ZNNT1/osteopontin/S100A9 feedback loop promote hepatocellular carcinoma progression via mediating crosstalk between hepatocellular carcinoma cells and macrophages

Background

Primary liver cancer is still one of the most common malignancies and the second leading cause of cancer-related death worldwide [1]. Hepatocellular carcinoma (HCC) is the major histological subtype of liver cancer and accounts for over 80% [2]. HCC has extremely poor prognosis, due to the rapid progression of HCC and insensitivity to routine chemotherapy and molecule-targeted therapy [3]. Thus, further uncovering the mechanisms driving HCC development is urgently needed [4, 5].

Accumulating evidences have revealed the importance of tumour microenvironment (TME) in tumour initiation and progression [6–12]. Macrophages constitute the critical components of TME [13–15]. Tumour infiltrated macrophages, also known as tumour-associated macrophages (TAMs) frequently presented oncogenic roles in various malignancies, including HCC [16–18]. The increased infiltration of TAMs is associated with worse survival in HCC [19]. Macrophages are routinely divided into the classical-activated macrophage (M1) and alternative-activated macrophage (M2) [20, 21]. M1 macrophages often show tumour suppressive roles, and while M2 macrophages often show oncogenic roles [22–24]. Most TAMs present M2 phenotype [25, 26]. The mechanisms underlying the oncogenic roles of TAMs have been preliminarily investigated [27]. S100 calcium-binding protein A9 (S100A9) was reported to be secreted by TAMs and exert oncogenic effects on HCC cells [28]. The recruitment, polarization, and roles of TAMs were also modulated by factors deriving from cancer cells and TME [29]. Osteopontin (OPN) is one of the factors secreted by cancer cells or myofibroblasts, which recruits macrophages and induces M2 polarization [30, 31]. Although several factors have been preliminarily uncovered to mediate the crosstalk between TAMs and cancer cells, the mechanisms underlying the crosstalk between TAMs and HCC cells are still largely unclear.

Transcriptomic sequencings have found that more than 80% of human genome transcribe RNAs and while only less than 2% of human genome encode proteins [32]. Thus, non-coding RNAs are remarkably more than protein-encoding mRNAs. Long noncoding RNAs (lncRNAs) are the major components of regulatory non-coding RNAs [33]. In various pathophysiological processes, lncRNAs exert diverse roles [34–38]. In malignancies, many lncRNAs show oncogenic or tumour-suppressive roles [39–41]. Most of reported cancer-related lncRNAs modulate the malignant behaviors of cancer cells, including cellular growth, cell cycle, apoptosis, migration, drug-resistance, and so on [42–45]. Furthermore, aberrant expressions of lncRNAs are frequently revealed in a variety of diseases [46–48]. Several lncRNAs were found to be correlated with prognosis of diseases and could be recognized as prognostic biomarkers [49, 50]. However, the potential involvement of lncRNAs in the crosstalk between HCC cells and macrophages still needs further investigation.

In this study, through analyzing the cancer genome atlas (TCGA) liver hepatocellular carcinoma (LIHC) dataset, we found that ZNNT1 was increased and associated with poor prognosis of HCC. Our results further confirmed that ZNNT1 was an HCC-related lncRNA, which promoted HCC development through mediating the crosstalk between HCC cells and TAMs.

Materials And Methods

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 14^th 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.

Results

ZNNT1 is upregulated and associated with poor prognosis of HCC

To search the differentially expressed lncRNAs in HCC, we analyzed TCGA LIHC dataset using the online in silico tool UALCAN (http://ualcan.path.uab.edu/analysis-lncRNA.html). We noted that lncRNA ZNNT1 was significantly upregulated in HCC tissues compared with normal liver tissues (Fig. S1A). In our cohort containing 98 pairs of HCC tissues and adjacent noncancerous liver tissues, we measured ZNNT1 expression and found that ZNNT1 was also significantly upregulated in HCC tissues compared with liver tissues (Fig. 1A). Analysis of TCGA LIHC dataset using another online in silico tool GEPIA (http://gepia.cancer-pku.cn/index.html) revealed that high ZNNT1 expression was associated with poor overall survival and disease-free survival (Fig. S1B, C). In our cohort, we also found that high ZNNT1 expression was associated with poor overall survival and recurrence-free survival (Fig. 1B, C). Correlation analyses of ZNNT1 expression levels and clinicopathological characteristics in these 98 HCC cases showed that high ZNNT1 expression was associated with poor grade, microvascular invasion, and advanced Barcelona Clinic Liver Cancer (BCLC) stage (Table S1). ZNNT1 expression was also upregulated in HCC cell lines SK-HEP-1, SUN-398, and HuH-7, compared with normal hepatic cell lines THLE-2 and THLE-3 (Fig. 1D).

