Fig. 4

miR-92b-3p directly targeted the 3′-UTR of GABRA3 to suppress its expression. a A heat map of the expression changes of 9 candidate genes predicted to be targets of miR-92b-3p in PC cells transfected with miR-92b-3p mimic, antagomir, or negative control. The scale from 0.2 to 4 indicates the intensity of the differential regulation of mRNAs: low expression (green), medium expression (yellow), and high expression (red). FC, fold change. b-d qPCR and immunoblotting analyses of the GABRA3 expression levels in PC cells transfected with miR-92b-3p mimic, antagomir, or negative control. e A putative miR-92b-3p-binding site (wild type, WT) existed in the 3′-UTR of Gabra3 mRNA, and a nucleotide mutation (mutant, MU) was created at the binding site. (F-G) The relative luciferase activities of either the WT or MU 3′-UTR of the GABRA3 reporter in combination with the miR-92b-3p mimic in AsPC-1 and SW1990 cells. h Pearson χ2 tests were used to analyze the association of miR-92b-3p levels with GABRA3 levels in 46 pairs of PC and CNP tissues. i-j qPCR and IHC analyses of the mRNA and protein levels of GABRA3 in 46 fresh and 82 FFPE paired PC and CNP tissues. k Representative images of IHC staining in the 82 FFPE paired PC and CNP tissues. Scale bars: 100 μm. l-n Association of the Gabra3 protein levels with tumor size, lymph node metastasis and TNM stage. o Kaplan-Meier analyses of postoperative survival in PC patients stratified by Gabra3 protein levels. The P value was assessed by log-rank test. GAPDH and RNU6B snRNAs were used to normalize the qPCR results. All n = 3; bar, SEM; n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test