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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: The RNA-binding protein GRSF1 promotes hepatocarcinogenesis via competitively binding to YY1 mRNA with miR-30e-5p

Fig. 5

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 (= 3). *< 0.05, **< 0.01

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