GATA6-AS1 via Sponging miR-543 to Regulate PTEN/AKT Signaling Axis Suppresses Cell Proliferation and Migration in Gastric Cancer

Figure 3

GATA6-AS1 binds to miR-543 in GC cells. (a, b) Cytoplasmic/nuclear fractionation and FISH assays were performed to detect the subcellular distribution of GATA6-AS1 in MKN-45 and AGS cells. U6 and GAPDH were used as control. Scale  μm. (c) LncBase database was used to screen out 14 putative target miRNAs which possibly bind to GATA6-AS1 and are confidently annotated. (d) The levels of 14 candidate miRNAs in GATA6-AS1-knockdown HGC-27 cells were detected by qRT-PCR. (e) miR-543 level in GATA6-AS1-overexpression MKN-45 and AGS cells was detected via qRT-PCR. (f) The starBase predicted the potential binding site between GATA6-AS1 and miR-543. The sequence of GATA6-AS1 was mutated based on the miR-543 binding site to obtain the GATA6-AS1-Mut plasmid. (g) The enrichment of GATA6-AS1 and miR-543 in anti-Ago2 bound complex was detected by RIP assays. (h) RNA pull-down assays followed by qRT-PCR analysis were carried out to detect the expression of GATA6-AS1 in the complexes pulled down by wild-type biotinylated miR-543 (Bio-miR-543-WT) or mutant biotinylated miR-543 (Bio-miR-543-Mut). (i) Luciferase reporter assays were conducted to further verify the interaction of GATA6-AS1 and miR-543. Luciferase activity of reporter vector containing wild-type GATA6-AS1 (GATA6-AS1-WT) or mutant type (GATA6-AS1-Mut) was detected post cotransfection with miR-543 mimics or NC mimics into HEK-293T cells. Student’s -test was used in (d), (e), (g), and (i) and one-way ANOVA and Dunnett’s test for (h). and .

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