Figure 4. MiR-30e inhibited tumor growth and RPS6KB1 expression, and higher levels of RPS6KB1 were associated with lower esophageal cancer survival rate. (A) Kyse30 cells were infected with lentivirus expressing miR-30e or miR-NC, and cells stably expressing miR-NC and miR-30e termed as Kyse30/miR-NC and Kyse30/miR-30e were obtained after puromycin selection. Cells (5 × 106 cells) in 100 μl of serum-free DMEM medium were subcutaneously injected into posterior flanks on both sides of the nude mice (n = 5). Tumors were measured every three days after they were visible starting on Day 12. Tumor volumes were calculated using the following formula: volume = 0.5 × (length × width2). (B) Representative pictures of tumors. Bar = 2 mm. (C) The weights of tumors from miR-30e and miR-NC groups were measured. (D) The total proteins were extracted from xenografts and subjected to Western blotting analysis to test levels of RPS6KB1. GAPDH levels were used as an internal control. (E) The expression levels of RPS6KB1 and PCNA in tumor tissues were analyzed by immunohistochemical (IHC) staining. (F) The Kaplan Meier plotter was used to detect the correlation between overall survival (OS) and RPS6KB1 expression levels in esophageal adenocarcinoma and esophageal squamous cell carcinoma tissues, respectively. Data represent mean ±SD of 3 replicates. *,**indicates significant difference at P < 0.05 and at P < 0.01, respectively.