Tissue samples
Ninety-eight pairs of HCC tissues and adjacent noncancerous liver tissues were collected from HCC patients who underwent surgical resection at the Affiliated Hospital of Youjiang Medical University for Nationalities (Baise, China). All tissue samples were examined by two experienced pathologists. The clinicopathological features of these 98 cases were shown in Table S1. This study was conducted following the Declaration of Helsinki and written informed consents were obtained from all participants. Youjiang Medical University for Nationalities Institutional Review Board reviewed and approved this study.
Cell culture and treatment
Human HCC cell lines SUN-398 and SK-HEP-1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human HCC cell line HuH-7 and monocytic THP-1 cell was purchased from National Collection of Authenticated Cell Cultures of Chinese Academy of Sciences (Shanghai, China). SUN-398, SK-HEP-1, HuH-7, and THP-1 cells were cultured in RPMI 1640 medium, Eagle's Minimum Essential Medium, Dulbecco’s modified Eagle’s medium, and RPMI 1640 medium respectively with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). All cells were maintained at 37°C with 5% CO2. Where indicated, cells were treated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma Aldrich, St. Louis, MO, USA), or 50μM Tasquinimod (Selleck, Houston, TX, USA).
RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA was extracted using the RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China). After quantification by UV-visible spectrophotometry, the RNA was reversely transcribed into complementary DNA (cDNA) using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). qRT-PCR was undertaken using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequences were as follows; 5'-CAAAGAGCAGGTAAGATTCA-3' (sense) and 5'-TCACCAGCCTAGAAAGAGC-3' (antisense) for ZNNT1, 5'-CGAGTCAGAGTCACCATCC-3' (sense) and 5'-GCTCAGCCTGTACTTATCCAT-3' (antisense) for iNOS, 5'-CTCAGCCTCTTCTCCTTCCT-3' (sense) and 5'-CTGGTTATCTCTCAGCTCCAC-3' (antisense) for TNF-α, 5'-AGCAGAGTTTGGTCAGGG-3' (sense) and 5'-GGCTTTTTGTGGGGTTTTC-3' (antisense) for CD163, 5'-GACGAGGAGTCCATTACAC-3' (sense) and 5'-TTACTGTCGCAGGTATCATC-3' (anti-sense) for CD206, 5'-CTAAGAAGTTTCGCAGAC-3' (sense) and 5'-GACTATCAATCACATCGG-3' (antisense) for SPP1, 5'-TGGAGAAATAGTAGATGGC-3' (sense) and 5'-GGTGAGGAAGTAAAAACAG-3' (antisense) for MALAT1, 5'-TCATCAACACCTTCCACCAA-3' (sense) and 5'-TTAGCCTCGCCATCAGCA-3' (antisense) for S100A9, 5'-GTCGGAGTCAACGGATTTG-3' (sense) and 5'-TGGGTGGAATCATATTGGAA-3' (antisense) for GAPDH. GAPDH was used as endogenous control for mRNAs and lncRNAs. For miRNAs quantification, TaqMan Advanced miRNA Assays (Thermo Fisher Scientific, Austin, TX, USA) were carried out on StepOnePlus Real-Time PCR System.
Constructions of plasmids and stable cell lines
ZNNT1 full-length sequences were PCR-amplified using the PrimeSTAR Max DNA Polymerase (Takara, Dalian, China) and the primers 5'-GGGGTACCCCCCATCTCTACTAAAAATAC-3' (sense) and 5'-CGGGATCCTTCATCAAAGGAAATGATTTTT-3' (antisense), followed by being cloned into the Kpn I and BamH I sites of pcDNA3.1(+) (Invitrogen) to construct ZNNT1 overexpression plasmid pcDNA3.1-ZNNT1. Furthermore, the PCR product was also cloned into the Kpn I and BamH I sites of pSPT19 (Roche, Basel, Switzerland) to construct ZNNT1 in vitro transcription plasmid. The MS2-12× fragment was PCR-amplified as we previously described and cloned into the EcoR V and Xho I sites of pcDNA3.1 or pcDNA3.1-ZNNT1 to construct pcDNA3.1-MS2-12× or pcDNA3.1-ZNNT1-MS2-12× [51]. SPP1 3'UTR sequences were PCR-amplified using the primers 5'-CTAGCTAGCCTCACTTTGCATTTAGTCAAAAG-3' (sense) and 5'-CCGCTCGAGTTAATTGCTGGACAACCGTG-3' (antisense), followed by being cloned into the Nhe I and Xho I sites of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to construct pmirGLO-SPP1. ZNNT1 promoter sequences were PCR-amplified using the primers 5'-GGGGTACCCTCCCACATTCATCTTCA-3' (sense) and 5'-CCCAAGCTTCTCTTGTTGTCCAGGCTA-3' (antisense), followed by being cloned into the Kpn I and Hind III sites of pGL3-Basic (Promega) to construct ZNNT1 promoter reporter pGL3-ZNNT1-pro.
