1. Data collection
Data on somatic mutation profiles, RNA expression profiles and clinical characteristics of HCC patients were obtained from The Cancer Genome Atlas (TCGA) database. Samples with missing information on lncRNA and mRNA expression profiles, clinical characteristics or somatic mutation were excluded. A total of 343 patients with HCC were enrolled. Table S1 listed all the websites that were accessed for this study. The GSE14520[19] and GSE76427[20] datasets were downloaded from the Gene Expression Omnibus (GEO) database.
2. Tissue samples and cell culture
Tumor and adjacent nontumor tissues from 80 HCC patients (age range: 15–91) were obtained from Zhongnan Hospital of Wuhan University and collected by surgery. Tissues collection dates ranged from April 2014 to May 2020. All patients provided written informed consent. This study conformed to the Declaration of Helsinki. The tissue samples were collected with the approval of the ethics committee of Zhongnan Hospital (KELUN2020100). Five liver cancer cell lines and a normal liver cell line were used: HepG2, Li-7, HCC-LM3, Huh-7, Hep3B and THLE-3. HepG2 is a human liver cancer cell line. All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or minimum essential medium (MEM) containing 10% foetal bovine serum (FBS, Gibco, USA). And they were obtained from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in a humidified incubator (5% CO2, 37°C). All cell lines were authenticated using STR (short tandem repeat) profiling.
3. DNA damage analysis
To detect DNA damage, an alkaline comet assay was performed using a reagent kit (ELK Biotechnology, Wuhan, China) according to the manufacturer's protocol. ImageJ-OpenComet software (ImageJ, v.1.53e, National Institutes of Health, USA) was employed to examine the comet experiment data. The extent of DNA damage was expressed as the tail moment, which corresponded to the fraction of the DNA in the tail of the comet.
4. RNA extraction and quantitative PCR (qPCR) analysis
Total RNA was extracted using the TRIzol method according to the manufacturer’s instructions (Vazyme, Nanjing, China). A standard SYBR Green PCR kit (Vazyme) was employed to perform qPCR as described previously[21]. Glyceraldehye-3-phosphate dehydrogenase (GAPDH) served as an endogenous control. Relative expression was calculated using the comparative cycle threshold (Ct) method (2 − ΔΔCt). Table S3 listed thermocycling conditions.
5. Plasmid, microRNA, and short hairpin RNA (shRNA) transfection
For transfection, HCC cells were plated in T25 flasks, one night prior to transfection. When the cell confluence reached 80%, the cells were transfected with ZFPM2-AS1 overexpression plasmid and a corresponding empty plasmid using Lipofectamine 3000 (Thermo Fisher, NY, USA). According to the manufacturers protocol, 5 µg of plasmid, 10 µL of P3000, 10 µL of Lipo3000, and 500 µL of Opti-MEM Medium (Gibco, USA) were mixed gently and then incubated at room temperature (RT) for 15 min. The mixture was then added to HCC cells slowly. After incubating at 37°C for 12h, the culture medium was subsequently replaced with fresh DMEM supplemented with 10% FBS. HCC cells were harvested at 48h after plasmid transfection. Total RNA was extracted and used to access transfection efficiency via qPCR. ZFPM2-AS1-wild-type (WT)/mutant (MUT) plasmids and XRCC4-WT/MUT plasmids were constructed by Genomeditech (Shanghai, China). shRNA, miR-3065-5p primers and miRNA mimic/inhibitor were designed by Wuhan Qingke Biotechnology Co., Ltd. (Wuhan, Hubei, China). HCC-LM3 and Huh7 cells were transfected with shRNA lentiviral plasmid (pLKO.1-purp). Puromycin (2 µg/ml) was used for sorting positive shRNA cells. TSnanofect (Wuhan Qingke) was used for transfection of miR-3065-5p mimic/inhibitor. All PCR primer, shRNA, and miRNA sequences are shown in Table S2.
