Fig. 6: Combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy via dual abrogation of MNK/eIF4E and mTOR in NSCLC.

Analysis of proteins of p-MNK1 ^Thr197/202 and p-eIF4E^S209 respectively extracted from cytosolic or nuclear in the indicated A549 cells was detected using Western blotting. GAPDH was used as a loading cytoplasmic control. And Histone H3 was used as a loading nuclear control. A A549 cells were treated with MNK inhibitor CGP57380 or mTORC1 inhibitor Everolimus alone or in combination. B The LV-miR-7-5p or LV-NC A549 cells were treated with or without Everolimus. C Treatment of A549 cells with Everolimus or miR-7-5p loaded exosomes alone or in combination. D Cells were transfected with indicated mimics with or without Everolimus for 48 h. Apoptotic cells were analyzed by flow cytometry using Annexin V/ PI staining. Columns, means of three replicate determinations; each bar represents mean ± SD. *P < 0.05, ** P < 0.01. E Representative microscopic images of liver metastatic lesions among all groups in previous abdominal metastasis models. The arrow showed the metastasis. F–H The stably LV-miR-7-5p of A549 cells were treated with Everolimus or DMSO for 48 h. Cell lysates were harvested for western blotting analysis to detect the indicated apoptotic proteins, and GAPDH was used as a loading control. I Schematic representation of a model for the major molecular mechanisms of “Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis” in NSCLC.