ZNNT1 promotes HCC cellular growth, migration, and invasion, and represses HCC cellular apoptosis in vitro

Due to the increased expression and clinical relevance of ZNNT1 in HCC, we further investigated the potential functions of ZNNT1 in HCC. We constructed SUN-398 and SK-HEP-1 cells with ZNNT1 stable overexpression (Fig. 2A). CCK-8 and EdU assays presented that both SUN-398 and SK-HEP-1 cells with ZNNT1 overexpression had faster cell growth compared with control SUN-398 and SK-HEP-1 cells (Fig. 2B, C). Caspase-3 activity assay and TUNEL assay presented that both SUN-398 and SK-HEP-1 cells with ZNNT1 overexpression had reduced cell apoptosis (Fig. 2D, E). Transwell migration and invasion assays presented that both SUN-398 and SK-HEP-1 cells with ZNNT1 overexpression had more migrated and invasive cells (Fig. 2F, G). Moreover, we constructed SUN-398 cells with ZNNT1 stable knockdown using two independent shRNA lentiviruses (Fig. S2A). CCK-8 and EdU assays presented that SUN-398 cells with ZNNT1 knockdown had slower cell growth compared with control SUN-398 cells (Fig. S2B, C). Caspase-3 activity assay and TUNEL assay presented that SUN-398 cells with ZNNT1 knockdown had increased cell apoptosis (Fig. S2D, E). Transwell migration and invasion assays presented that SUN-398 cells with ZNNT1 knockdown had less migrated and invasive cells (Fig. S2F, G). Thus, these data suggested that ZNNT1 exerted oncogenic roles in HCC in vitro.

ZNNT1 promoted HCC tumour growth in vivo

To further confirm the roles of ZNNT1 in HCC, we constructed liver orthotopic xenograft model using subcutaneous sections derived from luciferase labelled SUN-398 cells with ZNNT1 overexpression or knockdown. Bioluminescence detection presented that the xenografts derived from SUN-398 cells with ZNNT1 overexpression were significantly larger, and while the xenografts derived from SUN-398 cells with ZNNT1 knockdown were significantly smaller than those derived from control SNU-398 cells (Fig. 3A, B). Proliferation markers PCNA and Ki67 IHC staining presented that the orthotropic xenografts derived from SUN-398 cells with ZNNT1 overexpression had more proliferative cells, and while the xenografts derived from SUN-398 cells with ZNNT1 knockdown had less proliferative cells (Fig. 3C-F). Apoptosis markers cleaved caspase-3 IHC staining and TUNEL assays both presented that the orthotropic xenografts derived from SUN-398 cells with ZNNT1 overexpression had less apoptotic cells, and while the xenografts derived from SUN-398 cells with ZNNT1 knockdown had more apoptotic cells (Fig. 3G-J). Furthermore, TAM marker CD206 IHC staining presented that the orthotropic xenografts derived from SUN-398 cells with ZNNT1 overexpression had more TAMs, and while the xenografts derived from SUN-398 cells with ZNNT1 knockdown had less TAMs (Fig. 3K, L). These data suggested that ZNNT1 exerted oncogenic roles in HCC in vivo via regulating HCC cellular malignant phenotype and TAM.

ZNNT1 recruits and induces M2 polarization of macrophages via up-regulating OPN secretion

