Research Paper Volume 13, Issue 6 pp 8055—8067

Silencing SIX1 by miR-7160 inhibits non-small cell lung cancer cell growth

miR-7160 binds and silences SIX1 in NSCLC cells. miR-7160 (-3p) putatively targets 3’-UTR of SIX1 (position of 1061-1068) (A). Immuno-fluorescence results showed that miR-7160 localized in the cytoplasm in pCan-1 NSCLC cells (B). Biotinylated-miR-7160 immunoprecipitated with SIX1 mRNA in pCan-1 cells (C). Stable pCan-1 NSCLC cells, bearing a pre-miR-7160-expression lentiviral construct (“lv-pre-miR-7160”) or non-sense miRNA control lentiviral construct (“lv-miRC”), were established, expression of listed genes was tested by qPCR and Western blotting analyses (D, F, G), with SIX1 3’-UTR luciferase reporter activity tested as well (E). pCan-1 NSCLC cells were transfected with 500 nM of the wild-type (WT) or the mutant miR-7160 mimics (sequences listed in H), after 48h the SIX1 3’-UTR luciferase reporter activity (I) and its expression (J, K) were tested. A549 cells or primary NSCLC cells (pCan-2/pCan-3) were infected with lv-pre-miR-7160 or lv-miRC for 48h, expression of miR-7160 (L) and SIX1 mRNA (M) was tested by qPCR. “pare” stands for the parental cells (same for all Figures). Data were presented as mean ± SD (n=5), and results normalized. *PB).

Figure 1. miR-7160 binds and silences SIX1 in NSCLC cells. miR-7160 (-3p) putatively targets 3’-UTR of SIX1 (position of 1061-1068) (A). Immuno-fluorescence results showed that miR-7160 localized in the cytoplasm in pCan-1 NSCLC cells (B). Biotinylated-miR-7160 immunoprecipitated with SIX1 mRNA in pCan-1 cells (C). Stable pCan-1 NSCLC cells, bearing a pre-miR-7160-expression lentiviral construct (“lv-pre-miR-7160”) or non-sense miRNA control lentiviral construct (“lv-miRC”), were established, expression of listed genes was tested by qPCR and Western blotting analyses (D, F, G), with SIX1 3’-UTR luciferase reporter activity tested as well (E). pCan-1 NSCLC cells were transfected with 500 nM of the wild-type (WT) or the mutant miR-7160 mimics (sequences listed in H), after 48h the SIX1 3’-UTR luciferase reporter activity (I) and its expression (J, K) were tested. A549 cells or primary NSCLC cells (pCan-2/pCan-3) were infected with lv-pre-miR-7160 or lv-miRC for 48h, expression of miR-7160 (L) and SIX1 mRNA (M) was tested by qPCR. “pare” stands for the parental cells (same for all Figures). Data were presented as mean ± SD (n=5), and results normalized. *P< 0.05 vs. “lv-miRC”/“miRC” cells. Experiments in this figure were repeated five times with similar results obtained. Bar= 50 μm (B).