Fig 2: (A) The miR-124-3p level was downregulated in tumorous tissues with -?CT value of –9.7564 ± 2.7746 from HCC patients compared with that of –7.6357 ± 3.1093 from paracancerous normal liver tissue with statistical significance (P = 0.0188) by unpaired Student t-test analysis and presented as mean ± SD of 23 cases (n = 23). (B) Compared with LO2 cells, the miR-124-3p level was significantly reduced by 74% in HCCLM3 (P = 0.0130) and 75% in Huh7 cells (P = 0.0085) by unpaired Student t-test. (C) miR-124-3p downregulation and CRKL upregulation in HCC tumorous tissues were negatively correlated (R2 = 0.8954, P = 0.0001) by Spearman correlation test. (D) CRKL expressions at the mRNA level were increased by 23.844 ± 10.011 (P = 0.0164) and 20.821 ± 8.901 (P = 0.0178) folds, and (E) those at the protein level were elevated by 1.31 ± 0.165 (P = 0.0093) and 1.816 ± 0.321 (P = 0.0314) folds in HCCLM3 and Huh7 cells compared to LO2 cells. The data were presented as mean ± SD (standard deviation) of triplicate experiments for each assay. *, **, and *** refer to P-values below 0.05, 0.01, and 0.001.

Fig 3: miR-124-3p binds to 3'-UTR of CRKL. (A) Putative sites for binding of CRKL-3'-UTR to miR-124-3p. (B) Dual luciferase activity assay of miR-124-3p binding to 3'-UTR of CRKL. The co-transfection of 293T cells was done through miR-NC/miR-124-3p mimics and psiCHECK-2-CRKL-3'-UTR-WT/psiCHECK-2-CRKL-3'-UTR-MUT for 48 h. The data of triplicate experiments were presented as mean ± SD and analyzed by unpaired Student t-test for statistical differences. ns refers to non-statistical significance. * and *** refer to P-values below 0.05 and 0.001.

Fig 4: The schematic illustration of the miR-124-3p-CRKL axis in HCC malignancy. CRKL downregulation by miR-124-3p leads to suppressed C-JUN expression and decreased proliferations of HCC cells, whereas the CRKL suppression by miR-124-3p results in increased BAX levels and reduced BCL-2 and concomitant increased apoptosis of HCC cells. By binding to 3'-UTR of CRKL, miR-124-3p downregulates CRKL expression, which inhibits the activity of the RAF/MEK/ERK1/2 pathway and results in decreased EMT, ultimately leading to inhibited migration, invasion, and metastasis of HCC cells.