Given that HCC cells with ZNNT1 overexpression recruited more TAMs in vivo, we further investigated the potential effects of ZNNT1 on macrophages recruitment and polarization using in vitro co-culture assay. Co-culture with SNU-398 and SK-HEP-1 cells with ZNNT1 overexpression promoted macrophage migration compared with co-culture with control SNU-398 and SK-HEP-1 cells (Fig. 4A). Conversely, co-culture with SNU-398 cells with ZNNT1 knockdown inhibited macrophage migration compared with co-culture with control SNU-398 cells (Fig. 4B). Moreover, co-culture with SNU-398 and SK-HEP-1 cells with ZNNT1 overexpression down-regulated M1 markers iNOS and TNF-α expression, and up-regulated M2 markers CD163 and CD206 expression in macrophages compared with co-culture with control SNU-398 and SK-HEP-1 cells (Fig. 4C, D). Conversely, co-culture with SNU-398 cells with ZNNT1 knockdown up-regulated M1 markers iNOS and TNF-α expression, and down-regulated M2 markers CD163 and CD206 expression in macrophages compared with co-culture with control SNU-398 cells (Fig. 4E). In our cohort, we found that high ZNNT1 expression was associated with more CD206-positive M2-polarized macrophages infiltration (Fig. 4F). To explore the mechanisms mediating the recruitment and M2 polarization of macrophage, we measured secretory proteins expression which regulate macrophage recruitment and polarization. The results presented that SPP1 (encoding OPN protein) expression was significantly increased in SNU-398 and SK-HEP-1 cells with ZNNT1 overexpression compared with control SNU-398 and SK-HEP-1 cells (Fig. 5A), and significantly reduced in SNU-398 cells with ZNNT1 knockdown compared with control SNU-398 cells (Fig. 5B). Consistently, ELISA results showed that SNU-398 and SK-HEP-1 cells with ZNNT1 overexpression secreted more OPN than control SNU-398 and SK-HEP-1 cells (Fig. 5C), and while SNU-398 cells with ZNNT1 knockdown secreted less OPN than control SNU-398 cells (Fig. 5D). In vitro co-culture assays presented that depletion of SPP1 in SNU-398 cells reversed the increased macrophage migration caused by ZNNT1-overexpressed SNU-398 cells (Fig. 5E). Depletion of SPP1 in SNU-398 cells also reversed the downregulation of M1 markers iNOS and TNF-α, and the upregulation of M2 markers CD163 and CD206 in macrophages caused by ZNNT1-overexpressed SNU-398 cells (Fig. 5F). In our cohort, the expression of SPP1 was positively correlated with ZNNT1 in HCC tissues (Fig. 5G). The positive correlation between SPP1 and ZNNT1 expression was also verified in TCGA LIHC dataset (Fig. 5H). Furthermore, high SPP1 expression was associated with more CD206-positive M2-polarized macrophages infiltration in HCC tissues (Fig. 5I). Collectively, these data suggested that ZNNT1 highly expressed HCC cells induced recruitment and M2 polarization of macrophages through secreting OPN.

ZNNT1 upregulates SPP1 via sponging miRNAs

To investigate the mechanisms underlying the regulation of SPP1 by ZNNT1, we first assessed the subcellular distribution of ZNNT1 in HCC cells. RNA FISH presented that ZNNT1 was mainly distributed in the cytoplasm (Fig. 6A). Subcellular fractionation followed by qRT-PCR also presented that ZNNT1 was mainly distributed in the cytoplasm (Fig. 6B). One of the major mechanisms of cytoplasmic lncRNAs is to competitively sponge common microRNAs (miRNAs) and relieve the repressive roles of miRNAs on their targets [57]. To investigate whether ZNNT1 regulates SPP1 in such manner, we cloned SPP1 3'UTR into luciferase reporter. Dual-luciferase reporter assays presented that ZNNT1 overexpression upregulated the luciferase activity of the reporter containing SPP1 3'UTR (Fig. 6C), and while ZNNT1 knockdown downregulated the luciferase activity of the reporter containing SPP1 3'UTR (Fig. 6D). The online in silico tool miRcode (http://www.mircode.org/) predicted the miR-181a-5p, miR-181b-5p, miR-181c-5p, miR-181d-5p, miR-33a-5p and miR-33b-5p as the common miRNAs targeting ZNNT1 and SPP1 3'UTR (Table S2). To investigate whether ZNNT1 binds these miRNAs, we performed MS2-based RIP assays. The results showed that miR-181a/b/c/d-5p and miR-33a/b-5p were enriched in MS2-ZNNT1 group (Fig. 6E). RNA pull-down assay further verified the binding of miR-181a/b/c/d-5p and miR-33a/b-5p to in vitro transcribed ZNNT1 (Fig. 6F). Dual-luciferase reporter assays presented that miR-181a/b/c/d-5p and miR-33a/b-5p repressed the luciferase activity of the reporter containing SPP1 3'UTR (Fig. 6G), supporting SPP1 as the target of these miRNAs. Block of miRNAs generation using DICER depletion largely abolished the upregulation of SPP1 caused by ZNNT1 overexpression (Fig. 6H), supporting that the regulation of SPP1 by ZNNT1 was dependent on miRNAs.

