Cells and clinical tissues
Experiments were performed using one normal hepatocyte (HL-7702), three hepatocellular carcinoma cell lines (HepG2, Huh-7 and QGY-7703). All cell lines were purchased from the American Type Culture Collection (ATCC) cell bank and were frozen and recovered in Key Laboratory of Sichuan Academy of Medical Sciences & Sichuan Province People’s Hospital (Chengdu, China) from Augst 2017. No mycoplasma contamination was existed in the cultured cells. Cell lines used in this article was required ethics approval. HL-7702, HepG2 and Huh-7 were cultured in DMEM and QGY-7703 was cultured in RPMI-1640 containing 10% fetal calf serum 100 μg/mL penicillin and 100 units/mL streptomycin at 37 °C, 5% CO2. Primary tumor tissue and paired non-tumor tissue samples were obtained from Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital. Twenty patients with HCC were invited to participated in this research. All patients had not received treatment before surgery. Written informed consent has been obtained from each subject and the research method has been approved by the ethics committee of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital.
Plasmid construction and cell transfection
The full-length of human lncRNA CRNDE and POU2F1 was synthesized by Invitrogen (Shanghai, China) and cloned into the vector pmirGLO or pCDNA3 (Clontech Laboratories, Inc., San Francisco, CA). miR-539-5p mimics, inhibitor and negative control (NC) were synthesized by GenePharma (Shanghai, China). The cells were seeded in 6-well plates overnight (1 × 106/well), and then lncRNA (GenePharma), miRNA or plasmid were transfected with liposome Lipofectamine 2000 (Invitrogen) according to the instructions. The cells were changed to normal culture solution 6 h after transfection, 48 h and then proceed to the next test.
Cell viability was measured by MTT assay. Briefly, after transfection of cells 48 h, 96-well plates were seeded at a cell density of 4 × 103 cells/well, transfected at a density of 60–70% after adherence, and 20 μl MTT (5 mg/ml; Sigma-Aldrich) was added at 24 h, 48 h, and 72 h after transfection. After incubating for 4–6 h in a 37 °C incubator of % CO2, the culture solution was discarded, 100 μl of DMSO was added for 10 min, and OD450 was detected by a microplate reader. Three independent replicate experiments were performed.
QRT-PCR detection of cells 48 h after transfection. Total RNA extraction, RNA reverse transcription and qRT-PCR reaction were performed according to the kit instructions. The qRT-PCR reaction system was thoroughly mixed in a 96-well plate, centrifuged at 3000 r/min for 3 min, and then subjected to fluorescence quantitative PCR instrument for reaction. Each sample and the detected gene were set to 3 replicates. The qRT-PCR reaction was carried out with ACTB as the internal reference gene. The reaction conditions were: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 2 s, annealing at 60 °C for 20 s, and extension at 72 °C for 1 min for a total of 40 cycles. The ratio of the expression of the target molecule in the experimental group and the control group was expressed as a multiple = 2-ΔΔCt. Each experiment was repeated 3 times independently.
Forty-eight h after transfection of cells, trypsinize and collect the cells of each group, centrifuge at 700 r/min for 5 min, resuspend the cells in PBS for 2 times, add the cell lysate RIPA, thoroughly blow the cell pellet, and lyse on ice bath for 30 min. After centrifugation at 12000 r/min for 10 min, the supernatant was collected, and an appropriate amount of protein loading buffer was added in proportion, and boiled for 10 min to obtain a total protein sample of the cells. A 10% concentration of sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was prepared for loading, electrophoresis, and electroporation. After the electroporation, the PVDF membrane containing the target protein was placed in a TBST-containing 5% skim milk containing gel. In the solution, the cells were blocked at room temperature for 2 h, the primary antibody Bax(1:1000), Bcl-2(1:1000), Cleaved Caspase 3 (1:500), Caspase 3(1:500), E-cadherin(1:1000), Vimentin(1:1000), POU2F1(1:1000), p-IKB(1:500), IKB(1:1000), p-AKT(1:500), AKT(1:1000), p-ERK(1:300), ERK(1:1000), NF-KB(1:1000), GAPDH(1:2000) were incubated at 4 °C overnight, the TBST was eluted 3 times for 5 min, the second antibody was incubated for 2 h at room temperature, and then eluted 3 times with TBST for 5 min each time, and finally developed.
Transwell migration and invasion assay
After transfection of cells 48 h, the cells were inoculated into a 24-well plate. 8 × 104 cells were plated in each chamber. In the 24-well plate, medium without serum was added and 20% serum was added into each chamber. The Transwell chamber was taken out, washed once and fixed in anhydrous methanol for 20 min; After the fixation, the chamber was stained with 0.5% methyl violet dye solution for 15 min. Five different fields of view were randomly selected, photographed, and the number of cells migrated according to the records were counted, and finally statistical analysis was performed.
After transfection of cells 48 h, frozen sections of liver tissue (5 μm) were subjected to FCM staining using a commercially available kit (Beyotime, China).
After transfection of cells 48 h, the cells climbed on the slides were washed with PBS. Paraformaldehyde fixed cells and PBS washed. 0.2% Trinton X incubated cell. After wash with PBS, cell was incubated with POU2F1 antibody 4 °C overnight. Next day, cell was washed with 0.2% Trinton X-100 PBS wash and incubated with secondary antibody in room temperature dark. Slide was covered with tablet containing DAPI and photographed by fluorescence microscopy.
Luciferase reporter assay
We cloned the lncRNA CRNDE fragment containing the miR-539-5p target site or mutation site by PCR and then cloned into pmirGlO Dual-luciferase Vector (Promega) to construct the reporter vectors, lncRNA CRNDE Wt and lncRNA CRNDE Mut. The POU2F1 fragment containing the miR-539-5p target site or mutation site was amplified by PCR and cloned into pmirGlO Dual-luciferase miRNA Target Expression Vector (Promega) to construct the reporter vector POU2F1 3’UTR Wt and POU2F1 3 ‘UTR Mut. Cells were subjected to miR-539-5p mimic/inhibitors and lncRNA CRNDE wild type/mutant cotransformation or miR-539-5p mimic/inhibitors and POU2F1 Wt or Mut using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Fluorescence activity was measured using a dual luciferase assay as described after 48 h.
lncRNA CRNDE sequence was labelled in Biotin and performed RNA pulldown using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA). qRT-PCR assay was used to detect lncRNA CRNDE enrichment in the RNA fraction.
Subcutaneous xenografts in nude mice
The cells were seeded in T75 culture bottle and transfected with liposome Lipofectamine 2000 (Invitrogen) according to the instructions. After transfection, G418 was used the screen the cells which stably exoressed lncRNA CRNDE. Nude mice was usd to purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Stably expressed cells (2 × 106 cells per mice) was injected into subcutaneous of right scapula. Four weeks later, tissues was taken off, measured tumor size and weight.
All analyses were carried out using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Paired Student’s t-test, Mann-Whitney U test, and one-way analysis of variance were used in the study to evaluate statistical differences. p < 0.05 was considered statistically significant.