Figure 5.

GJB2 is a direct target of miR-892a. (A) Potential miR interaction partners of CAR10 and GJB2 were predicted using the TargetScan, miRDB, RegRNA and miRWalk online platforms. (B) Expression of miR-892a in normal lung and NSCLC tissues was determined by analyzing the GSE19945 dataset. *P=0.0255. (C) miR-892a was downregulated in the majority of NSCLC tissue samples (39/50; 78.00%), as confirmed using RT-qPCR. (D) Expression of miR-892a was inversely correlated with that of GJB2 in 50 NSCLC tissue samples. P=0.0210. (E) miR-892a expression following transfection with miR-892a mimics and inhibitors was quantified using RT-qPCR; NC mimics and NC inhibitors were used as the corresponding controls. **P<0.01 and ****P<0.00001. (F). GJB2 protein expression was determined using Western blot analysis. (G) miR-892a upregulation suppressed A549 cell migration and invasion abilities, and the suppressive effect was reversed by co-overexpression with GJB2. Not significant, P>0.05, and ***P<0.001. (H) miR-892a knockdown enhanced the metastatic abilities of the H1299 cells, and this promotive effect was abolished by GJB2 inhibition. Not significant, P>0.05, and ***P<0.001. Scale bar, 100 μm. (I) GJB2 3ʹUTR possesses a binding site for miR-892a at position 975–982 (upper panel). The schematic diagrams of wild-type and mutant binding sequences of GJB2 reporter plasmids are displayed in the lower panel. Relative luminescence was determined using a luciferase assay in (J) A549 and (K) H1299 cells co-transfected with wild-type or mutant GJB2 reporter plasmids and miR-892a mimics. Not significant, P>0.05, and **P<0.01. Data are presented as the mean ± SD from three independent experiments.

Abbreviations: GJB2, gap junction protein beta 2; miR, microRNA; CAR10, chromatin-associated RNA Intergenic 10; NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; UTR, untranslated region.