OPN mediates the oncogenic effects of ZNNT1 on HCC cells

Except the effects on macrophage recruitment and polarization, OPN has been frequently reported to exert oncogenic roles in cancer cells [58, 59]. Therefore, we further investigated whether OPN mediates the oncogenic effects of ZNNT1 on HCC cells. CCK-8 and EdU assays presented that depletion of SPP1 largely reversed the faster cell growth caused by ZNNT1 overexpression (Fig. S3A, B). Caspase-3 activity assay and TUNEL assay presented that depletion of SPP1 largely reversed the reduced cell apoptosis caused by ZNNT1 overexpression (Fig. S3C, D). Transwell migration and invasion assays presented that depletion of SPP1 largely reversed the increased cell migration and invasion caused by ZNNT1 overexpression (Fig. S3E, F). These data suggested that ZNNT1 exerted oncogenic roles in HCC cells at least partially through upregulating OPN.

M2 macrophages recruited by ZNNT1 enhanced malignant phenotypes of HCC cells

TAMs have been frequently reported to exert oncogenic influences on cancer cells [27]. Thus, we further investigated the potential effects of M2-polarized macrophages recruited by ZNNT1 on HCC cells using in vitro co-culture assays (Fig. 7A). In vitro co-culture assay followed by CCK-8 and EdU assays presented that macrophage-educated by SNU-398 cells promoted SNU-398 cell growth (Fig. 7B, C). The increased cell growth was further enhanced by macrophage-educated by ZNNT1-overexpressed SNU-398 cells (Fig. 7B, C). Conversely, the increased cell growth was attenuated by macrophage-educated by ZNNT1-knocked down SNU-398 cells (Fig. S4A, B). In vitro co-culture assay followed by caspase-3 activity assay and TUNEL assay presented that macrophage-educated by SNU-398 cells inhibited SNU-398 cell apoptosis, which were further enhanced by macrophage-educated by ZNNT1-overexpressed SNU-398 cells (Fig. 7D, E). The inhibited cell apoptosis was attenuated by macrophage-educated by ZNNT1-knocked down SNU-398 cells (Fig. S4C, D). In vitro co-culture assay followed by transwell migration and invasion assays presented that macrophage-educated by SNU-398 cells promoted SNU-398 cell migration and invasion, which were further enhanced by macrophage-educated by ZNNT1-overexpressed SNU-398 cells (Fig. 7F, G). The increased cell migration and invasion was attenuated by macrophage-educated by ZNNT1-knocked down SNU-398 cells (Fig. S4E, F). Thus, these data suggested that macrophages-recruited by ZNNT1-overexpressed HCC cells promoted malignant phenotypes of HCC cells.

S100A9 secreted by M2 macrophages upregulated ZNNT1 expression in HCC cells via activating AGER/NF-κB signaling

