Fig. 4

The suppression of malignant behaviors in ESCC cells was observed following PIM1 knockdown mediated by miR-3619-5p. The knockdown effects varied depending on the TP53 mutation status, highlighting the potential influence of TP53 alterations on the functional impact of miR-3619-5p in targeting PIM1. (a) Effects of miR-3619-5p-mediated PIM1 knockdown on downstream signaling pathways. KYSE70 (TP53 wild-type) and KYSE170 (TP53 mutant) cells were transfected with miR-3619-5p or miR-NC mimics at 12 nM. At 72 h post-transfection, protein lysates were analyzed by western blot. In both cell lines, miR-3619-5p overexpression suppressed phosphorylation of AKT (p-AKT), upregulated p21, and enhanced phosphorylation of retinoblastoma protein (p-Rb), suggesting inhibition of cell cycle progression. Notably, cleaved caspase-3 and cleaved PARP—markers of apoptosis—were observed only in TP53 wild-type cells, indicating a TP53-dependent apoptotic response. β-actin was used as a loading control. (b) Schematic illustration of a proposed model. Loss of miR-3619-5p in ESCC results in de-repression of PIM1, promoting downstream oncogenic signaling via AKT activation and inhibition of p21-mediated cell cycle arrest. Restoration of miR-3619-5p expression reverses this phenotype by suppressing PIM1 and downstream pathways, particularly in TP53-competent cells. (c) Restoration of plasma miR-3619-5p levels was shown to inhibit tumor growth in vivo. BALB/c nude mice (n = 4 per group) were inoculated with 2 × 10⁶ KYSE170 cells into the left lower flank. Once tumors were established (day 7), mice received subcutaneous injections of either miR-3619-5p mimic or miR-NC mimic (each 5 nmol) mixed with 20 µL AteloGene Local Use Quick Gelation into the right lower flank, away from the tumor site. Treatments were conducted weekly for three weeks following the initial injection. (d) Treatment with the miR-3619-5p mimic led to a marked reduction in tumor growth relative to the negative control mimic (P < 0.01, Student’s t-test). PIM1 expression in tumor tissues was evaluated using western blotting and immunohistochemistry. Tumors from mice treated with the miR-3619-5p mimic exhibited significantly lower PIM1 expression levels than those treated with the negative control mimic. β-actin was used as a loading control. Data are presented as mean ± SD from four independent experiments. (e) Plasma miR-3619-5p levels were compared between mice receiving the miR-3619-5p mimic and those treated with the negative control mimic. Mice treated with the miR-3619-5p mimic showed significantly higher plasma levels of miR-3619-5p as opposed to those treated with the negative control mimic (P < 0.01). Data are presented as mean ± SD from four independent experiments. (f) Analysis of blood parameters was executed to investigate potential side effects of miR-3619-5p treatment. No signs of organ dysfunction were observed, as indicated by normal levels of creatinine (Cre), aspartate aminotransferase (AST), alanine aminotransferase (ALT), amylase (AMY), and total bilirubin (T-Bil). Data are presented as mean ± SD from four independent experiments.