Research Paper Volume 13, Issue 6 pp 8055—8067

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

Altering miR-7160 expression is ineffective on the function of SIX1 KO NSCLC cells. The pCan-1 NSCLC cells with the CRISPR/Cas9-SIX1-KO-GFP construct (SIX1 KO cells) were further infected with pre-miR-7160-expression lentivirus (lv-pre-miR-7160), pre-miR-7160 anti-sense lentivirus (lv-antagomiR-7160) or the control non-sense miRNA lentivirus (lv-miRC), control cells were transduced with CRISPR/Cas9 empty construct (Cas9-C), stable cells were established; Expression of listed proteins was tested by Western blotting assays (A); Cells were further cultured for applied time periods, cellular functions, including cell proliferation (nuclear EdU ratio, B), migration and invasion (“Transwell” assays, C, D), as well as cell apoptosis (nuclear TUNEL staining, E), were tested, with results quantified; Expression of miR-7160 was tested by qPCR (F). The pCan-1 cells bearing the pre-miR-7160-expression lentiviral construct (“lv-pre-miR-7160”) were transfected with or without a lentiviral 3’-UTR-null SIX1 expression construct: +SIX1 (UTR-null); Control cells were transfected with the control non-sense miRNA lentivirus (lv-miRC); Expression of listed proteins was tested by Western blotting (G); Cells were further cultured for applied time periods, cell proliferation (H), migration and invasion (I, J), as well as cell apoptosis (K) were tested, with results quantified; Expression of miR-7160 was tested by qPCR (L). Data were presented as mean ± SD (n=5), and results normalized. *PB–F). n.s. stands for no statistic difference (B–E, L). Experiments in this figure were repeated five times with similar results obtained.

Figure 5. Altering miR-7160 expression is ineffective on the function of SIX1 KO NSCLC cells. The pCan-1 NSCLC cells with the CRISPR/Cas9-SIX1-KO-GFP construct (SIX1 KO cells) were further infected with pre-miR-7160-expression lentivirus (lv-pre-miR-7160), pre-miR-7160 anti-sense lentivirus (lv-antagomiR-7160) or the control non-sense miRNA lentivirus (lv-miRC), control cells were transduced with CRISPR/Cas9 empty construct (Cas9-C), stable cells were established; Expression of listed proteins was tested by Western blotting assays (A); Cells were further cultured for applied time periods, cellular functions, including cell proliferation (nuclear EdU ratio, B), migration and invasion (“Transwell” assays, C, D), as well as cell apoptosis (nuclear TUNEL staining, E), were tested, with results quantified; Expression of miR-7160 was tested by qPCR (F). The pCan-1 cells bearing the pre-miR-7160-expression lentiviral construct (“lv-pre-miR-7160”) were transfected with or without a lentiviral 3’-UTR-null SIX1 expression construct: +SIX1 (UTR-null); Control cells were transfected with the control non-sense miRNA lentivirus (lv-miRC); Expression of listed proteins was tested by Western blotting (G); Cells were further cultured for applied time periods, cell proliferation (H), migration and invasion (I, J), as well as cell apoptosis (K) were tested, with results quantified; Expression of miR-7160 was tested by qPCR (L). Data were presented as mean ± SD (n=5), and results normalized. *P< 0.05 vs. “Cas9-C” cells (BF). n.s. stands for no statistic difference (BE, L). Experiments in this figure were repeated five times with similar results obtained.