Fig 1: MEG3 inhibits the CLU through directly binding to CLU protein. (A) MEG3 directly binds to CLU protein as the CLU antibody formed RNA-protein-antibody complexes suggested by EMSA assay; (B) The MEG3 spatial structure predicted by RNA structure (version 3.7). MEG3 was divided into four segments according to prediction, F1:1–389, f2: 390–731, F3: 732–1174, F4: 1175–1598; (C) C expression of LU mRNA and protein in SW1116 transfected with CLU siRNAs or not etermined by RT-PCR and western-blot (*P < .05); (D) Representative image of the trans-well assay to investigate migration ability (upper) and invasion ability (lower) of CRC cells with CLU down-regulation(si-CLU2), MEG3 over-expression (MEG3), MEG3 over-expression combinated with CLU over-expression (MEG3/CLU) or not (Blank).
Fig 2: MEG3 interacts with Clusterin and inhibites proliferation and metastasis. (A) The represent image of SDS-PAGE with precipitated proteins from MEG3 RNA pull down. Gel indicated by arrow was obtained for mass spectrometry analysis; (B) The represent image of western blotting for detection of LYAR and CLU in precipitated proteins from MEG3 RNA pull down; (C) The represent image of agarose gel for MEG3 detection in the precipitation from RIP by CLU antibody; (D) The represent image of EMSA to investigate the binding site of CLU protein in MEG3.MEG3 was truncated into four fragments: F1–F4; (E) Up-regulation of CLU by MEG3 over-expression (**P < .01); (F and G) The cellular proliferation, migration and invasion abilities of SW1116 cells transfected with CLU siRNAs (SW1116/si-CLU), SW1116 cells with MEG3 over-expression (SW1116/MEG3), SW1116 cells with MEG3 and CLU over-expression (SW1116/MEG3/CLU) and blank control cells detected by CCK8 (F) and trans well assays (G). The data are representative of at least three independent experiments.
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