Cell culture and transfection
The human HCC cell line Hep3B and 293T cells were purchased from ATCC. The normal human liver cell line MIHA was purchased from YaJi Biological (Shanghai, China). HCC cells Bel-7402 and Bel-7404 were obtained from Beina Biological (Beijing, China) in 2017. The SMMC-7721 was bought from Fenghui Biotechnologies Inc (Hunan, China). MIHA, SMMC-7721, Bel-7402, and Bel-7404 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Invitrogen). Hep3B and 293T were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) containing 10% FBS. All cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Dimethyl sulfoxide was purchased from Sigma-Aldrich (St. Louis, MO).
Scrambled siRNA of URHC (siRNA-Con) and URHC siRNAs were purchased from GenePharma (Shanghai, China). miR-5007-3p inhibitor, inhibitor negative control (NC inhibitor), miR-5007-3p mimic and NC mimic were also purchased from GenePharma (Shanghai, China). Full-length URHC cDNA was subcloned into GV230 lentiviruses (Genechem, Shanghai, China) and infected into Bel-7404 and Hep3B cells to generate URHC-overexpressing cells. Lipofectamine 2000 Reagents (Invitrogen Co, USA) were used for cell transfections.
Human tissue samples
We obtained 26 pairs of primary HCC and adjacent non-tumor tissues from patients undergoing surgery at Xijing Hospital, the Fourth Military Medical University; all diagnoses were based on a biopsy. These tissues were immediately frozen in liquid nitrogen after surgical resection. All patients provided written informed consent, and these studies were approved by the Ethics Review Committees of Xijing Hospital.
Reverse transcription and quantitative real-time PCR.
Total RNA was prepared from the indicated cells using TRIzol reagent (Invitrogen) based on the manufacturer’s instructions. One microgram of total RNA was converted to cDNA using PrimeScript® RT Master Mix Perfect Real-Time (Takara, Inc). SYBR Premix EX Taq II (TaKaRa) was used based on the manufacturer’s instructions under the following conditions: 95°C for 2 min, 95°C for 15 s, and 60°C for 60 s for 40 cycles. miR-5007-3p expression levels were quantified using MicroRNA First-Strand Synthesis and miRNA Quantitation Kits including U6 control primer (Takara) based on the manufacturer’s instructions. The results were analyzed based on the 2−ΔΔCq formula. The primers used for RT-qPCR were as follows: b-actin: sense 5′-CTGGAACGGTGAAGGTGACA-3′, and antisense 5′- CGGCCACATTGTGAACTTTG-3′; URHC: sense 5′- TGTTTATGTGAGAGGAGAAAGGAAG-3′; and antisense 5′- CACTAGAGGTCTGCAAATAAAGTGA-3′; DNAJB9: sense 5′- TAGGCACACACCACCACATC-3′; and antisense 5′- CTTTGGGAGGCCAAGGTAGG-3′; and miR-5007-3p 5′-CCATATGAACCAAACTCTAATA-3′. The internal control genes were b-actin for URHC, DNAJB9, and U6 snRNA for miR-5007-3p.
Fluorescence in situ hybridization
SMMC-7721 and Hep3B cells were plated to achieve 70% confluency for staining. Frozen sections of HCC tissues were treated with 4% DNase/RNase-free paraformaldehyde. After fixation, the sections were treated with Proteinase K (20 mg/ml) for 5 min, and then washed three times with phosphate-buffered saline (PBS). The cells were incubated with a prehybridization solution for 1 h at 37°C. Then, the prehybridization solution was removed, and the cells were covered with a URHC probe (8 ng/μl) hybridization solution overnight in a 37°C incubator. The slices were washed three times using Wash Buffer Solution. The cells were extensively washed three times using Wash Buffer Solution, counterstained with DAPI, and mounted with an anti-fade reagent (Invitrogen). The URHC probe for fluorescence in situ hybridization (FISH) was: 5′-GFP-AGTACATACTCACTACACTAGAGGTCTGCA-GFP-3′ (Servicebio, China). The mir-5007-3p probe for FISH was: 5′-Cy3-ATTAGAGTTTGGTTCATATGAT-Cy3-3′ (Servicebio).
