Research Paper Volume 12, Issue 4 pp 3936—3949

The circular RNA NT5E promotes non-small cell lung cancer cell growth via sponging microRNA-134

Figure 5. miR-134 inhibition attenuates circNT5E silencing-induced anti-NSCLC cell activity. Expression of mature miR-134 in stable A549 cells with the lentivirus-packaged circNT5E shRNA (“sh-circNT5E-Seq-1/2”), the non-sense control shRNA (“sh-c”) as well as in A549 cells with the lentivirus-packaged circNT5E expression construct (LV-circNT5E-Line-1/2, two lines) or the empty vector (LV-C) was shown (A and B); RNA-Pull down assay confirmed the direct association between biotinylated-miR-134 and circNT6E in A549 cells (C). Stable A549 cells with the lentivirus-packaged circNT5E shRNA (“sh-circNT5E-Seq-2”) or sh-c were further transfected with the applied microRNA inhibitors (500 nM, 48h), cells were cultured for applied time periods, cell growth, proliferation and apoptosis were tested by cell counting (D), EdU incorporation (E) and TUNEL staining (F) assays, respectively. A549 cells with the pre-miR-134 expression construct (LV-miR-134) were further infected with/without lentivirus-packaged circNT5E shRNA (“sh-circNT5E-Seq-2”), control cells were transduced with lentiviral empty vector (“LV-C”); Cells were further cultured for applied time periods, mature miR-134 and circNT5E expression was tested by qPCR assays (G and H); Cell growth (I), proliferation (J) and apoptosis (K) were tested. Stable primary human NSCLC cells, Pri-Can-1, with the lentivirus-packaged circNT5E expression construct (LV-circNT5E) or the lentivirus-packaged circNT5E shRNA (“sh-circNT5E-Seq-2”) were cultured, mature miR-134 expression was tested (L). Stable Pri-Can-1 cells with circNT5E shRNA were further transfected with miR-134 inhibitor (500 nM, 48h), cells were further cultured for applied time periods, cell growth (M) and apoptosis (N) were tested. Error bars stand for mean ± standard deviation (SD, n=5). “miRi” stands for non-sense control miRNA inhibitor. * P <0.05 vs. “sh-C”/“Pare”/“LV-C” cells. #P <0.05 vs. “miRi-C” cells. Experiments in this figure were repeated five times, and similar results were obtained. Bar= 100 μm (J and K).