6. Western blotting (WB)
WB was performed using standard methods as described previously [22]. The adherent cells were washed twice with cold phosphate buffer solution (PBS), trypsinized to collect the cell precipitate and resuspended in PBS. Cell suspension was then centrifuged at 4℃ and 500 g for 3 min and cell precipitate was harvested. Cells were lysed for 15 min on ice in RIPA buffer (Beyotime, Shanghai, China). Cell lysates were centrifuged at 14,000 g for 10 min to collect supernatant. Protein samples (supernatant) were denatured at 95°C for 10 min. Quantification of protein levels was performed using the BCA kit (Beyotime). 50ug protein was separated by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred into 0.45µm polyvinylidene difluoride (PVDF) membranes by electro-transblotting, under 300 mA for 1h. Electro‐transblotting was performed by using transblotting buffer (192 mM Glycine, 25 mM TrisBase, 20% methanol). Then 5% skim milk was used to block PVDF membranes for 1 hour at RT. The membranes were incubated with primary antibodies overnight at 4°C. They were then washed thrice with tris-buffered saline/Tween 20 (TBST) for 30 min and incubated with secondary antibody for 1 hour at RT. Finally, the membranes were washed thrice with TBST for 30 min again. The antibodies and antibody dilution ratio used in this experiment are listed in Table S4. Protein expression was identified using ECL reagents (Thermo Fisher) and an imaging system (BioRad). WB band integrated density was quantified using ImageJ software. GAPDH served as loading control for normalization.
7. In vivo experiments
Male nude mice (BALB/c Nude mice, 5 weeks old) with body weights of 18-20g were housed under specific pathogen free (SPF) conditions and ear notched for identification. For the xenograft model, negative control or HCC-LM3 cells with stable ZFPM2-AS1 knockdown (0.1ml, 2×106 cells/mouse, 5 mice/group) were subcutaneously injected into the axillary tissue. To establish the lung metastasis model, negative control or HCC-LM3 cells with stable ZFPM2-AS1 knockdown (0.2ml, 1×106 cells/mouse, 5 mice/group) were intravenously injected into nude mouse tails vein. Tumor volume (mm3) of each mouse was measured by Vernier caliper every 3 days and calculated as tumor volume = tumor length * width2/2. Mice were euthanized following a 21-day xenograft study and a 50-day lung metastasis research, respectively. The following humane endpoints were established: tumor diameter > 2.0 cm, weight loss > 25% and poor overall condition. None of the mice reached the humane endpoints in this study. To reduce suffering, mice were anesthetized with 4% isoflurane inhaled anesthesia and anesthesia was maintained with 1% inhaled isoflurane. Mice were then euthanized by cervical dislocation rapidly. Death verification included cardiac and respiratory arrest, lack of reflection and changes in mucosal color.
8. Chromosomal aberration assay
Stable ZFPM2-AS1 knockdown HCC cells were washed and treated with a demecolcine solution (D1925, Sigma, St Louis, MO) for 2 h. The cells were collected and suspended in a hypotonic solution (0.075 M KCl) for 20 min at room temperature. The cells were then fixed in cold Carnoy's fixative (methanol:acetic acid, 3:1), placed on slides for staining with Giemsa (G146–10, Fisher, USA) and examined.
9. RNA pull down
The interaction between ZFPM2-AS1 and miR-3065-5p was validated using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, CA, United States). The miR-3065-5p-Wt/Mut and control were synthesized and biotin labelled. cell lysates extracted from HCC cells were cultivated after 2 d and mixed with the biotinylated probe and magnetic beads for 1h. We used qPCR to the miR-3065-5p targets from the pull-down reaction mixture.
10. Immunohistochemistry
For immunohistochemical staining, 4 µm-thick slices of mouse tumor tissues were used. Paraffin sections were placed in an oven at 65°C for 2h, dewaxed, and washed three times with PBS (5 min each time). Primary antibody incubation was overnight at 4°C and secondary antibody incubation 50min at 37°C. After labeling proteins, the staining was visualized using diaminobenzidine (DAB) staining followed by hematoxylin counterstaining. Antibody information and dilution ratio were listed in Table S4. A microscope (IX51, Olympus) scanned the slides at 400X or 200X after alcohol dehydration. The mean optical density (MOD) value of IHC images were determined using the IHC Toolbox plugin for ImageJ.
11. Luciferase reporter assay
A luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega) with the pmirGLO dual-luciferase miRNA target expression vector (Promega). HCC-LM3 cells were seeded in a 96-well plate and cultured overnight. They were cotransfected with miR-3065–5p mimic and ZFPM2-AS1 3′ untranslated region (UTR), XRCC4 3′ UTR, ZFPM2-AS1 3′ UTR-mut or XRCC4 3′ UTR-mut. Luciferase activities were measured using a dual-luciferase reporter gene assay system (Promega) and normalised to Renilla activity.
12. Statistical analyses
All statistical analyses were performed using SPSS 25.0 software, GraphPad Prism 8.0, and R software (R version 3.5.2). Quantitation of cell number, colony number and wound healing closure were performed with ImageJ software. Survival analysis was performed by log rank test. The Renyi test was performed to generate the P-values when survival curves crossed over. All experiments were performed three times, and P < 0.05 was considered statistically significant.
Other experimental procedures are detailed in the Supplementary Materials and Methods (Doc. S1).