CCK-8 and colony formation assays
The CCK-8 assay was used to assess HCC cell proliferation. HCC cells were seeded in a 96-well plate at a density of 2 × 103 cells per well. Cell proliferation was measured at 24, 48, 72, and 96 h. CCK-8 solution (10mL) was added to each well, and incubated for 1 h at 37°C, in a 5% CO2 incubator. After incubation, the optical density (OD) 450 value was read using an Epoch Spetrophotometer (USA). Experiments were repeated at least three times.
The colony formation assay was performed to measure the capacity of cell proliferation. SMMC-7721 and Hep3B cells (300 cells/well) were seeded in a six-well plate and cultured for 10 days. Colonies were then fixed with methanol for 15 min and stained for 10 min with 0.5% crystal violet. Then, colonies were imaged with a Phone and analyzed by ImageJ software. The assays were repeated at least three times.
5-Ethynyl-2′-deoxyuridine (EdU) assay
Cell proliferation was also determined by the 5-ethynyl-2′-deoxyuridine assay using an Apollo®488 EdU Kit (RIBOBIO, Guangzhou, China). The EdU assay was performed based on the manufacturer’s instructions. The cells were then visualized under a fluorescence microscope (20 × 10). To assess cell proliferation, the ratio of EdU-stained cells (with red fluorescence) to Hoechst-stained cells (with blue fluorescence) was calculated. Experiments were repeated at least three times.
Luciferase reporter assay
The luciferase reporter assay was performed in 293T cells. The 293T cells were seeded in a six-well plate and cotransfected with luciferase reporter constructs encoding the wild-type 3′-UTR region of DNAJB9 or URHC (DNAJB9/URHC-WT-3′-UTR) or a mutated 3′-UTR region of DNAJB9 or URHC 3′-UTR region (DNAJB9/URHC-MUT-3′-UTR) (RIBOBIO, Guangzhou, P.R. China) and miR-5007-3p mimic or mi-NC using Lipofectamine 2000 (Invitrogen). After 48 h of incubation, the cells were washed with PBS and lysed with Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (Promega) based on the manufacturer’s protocol.
Western blot assay
Cells were lysed using RAPI buffer (Beytime, China). Total proteins were quantified using the BCA Protein Assay Kit (Beytime, China). Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membranes (Milipore, Inc). The membranes were washed three times for 10 mins with Tris-buffered saline-0.5% Tween 20 (TBS-T) and blocked with 5% nonfat milk-TBS-T at room temperature for 1 h. Subsequently, they were incubated with primary antibodies (anti-DNAJB9 and anti-GAPDH obtained from Abcam and Cell Signaling Technology, respectively) at 4°C overnight. Following, incubation with corresponding secondary antibodies, the signal were detected with ECL Western Blotting Detection Reagents (Milipore, Inc). GAPDH was used as an endogenous reference.
Immunohistochemical (IHC) assay
DNAJB9 and Ki67 were detected in xenograft tumor specimens of nude mice, which were fixed with paraformaldehyde, embedded with paraffin and cut into 4 mm sections. The subsequent steps accomplished with the biotin-streptavidin peroxidase method (SPlink Detection Kit, ZSGB-Bio, Beijing, China) were followed based on the manufacturer’s instructions. Briefly, paraffin-embedded samples were deparaffinized, dehydrated in a graded series of ethanol and blocked with endogenous peroxidase for 10 min, and incubated in goat serum for 30 min. The slides were incubated with the corresponding primary antibodies at 4°C overnight. Then, the slides were washed with PBS, incubated with biotinylated goat anti-rabbit IgG, and then incubated with horseradish peroxidase (HRP) conjugated streptomycin. Diaminobenzidine (ZSGB-Bio) was added to the slides for chromogenic reaction. The slides were observed with an optical microscope (Olympus, Tokyo, Japan).
In vivo proliferation assay
Six week-old male BALB/c nude mice were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). Animal studies were approved by the Institutional Animal Care and Use Committee of Fourth Military Medical University. The mice were housed in pathogen-free conditions. To evaluate the in vivo tumorigenic effects, mice were randomly divided into the following two groups with five mice in each group: the control group and the LncRNA URHC group. siRNA-URHC and URHC-NC cells (1 × 107) were inoculated subcutaneously in the right flank of nude mice. Tumors were measured every 7 days, and tumor volumes were calculated. Five weeks after HCC cell inoculation, the mice were sacrificed, and the tumors were collected.
All experimental data were reported as means ± experimental standard deviations (SD). Student’s t-test was used to determine the significance of the results (significance: *P < 0.05; **P < 0.01, ***P < 0.001).