Fig. 5

MALAT1 is a molecular sponge for miR-23b-3p. a Illustration of the base pairing between miR-23b-3p and MALAT1. The base pairing between miR-23b-3p and ATG12 3’UTR is also shown. b Schematic representation of psicheck2-based luciferase reporter plasmid containing wild-type MALAT1 (psicheck2-MALAT1-wt) and a mutant reporter construct in which two putative miR-23b-3p binding sites were mutated (psicheck2-MALAT1-mut), and mutated bases are indicated in red. miR-23b-3p or control mimics were transfected into SGC7901/VCR cells together with the indicated psicheck2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. c miR-23b-3p can bind directly to MALAT1. SGC7901/VCR cells were transfected with biotinylated wild-type miR-23b-3p (Bio-23b-3p-wt) or biotinylated mutant miR-23b-3p (Bio-23b-3p-mut). A biotinylated miRNA that is not complementary to MALAT1 was used as a negative control (Bio-NC). Forty-eight hours after transfection, cells were harvested for biotin-based pull-down assay. MALAT1 expression levels were analysed by real-time PCR. *, p<0.05 versus Bio-NC. d Lysates from SGC7901/VCR cells were incubated with in vitro-synthesized biotin-labeled sense or antisense DNA probes against MALAT1 for biotin pull-down assay, followed by real-time RT–PCR analysis to examine miR-23b-3p levels. e Lysates from SGC7901/VCR cells were incubated with in vitro-synthesized biotin-labeled MALAT1 and antisense RNA for biotin pull-down assay, followed by real-time RT–PCR analysis to examine miR-23b-3p and miR-218-5p levels. f SGC7901/VCR cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro-synthesized biotin-labeled sense or antisense DNA probes of MALAT1 for biotin pull-down assay, followed by real-time RT–PCR analysis to examine miR-23b-3p levels. Data shown are means ± S.D. (n = 3; *, p < 0.05, two-tailed t-test). *, p < 0.05