1 in HGC27 cells with or without ARGLU1 knock-down. (d) Relative luciferase activity of MLH3 in shARGLU1, siSP1 or siYY1 treated HGC27 and (e) MGC803 cells. (f) Schematic diagram of the molecular mechanism of ARGLU1 enhancing the transcriptional activity of SP1 and YY1 on MMR genes in GC. The data are the means ± SDs of three independent experiments (two-tailed t-test, *, p < 0.05, **, p < 0.01, ***, p < 0.001, NS, not significant).

Fig 4: GRSF1 and miR-30e-5p competitively regulate YY1. A, B Wild-type (WT) fragments of YY1 3`UTR 2663-2847 and four mutants were constructed and used in RNA pull-down assays. C Luciferase reporter assays showed that the M1, M2 and M3 mutants partially lost the capacity to associate with GRSF1, while the M4 mutant essentially lost all ability to interact with GRSF1. D The binding site of YY1 for miR-30e-5p partially overlaps with GRSF1 binding sites. E YFP reporter construct containing only YFP (YFP Ctrl), wild-type YY1 3`UTR (YFP-YY1 WT), a mutated miR-30e-5p-binding site (YFP-YY1-miR-30e-5p MT) or a mutated GRSF1-binding site (YFP-YY1-GRSF1 MT). The underlined sequences are the mutated sites. F The AGO2 IP/IgG IP ratio of YY1 was increased in the YFP-YY1 WT group compared to the YFP-YY1-miR-30e-5p MT group. YFP-YY1-miR-30e-5p MT disrupts binding with miR-30e-5p, resulting in decreased RNA compared to YFP-YY1 WT. However, YFP-YY1-GRSF1 MT resulted in more enrichment in the AGO2/IP fraction than YFP-YY1 WT, suggesting that miR-30e-5p bound to the YY1 3`UTR 2663-2847 region and that the binding function was impaired by transfection with YFP-YY1-miR-30e-5p MT. GAPDH was used as a negative control. G miR-30e-5p expression in MHCC-97H cells was not affected by GRSF1 knockdown. H Western blotting assays showing that miR-30e-5p inhibits YY1 expression. I Western blotting assays showing that anti-miR-30e-5p promotes YY1 expression, while the promotion function was disrupted by GRSF1 knockdown. J miR-30e-5p expression in MHCC-97H cells was decreased by transfection with anti-miR-30e-5p but not further inhibited by GRSF1 knockdown. K pre-miR-30e-5p enhanced the AGO2 IP/IgG IP ratio of YY1, which was further enhanced by GRSF1 knockdown. GAPDH was used as a negative control. Values are the mean± SEM (n = 3). *p < 0.05, **p < 0.01

Fig 5: VE821 inhibits HCC by repressing the GRSF1/YY1 pathway. A Chemical structure of VE821. B Cytotoxicity analysis of VE821 in MHCC-97H and Hep3B cells. C GRSF1 and YY1 expression levels were decreased in MHCC-97H and Hep3B cells treated with VE821. D VE821 markedly inhibited the proliferation, migration, and colony formation ability of MHCC-97H and Hep3B cells and enhanced apoptosis (n=5). Values are the mean± SEM (n=3); *p<0.05, **p<0.01