To construct HCC cells with ZNNT1 stable overexpression or control, pcDNA3.1-ZNNT1 or empty plasmid pcDNA3.1 was transfected into SNU-398 and SK-HEP-1 cells using Lipofectamine 3000 (Invitrogen), followed by being treated with 800 µg/ml neomycin for 4 weeks to select ZNNT1-overexpressed cells. Two shRNA lentiviruses targeting ZNNT1 were purchased from GenePharma (Shanghai, China). Scrambled non-targeting shRNA lentivirus was used as negative control (NC). The shRNA oligonucleotide sequences were 5'-GATCCGCTTCACTTTCTCCACTTATATTCAAGAGATATAAGTGGAGAAAGTGAAGCTTTTTTG-3' (sense) and 5'-AATTCAAAAAAGCTTCACTTTCTCCACTTATATCTCTTGAATATAAGTGGAGAAAGTGAAGCG-3' (antisense) for shRNA-ZNNT1-1, 5'-GATCCGCGACAACGTGATGAGAATAATTCAAGAGATTATTCTCATCACGTTGTCGCTTTTTTG-3' (sense) and 5'-AATTCAAAAAAGCGACAACGTGATGAGAATAATCTCTTGAATTATTCTCATCACGTTGTCGCG-3' (antisense) for shRNA-ZNNT1-2, 5'-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTG-3' (sense) and 5'-AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACG-3' (anti-sense) for shRNA-NC. To construct HCC cells with ZNNT1 stable knockdown or control, SNU-398 cells were infected with shRNA lentiviruses targeting ZNNT1 or scrambled non-targeting shRNA lentivirus, followed by being treated with 2 µg/ml puromycin for 4 weeks to select ZNNT1-kncoked down cells.
miRNAs, siRNAs, and transfection
miR-NC, miR-181a-5p, miR-181b-5p, miR-181c-5p, miR-181d-5p, miR-33a-5p and miR-33b-5p mimics (mirVana miRNA mimic) were purchased from Thermo Fisher Scientific. ON-TARGETplus Human SPP1 siRNA SMARTpool and DICER1 siRNA SMARTpool was purchased from Dharmacon (Cambridge, England). Transfection of miRNAs and siRNAs was performed using Lipofectamine 3000 (Invitrogen).
Cell growth, apoptosis, migration, and invasion assays
Cell growth was measured using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays as we previously described [51]. Cell apoptosis was measured using caspase-3 activity assay and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Caspase-3 activity assay was undertaken using the Caspase-3 Activity Assay Kit (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instruction. TUNEL assay was undertaken using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme). Cell migration and invasion were measured using transwell migration and invasion assays as we previously described [52].
Hepatic orthotopic xenografts
Six-week-old male BALB/C athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China) and maintained in Specific Pathogen Free condition. Luciferase-labelled SNU-398 cells were subcutaneously inoculated into the nude mice. When the subcutaneous xenografts grew to about 5mm in diameter, they were removed, cut into small pieces, and then transplanted into the liver of nude mice. At the 14th day after transplantation, the hepatic xenografts were detected by bioluminescence imaging using IVIS@ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA). The hepatic xenografts were resected and subjected to immunohistochemistry (IHC) staining using primary antibodies against PCNA (#13110, 1:4000; Cell Signaling Technology), Ki67 (#9027, 1:400, Cell Signaling Technology), or cleaved caspase-3 (#9664, 1:1000, Cell Signaling Technology). TUNEL assays were performed using the hepatic xenografts and the TUNEL BrightRed Apoptosis Detection Kit (Vazyme). The hepatic xenografts were also subjected to immunofluorescence (IF) staining using primary antibodies against CD206 (#24595, 1;200, Cell Signaling Technology). Youjiang Medical University for Nationalities Institutional Review Board reviewed and approved the use of mice.
Enzyme linked immunosorbent assay (ELISA)
OPN and S100A9 concentrations in cell culture supernatant were measured by ELISA using the Human Osteopontin ELISA Kit (ab100618, Abcam, Cambridge, MA, USA) and the FastScan Total S100A9 ELISA Kit (#80235, Cell Signaling Technology) following the manufacturers’ manuals.
