Collection of clinical samples
From 2016 to 2018, 50 paired fresh liver tumor and adjacent normal tissues were harvested in Zhongnan University Affiliated Xiangya Second Hospital. We snap-frozen these tissues at -80°C. All included subjects offered an informed consent and the research got approval from the Institutional Review Board of Zhongnan University Affiliated Xiangya Second Hospital.
HB611, HHCC, H-97, HuH-7, Li-7, and LO2 cell line were acquiredfrom the American Type Culture Collection (ATCC, Manassas VA, USA) and the Cell Bank of Chinese Academy of Science (Shanghai, China).The human immortalized liver LO2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA). We cultured HCC cells in Dulbecco's modified Eagle's medium (DMEM) with high glucose concentration (25 mM), 1% of penicillin-streptomycin, 10% of fetal bovine serum (FBS), and maintained them in a 5% CO2 humidified incubator.
From BioVector NTCC Inc., Guangzhou, China, we purchased plasmid vector PLKO.1-puro. Through chemical synthesis, we designed the related TMEM220-AS1 short hairpin RNA(shRNA) sequences and its negative control. These synthetic related sequences were inserted into PLKO.1-puro vector. From RIBOBIO, Guangzhou, China, we purchased the microRNA mimics and its inhibitor. We cultured the cells for 24 h before transfection. Then, using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA), we transfected the cells transiently with corresponding vector, during which we referred to the manufacturer’s instructions. We harvested cells that were transfected with corresponding vector and operated quantitative real-time polymerase chain reaction(qRT-PCR) after 48 h. Each experiment was carried out three times.
RNA isolation and qRT-PCR
With TRIzol reagent (Invitrogen, Carlsbad, CA, USA), total RNA from cells samples was extracted. Referring to the manufacturer's instructions, RNA was reverse transcribed via PrimerScript RT-PCR kit (Takara). The RNA level was determined using qRT-PCR analysis via TaqMan MicroRNA Assay Kit (Applied Biosystems). We measured their relative level of predicted targets in triplicate on an ABI 7500 real-time PCR machine (Applied Biosystems). U6 or β-actin was used as reference gene to normalize the expression levels of miRNA or mRNA. The delta Ct method was used to calculate the relative expression. Primers using in this study were shown in Supplementary Table 1.
Cell proliferation, invasion, cycle and apoptosis detection
These methods are shown in the Supplementary Methods.
Western blot evaluation
We prepared total cell lysates in a 1× sodium dodecyl sulfate buffer. Afterwards, we separated protein with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and onto nitrocellulose membranes total protein was transferred. Then, with 5% non-fat milk the membrane was blocked and with primary antibodies it was cultured at 4 °C overnight. After incubating with antibodies which is specific for BMI-1, E-cadherin, vimentin andsnail, the blots shared incubation with goat anti-rabbit secondary antibody (Abcam, Hong Kong, China) and via enhancedchemiluminescence they were visualized. Each experiment was carried out three times.
RNA fluorescent in situ hybridization (FISH)
With Ribo™ Fluorescent in Situ Hybridization Kit (Ribobio Company, China), FISH assay was implemented. TMEM220-AS1 probe was labeled with FITCfluorescent dye, the design and synthesis were implemented by Ribobio Company. RNA FISH were implemented utilizing fluorescent in situ hybridization kit (RiboBio)following the manufacturer’s instructions. With a confocal laser-scanning microscope (Leica, Germany), fluorescence detection was implemented.
Following the product specifications, we adopted the EZ-magna RIP kit (Millipore, United States) to carry out the RIP assay. We collected the HB611 and HuH-7 cells and lysed them in a full RIP lysis buffer. We incubated cell extracts with RIP buffer which contains magnetic beads conjugated to human AGO2 antibodies (ab32381, abcam, Cambridge, United Kingdom), and we used the IgG antibody (ab6702, abcam, Cambridge, United Kingdom) as controls. We incubated the samples with protease K and we oscillated them for digesting the protein and isolating the immunoprecipitated RNA. Via a NanoDrop spectrophotometer, we measured the concentration of RNA and performed real-time PCR analysis of the purified RNA.
Dual luciferase reporter gene assay
First, we manufactured TMEM220-AS1 Wt and MAGI1 Wt. In brief, TMEM220-AS1 and MAGI1 fragments containing miR-484 bindingsites were amplified by PCR and cloned into the downstreamof luciferase reporter gene in pmirGLO vector, which werenamed TMEM220-AS1Wt and MAGI1 Wt.Usingthe Quickchange XL Site-Directed Mutagenesis Kit (Stratagene), we generated TMEM220-AS1 Mut and MAGI1 Mut (mutations within the binding sites). With TMEM220-AS1 Wt or TMEM220-AS1 Mut as well as MAGI1 Wt or MAGI1 Mut, MiR-NC and miR-484 mimics were co-transfected into HEK293T cells, respectively. Cells were harvested after 48 h of transfection and we utilized the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) to perform luciferase assay.
To detect Ki67 staining in tumor tissue samples, sections of 5 μm were cut. After series of dewaxing and hydration, the slides were rinsed in PBS, followed by boiling in 10 mM of sodium citrate when pH was 6. Then, the slides were incubated in 3% of H2O2 for 25 minutes to remove the horseradish peroxidase. The slides were blocked with 10% of BSA after washing with 1 × PBS for three times, followed by incubation for primary anti-Ki67 antibody (ab92742) at 4°C overnight. The slides were incubated with a second antibody labeled by HRP (rabbit) at room temperature for 45 minutes and with 3,3′-diaminobenzidine tetrahydrochloride (DAB) the immunoreactivity was visualized the next day. Finally, the slides were dehydrated and mounted with neutral gum.
Tumor xenograft implantation in nude mice
We divided six-week-old nude mice into two groups (3 mice per group) randomly, cultured them with continuous access to sterile food and water in pathogen-free sterile conditions. To establish the HCC xenograft model, we subcutaneously injected HuH-7 cells into nude mice. We monitored tumor growth every week and calculated it as equation: Volume = (Length) × (Width)2/2.The study was approved by the Ethics Committee of Zhongnan University Affiliated Xiangya Second Hospital,, and experiments were performed following the NIH guidelines on animal welfare.
For normally distributed data with equal variance, the difference was evaluated by 2-tailed Student t test (2‐group comparisons) or ANOVA followed by the post hoc Bonferroni test (multigroup comparisons) as appropriate. For nonnormally distributed data or data with unequal variances, the difference was evaluated by a nonparametric Mann-Whitney U test (2‐group comparisons) or the Kruskal-Wallis test followed by the post hoc Bonferroni test (multigroup comparisons). The standard we used to determine statistical significance was that P<0.05. We carried out all tests via SPSS 22.0 (SPSS, Chicago, IL, USA).