TAMs have been reported to secrete S100A9 to exert oncogenic roles [28]. S100A9 binds to its receptor AGER on HCC cells and further activates NF-κB signaling [28]. We also found that PMA-stimulated THP-1 cells had significantly higher expression of S100A9 than SNU-398 cells (Fig. 8A). Co-culture with SNU-398 cells increased S100A9 expression in THP-1 cells (Fig. 8A). Co-culture with ZNNT1-overexpressed SNU-398 cells further increased S100A9 expression in THP-1 cells (Fig. 8A). ELISA results presented that THP-1 cells secreted significantly more S100A9 than SNU-398 cells (Fig. 8B). Co-culture with SNU-398 cells increased S100A9 secretion from THP-1 cells (Fig. 8B). Co-culture with ZNNT1-overexpressed SNU-398 cells further increased S100A9 secretion from THP-1 cells (Fig. 8B). To investigate whether macrophages-recruited by ZNNT1 modulate NF-κB activation in HCC cells, mock SNU-398 cells were treated with conditioned medium (CM) from PMA-stimulated THP-1 cells co-culture with ZNNT1 overexpressed or control SNU-398 cells. NF-κB reporter assays showed that CM from SNU-398-eductaed THP-1 cells increased NF-κB transcriptional activity, which was further increased by CM from THP-1 cells co-culture with ZNNT1-overexpressed SNU-398 cells (Fig. 8C). S100A9 inhibitor Tasquinimod abolished the increasing of NF-κB transcriptional activity (Fig. 8C). p50 and p65 transcription factor assays presented that CM from SNU-398-eductaed THP-1 cells promoted NF-κB (p50 and p65) activation, which was further activated by CM from THP-1 cells co-culture with ZNNT1-overexpressed SNU-398 cells and abolished by Tasquinimod (Fig. 8D). The online in silico tool JASPAR (https://jaspar.genereg.net/) predicted three potential NF-κB binding sites on ZNNT1 promoter (Fig. 8E). ChIP assays confirmed the binding of NF-κB to ZNNT1 promoter (Fig. 8E). CM from SNU-398-eductaed THP-1 cells promoted NF-κB binding to ZNNT1 promoter, which was further enhanced by CM from THP-1 cells co-culture with ZNNT1-overexpressed SNU-398 cells and abolished by Tasquinimod (Fig. 8F). To investigate whether ZNNT1-recruited macrophages regulate ZNNT1 expression via AGER/NF-κB signaling, we constructed ZNNT1 promoter reporter plasmid. Dual-luciferase reporter assays presented that CM from SNU-398-eductaed THP-1 cells upregulated ZNNT1 promoter activity, which was further enhanced by CM from THP-1 cells co-culture with ZNNT1-overexpressed SNU-398 cells and abolished by Tasquinimod (Fig. 8G). Consistently, CM from SNU-398-eductaed THP-1 cells upregulated ZNNT1 expression, which was further enhanced by CM from THP-1 cells co-culture with ZNNT1-overexpressed SNU-398 cells and abolished by Tasquinimod (Fig. 8H). Collectively, these data suggested that S100A9 secreted from ZNNT1-recruited macrophages upregulated ZNNT1 expression in HCC cells via activating AGER/NF-κB signaling. ZNNT1 expression was positively associated with S100A9 expression in HCC tissues (Fig. 8I). SPP1 expression was also positively associated with S100A9 expression in HCC tissues (Fig. 8I), supporting the ZNNT1/SPP1/S100A9 regulatory loop in vivo.

S100A9 mediates the oncogenic effects of M2 macrophages on HCC cells

S100A9 has been revealed to exert oncogenic roles in HCC [28, 60]. Therefore, we further investigated whether S100A9 mediates the oncogenic effects of M2 macrophages on HCC cells using in vitro co-culture assays as described in Fig. 7A. CCK-8 and EdU assays presented that adding Tasquinimod in the in vitro co-culture system reversed the faster cell growth caused by macrophages-educated by ZNNT1-overexpressed SNU-398 cells (Fig. S5A, B). Caspase-3 activity assay and TUNEL assay presented that adding Tasquinimod in the in vitro co-culture system reversed the reduced cell apoptosis caused by macrophages-educated by ZNNT1-overexpressed SNU-398 cells (Fig. S5C, D). Transwell migration and invasion assays presented that adding Tasquinimod in the in vitro co-culture system reversed the increased cell migration and invasion caused by macrophages-educated by ZNNT1-overexpressed SNU-398 cells (Fig. S5E, F). These data suggested that S100A9 at least partially mediated the oncogenic effects of M2 macrophages on HCC cells.

Discussion

ZNNT1 is a recently reported lncRNA, which suppressed tumorigenesis of uveal melanoma via inducing autophagy [61]. ZNNT1 has 3435 nucleotides in length, with the NCBI Reference Sequence number NR_164368.1. ZNNT1 is located at chromosome 8q22.3. ZNNT1 was also annotated as KB-1460A1.5. A recent report showed that ZNNT1 (KB-1460A1.5) suppressed tumorigenesis of glioma via miR-130a-3p/TSC1/mTOR/YY1 feedback loop [62]. However, in this study we found that ZNNT1 functioned as an oncogene in HCC. Both TCGA LIHC dataset and our own cohort presented that ZNNT1 was upregulated in HCC, and increased expression of ZNNT1 was correlated with worse overall survival and recurrence-free survival of HCC. Furthermore, in vitro and in vivo gain- and loss-of-function assays demonstrated that ZNNT1 promoted HCC cellular growth, migration, and invasion, and repressed HCC cellular apoptosis in vitro, and also promoted HCC xenografts growth in vivo. Intriguingly, besides the oncogenic roles of ZNNT1 in HCC cells, ZNNT1 was found to recruit and induce M2 polarization of macrophages in vitro and in vivo. Consistent with previous reports about the oncogenic roles of M2 macrophages [21], our data also showed that the microphages-recruited and M2 polarized by ZNNT1 exerted oncogenic effects on HCC cells. Thus, ZNNT1 promoted HCC development via modulating both HCC cells and TAMs. Collectively, our findings suggested that ZNNT1 could be a potential prognostic biomarker in HCC and targeting ZNNT1 represents potential strategy for HCC therapy.

