lncRNA OXCT1-AS1 Promotes Metastasis in Non-Small-Cell Lung Cancer by Stabilizing LEF1, In Vitro and In Vivo

Figure 5

lncRNA OXCT1-AS1 stabilizes LEF1 by inhibiting NARF-mediated LEF1 ubiquitination. (a) The ubiquitination sites of LEF1 were predicted using Protein Lysine Modifications Database. (b, c) A549 cells were transfected with shRNA against lncRNA OCT1-AS1, followed by MG132 treatment. Then, LEF1 expression was detected by western blot analysis. (d, e) A549 cells were transfected with shRNA against lncRNA OCT1-AS1, followed by CHX treatment (100 μg/mL) for different times (0, 1, and 2 h). Then, LEF1 expression was detected by western blot analysis. (f) After transfection with HA-UB, Flag-LEF1, or lncRNA OCT1-AS1 in combination or separately, and one day posttranscription, 293T cells were treated with MG132 (6 h), followed by CO-IP using the anti-LEF1 antibody. Then, western blot analysis was performed. (g) lncRNA OXCT1-AS1 stably depleted A549 cells were treated with MG132 for 6 h, followed by CO-IP using the anti-LEF1 antibody; then, western blot was performed. (h–j) Lysates from A549 cells with NARF knockdown or NARF overexpression were used to detect the levels of NARF and LEF1 by western blot analysis. (k, l) 293T cells were transfected with Flag-NARF, HA-LEF1, and sh-lncRNA, followed by treatment with MG132 (6 h); next, cell lysates were immunoprecipitated using the antibody against Flag or HA. The precipitates and inputs were analyzed by western blot analysis. (m, n) NARF expression in H1299 and A549 cells with lncRNA OXCT1-AS1 knockdown was detected by western blot.

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