Fig. 7: MIR200CHG stabilizes AGO2 from proteasome degradation via competitively binding to the target of miR-200c. | Nature Communications

Fig. 7: MIR200CHG stabilizes AGO2 from proteasome degradation via competitively binding to the target of miR-200c.

From: LncRNA MIR200CHG inhibits EMT in gastric cancer by stabilizing miR-200c from target-directed miRNA degradation

Fig. 7: MIR200CHG stabilizes AGO2 from proteasome degradation via competitively binding to the target of miR-200c.The alternative text for this image may have been generated using AI.

a The sequence match between miR-200c and MIR200CHG or between miR-200c and ZEB1. The seed sequence was highlighted. b Gradient doses of MIR200CHG oligos containing the miR-200c binding site (MIR-S) were incubated with miR-200c mimics, and the biotin-labeled ZEB1 mRNA probes (ZEB1-S) were used for RNA-RNA pulldown. Subsequent RT-qPCR analysis showed the interaction of miR-200c with ZEB1. c MIR200CHG overexpression plasmid was transfected into Hs746T cells. AGO2-RIP assay and RT-qPCR showed that less ZEB1 occupied the same AGO2 protein when MIR200CHG was present. d Western blotting was used to measure the expression of AGO2 in NCI-N87 and Hs746t. e Western blotting was used to measure the expression of AGO2 in NCI-N87 following treatment of 20 ug/ml with CHX after the knockdown of MIR200CHG. f Western blotting was used to measure the expression of AGO2 in Hs746t following treatment of 20ug/ml with CHX after overexpression of wild-type (MIR200CHG) or mutated MIR200CHG (MIR-Mut). g, h Western blotting was used to measure the expression of AGO2 in NCI-N87 and Hs746t that were treated with MG132 after the knockdown or overexpression of wild-type or mutated MIR200CHG. Cells were treated with MG132 (20 μM) for 6 h. i Western blot analysis of ubiquitinated AGO2 immunoprecipitated from Hs746t cells with or without wild-type MIR200CHG or MIR200CHG mutation overexpression. The cells were treated with MG132 to inhibit the proteasome. j Western blot analysis of ubiquitinated AGO2 immunoprecipitated from NCI-N87 cells with or without MIR200CHG knockdown. The cells were treated with MG132 to inhibit the proteasome. k The schematic illustration of the mechanism by which MIR200CHG stabilized miR-200c by inhibiting TDMD (Created with BioRender.com). The experiments were repeated three times independently with similar results (d–j). Each bar in bar plots represents the mean ± standard deviation of three biologically independent samples (b, c). P-values were determined by two-sided Student’s t-tests (b, c). Source data are provided as a Source Data file.

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