Mechanistic investigations identified OPN as the critical downstream target of ZNNT1. As a cytoplasmic lncRNA, ZNNT1 functioned as a competing endogenous RNA (ceRNA) to sponge miR-181a-5p, miR-181b-5p, miR-181c-5p, miR-181d-5p, miR-33a-5p and miR-33b-5p, relieving the repressive roles of these miRNAs on SPP1 mRNA. As another class of regulatory non-coding RNAs, miRNAs generally bind to 3'UTR of target mRNAs, induce degradation and/or inhibit translation of target mRNAs [63, 64]. One of the major mechanisms of cytoplasmic lncRNAs is to function as ceRNA to block the roles of miRNAs [57, 65–67]. ZNNT1 was shown as another example of cytoplasmic ceRNA. The positive correlation between ZNNT1 and SPP1 expression in HCC tissues supported the modulation of SPP1 by ZNNT1. SPP1 encodes OPN protein. Thus, through upregulating SPP1 expression, ZNNT1 upregulated OPN secretion. Consistent with previously reported roles of OPN in recruiting and inducing M2 polarization of macrophages [30, 31], ZNNT1 highly expressed HCC cells had stronger ability to recruit and induce M2 polarization of macrophage via increased secretion of OPN. Analysis of single cell RNA sequencing (scRNA-seq) dataset using Tumor Immune Single-cell Hub (TISCH) (http://tisch.comp-genomics.org/home/) reveled that SPP1 is mainly expressed in hepatic progenitor cells and HCC cells in LIHC. Enhanced expression of OPN showed pro-tumorigenic roles in HCC [59]. Here, we also found that besides the roles in mediating the recruitment and M2 polarization, OPN also mediated the oncogenic effects of ZNNT1 on HCC cells. Functional rescue assays showed that depletion of OPN largely reversed the roles of ZNNT1 in HCCs.

The macrophages recruited and M2 polarized by ZNNT1-overexpressed HCC cells exerted oncogenic effects on HCC cells. We further explored the factors mediating the oncogenic roles of macrophages-educated by ZNNT1-overexpressed HCC cells. We found that S100A9 was mainly secreted by macrophages, and macrophages secreted more S100A9 after co-culture with ZNNT1-overexpressed HCC cells. Analysis of scRNA-seq dataset using TISCH also reveled that S100A9 is mainly expressed in monocytes and macrophages in LIHC. Functional rescue assays showed that S100A9 inhibitor Tasquinimod abolished the oncogenic effects of macrophages-educated by ZNNT1-overexpressed HCC cells, supporting that S100A9 at least partially mediated the oncogenic roles of macrophages-educated by ZNNT1-overexpressed HCC cells.

Secreted S100A9 was reported to bind the AGER receptor on HCC cells and further activated NF-κB signaling in HCC cells [28]. Intriguingly, here we identified ZNNT1 as the direct target of NF-κB transcription factor. Through activating NF-κB, S100A9 upregulated ZNNT1 promoter activity and ZNNT1 expression. Macrophages-educated by ZNNT1-overexpressed HCC cells upregulated ZNNT1 expression in HCC cells through S100A9. Therefore, ZNNT1/OPN/S100A9 formed a positive feedback loop (Fig. 9). ZNNT1 upregulated SPP1 expression in HCC cells and induced OPN secretion from HCC cells. Secreted OPN recruited and induced M2 polarization of macrophages, which secreted S100A9. S100A9 upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling.

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ZNNT1/osteopontin/S100A9 feedback loop promote hepatocellular carcinoma progression via mediating crosstalk between hepatocellular carcinoma cells and macrophages

Abstract

Background

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Conclusions

Background

Materials And Methods

Results

Discussion

Conclusion

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References