RNA fluorescence in situ hybridization (FISH)
To detect the subcellular distribution of ZNNT1 in HCC cells, the ZNNT1 probes were purchased from Advanced Cell Diagnostics (Newark, CA, USA). RNA FISH was conducted using the probes and the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics) following the provided manual.
Isolation of cytoplasmic and nuclear RNA
Cytoplasmic and nuclear RNA were isolated and purified using the PARIS Kit (Thermo Fisher Scientific) following the provided protocol. Isolated RNA was detected using qRT-PCR as above described.
Dual-luciferase reporter assays
pmirGLO or pmirGLO-SPP1 was transfected or co-transfected with miRNA mimics into SNU-398 cells. pGL6 (Beyotime, Nantong, Jiangsu, China), pNFκB-luc (Beyotime), pGL3-basic, or pGL3-ZNNT1-pro was co-transfected with pRL-TK (Promega) into SNU-398 cells. pRL-TK encodes renilla luciferase and was employed as endogenous control. 48 hours after transfection, the firefly luciferase and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Results were calculated as the ratio of firefly luciferase activity to renilla luciferase activity.
Quantitative measurement of NF-κB activation
Nuclear proteins were extracted from SNU-398 cells with the Nuclear Extraction Kit (Ab113474, Abcam). NF-κB (p50 and p65) activation in these nuclear extracts were measured using the NF-κB p50 Transcription Factor Assay Kit (ab207217, Abcam) and NF-κB p65 Transcription Factor Assay Kit (ab133112, Abcam).
RNA immunoprecipitation (RIP)
MS2-based RIP assays were undertaken as we previously described [51]. Briefly, pcDNA3.1-MS2-12× or pcDNA3.1-ZNNT1-MS2-12× was co-transfected with pMS2-GFP (Addgene, Watertown, MA, USA) into SNU-398 cells. 48 hours after transfection, the cells were subjected to RIP assays using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA) and the primary antibody against GFP (11814460001, 5µg per reaction, Roche). The enrichment of miRNAs was detected using qRT-PCR as above described.
RNA pull-down
ZNNT1 was in vitro transcribed from pSPT19-ZNNT1 using the MEGAscript Kit (Thermo Fisher Scientific) and SP6 RNA polymerase, followed by being labeled with the Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo Fisher Scientific). SNU-398 cells were subjected to RNA pull-down assays using the above described labeled ZNNT1 and the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). The miRNAs present in the pull-down material were detected using qRT-PCR as above described.
Chromatin immunoprecipitation (ChIP)
SNU-398 cells were subjected to ChIP assays using the EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit (17-10086, Millipore) and the primary antibodies against p50 (#12540, Cell Signaling Technology) or p65 (#8242, Cell Signaling Technology). The enrichment of DNA was detected using qPCR and the primers 5'-GTGGCTCACGCCTGTAAT-3' (sense) and 5'-CCATGTCTGCCTAATTTTG-3' (antisense) for P1, 5'-GTAAAAGCTCTACAGATGT-3' (sense) and 5'-AAGGTAACTGGAAAGCAA-3' (antisense) for P2, 5'-CTAGAACCCAGGTCTGTG-3' (sense) and 5'-CTGTTAGATGGCAGCAATG-3' (antisense) for P3, 5'-TGGGTGTTGTTCTTGTATC-3' (sense) and 5'-GGAATGTATGTGGGTTTGT-3' (antisense) for P4. P4 was used as negative control, which did not occupy NF-κB binding site.
Bioinformatics analyses
Expression of lncRNAs in TCGA LIHC dataset was analyzed using the online in silico tool UALCAN (http://ualcan.path.uab.edu/analysis-lncRNA.html) [53]. The correlation between ZNNT1 expression and prognosis based on TCGA LIHC dataset was analyzed using the online in silico tool GEPIA (http://gepia.cancer-pku.cn/) [54]. miRNAs binding sites on ZNNT1 and SPP1 were predicted using the online in silico tool miRcode (http://www.mircode.org/) [55]. NF-κB binding sites on ZNNT1 promoter were predicted using the online in silico tool JASPAR (https://jaspar.genereg.net/) [56].
Statistical analyses
Statistical analyses were performed using the GraphPad Prism 6.0 Software. The detailed statistical methods were described in the figure and table legends. P < 0.05 was considered as statistically significant.