We included 12 cases of hepatoblastoma who were operated on and confirmed by pathology in 2015 to 2016. All the patients were operated on before receiving radiotherapy, chemotherapy, or immunotherapy. The sample consisted of 7 boys and 5 girls, and their average age was 1.85 ± 1.46. The adjacent tumor tissues from these patients were collected for controls. These hepatoblastomas were classified using the PRETEXT system 20. The expression level of miR-125b-5p was determined by ROC curves. Higher than the median was defined as high expression, lower was defined as low expression. All the patients were followed-up after treatment through December 2019. The survival time was calculated in months.
Cell proliferation assay
When the HepG2 and HuH-6 cells reached a logarithmic growth phase, we adjusted the cell concentration to approximately 1 × 104/well in 96-well plates after digestion. The following day, the cells were transfected and then cultured for 24 h, 48 h, and 72 h. We added 10 µl CCK-8 solution and incubated the cells in the incubator for 2 h. The absorbance at 450 nm of each well was measured to draw the cell growth curve.
Cell invasion and wound healing assays
Cell invasion and wound healing assays were performed referenced to 8.
Dual luciferase reporter assay
Wildtype (WT) or mut sequences of the NEAT1 or YES1 3' UTR were synthesized and cloned into the pGL3-reporter vector. HepG2 or HuH-6 cells were co transfected with wild type or mutant plasmids and mir-125b-5p mimic or mimic NC vectors. After 48 h, we used a luciferase detection kit (#D0010, Solarbio, Beijing, China) and a dual fluorescent enzyme reporter gene analysis system (Promega, Madison, WI, USA) to detect luciferase activity.
RNA pull down assay
The RNA pull-down experiment was performed as described previously 21. The M-280 streptavidin beads were purchased from Thermo Fisher (#11205D, Thermo Fisher Science, Waltham, Massachusetts, USA). Purified RNA was used to detect the expression of NEAT1.
In vivo experiment
24 male BALB/c nude mice (purchased from Guangdong Medical Laboratory Animal Center) aged 5–6 weeks were randomly divided into a mimic NC group and a miR-125b-5p mimic group. Then, 0.2 ml 1 × 106 transfected cells were injected subcutaneously into each mouse’s right armpit. We observed the mental diet and the body weight of the nude mice and recorded the tumor volume. When the tumor diameter reached 1.5 cm, the nude mice were killed, and the tumor was removed and weighed.
Total RNA was extracted from tissues and cells by TRIzol (#15596906, Thermo Fisher Science) and reversed transcribed into cDNA. The cDNA was used to apply qRT-PCR and U6 was set as the internal reference. Gene mRNAs were detected according to the TaqMan Gene Expression Assays protocol (#4331182, Thermo Fisher Science). GAPDH was used as the internal reference. The primer sequences are shown in Table 1. The relative expression levels were calculated using the 2-∆∆CT method.
Western blot analysis
Lysis buffer (#P0013, Beyotime, Shanghai, China) was used to extract the total protein. Then, 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) were prepared, and the proteins were separated by electrophoresis and electro-transferred to polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk for 1 h. We added the rabbit anti-YES1 primary antibody (#ab109744, 1:1000, Abcam, UK) and the anti-β-actin primary antibody (#ab8227, 1:5000, Abcam) separately and incubated them at 4 ℃ overnight. The secondary goat anti-rabbit IgG H&L antibody (#ab205718, 1:2000, Abcam) was added and incubated at 37 ℃ for 1 h. The results were shown by enhanced chemiluminescence (Thermo Fisher Science).
Data in our study were analyzed by SPSS 21.0 (SPSS, Inc, Chicago, IL, USA). Survival curve was drawn using Kaplan-Meier method and analyzed by Log-rank test. Data was shown in mean ± standard. Compared between two groups using t-test and multiple groups using ANOVA with Tukey’s test for post hoc test. P < 0.05 indicated the difference was statistically significant.