Fig 2: The effect of miR-124-1 on tumor formation in a nude mice xenograft modelNude mice were injected subcutaneously in opposite flanks with 5×106 control lentiviral vector-infected cells and miR-124-1 vector-infected cells. After 2 weeks, the mice were sacrificed when the tumors reached 1.0 cm in diameter and the subcutaneous tumors were cut into 1.0 mm3 sections, which were then inserted into the livers of another 10 nude mice. The mice were followed for 30 days and then killed by cervical dislocation. Livers and lungs were resected and imaged with a high-definition digital camera. Each group was composed of 5 mice. The weight of the tumors in the two groups was compared using the Student's t-test. A. control lentiviral vector groups; B. miR-124-1 vector groups; C. Sh-CASC3 groups.

Fig 3: miR-124-1 inhibits tumorigenesis via the p38-Akt-JNK pathwayA, B. The activity of the JNK pathway was evaluated in MHCC-LM3 (A) and Huh7 (B) cells transfected with si-CASC3. The activity of the JNK pathway in HCC cells infected with a control lentiviral vector or an miR-124-1expression lentiviral vector was assessed after transduction with a lentiviral vector encoding CASC3 (open reading frame without the 3′-UTR).

Fig 4: MicroRNA-124-1 downregulates CASC3 expression by directly targeting its 3′-UTRA. Putative miR-124-1 binding sequence in the 3′-UTR of CASC3 mRNA. A human CASC3 3′-UTR fragment containing the wild-type or mutant miR-124-1 binding sequence was cloned downstream of the luciferase reporter gene. HEK293 cells were cotransfected with miR-124 or a control vector and a luciferase reporter construct containing the wild-type or mutant CASC3 3′-UTR. Luciferase activity was assayed 48 h after transfection. Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. Data were normalized to the luciferase activity detected in cells transfected with the control vector, and the luciferase activity of the control vector was not significant. B. Effects of miR-124-1 overexpression on endogenous CASC3 expression as measured by real-time PCR and western blotting. β-actin served as the internal control. C. miR-124-1 and CASC3 protein expression were examined by in situ hybridization and immunohistochemistry, respectively.

Fig 5: CASC3 mediates the tumor-suppressive function of miR-124-1A. MHCC-LM3 and Huh7 cells were transfected by siRNA for CASC3. B. MHCC-LM3 and Huh7 transfectants stably expressing CASC3, miR-124-1 and control vector were generated using a lentiviral infection system. CASC3 protein expression levels were confirmed by western blotting (A-a, B-a). HCC cell growth was measured by CCK-8 (A-b, B-b) and colony formation assays (A-c, B-c). The results are presented as the mean±sd of values obtained in three independent experiments. Statistical significance was calculated using the Student's t-test. * P<0.05.