Long non-coding RNA HOMER3-AS1 drives hepatocellular carcinoma progression via modulating the behaviors of both tumor cells and macrophages

The crosstalk between cancer cells and tumor microenvironment plays critical roles in hepatocellular carcinoma (HCC). The identification of long non-coding RNAs (lncRNAs) mediating the crosstalk might promote the development of new therapeutic strategies against HCC. Here, we identified a lncRNA, HOMER3-AS1, which is over-expressed in HCC and correlated with poor survival of HCC patients. HOMER3-AS1 promoted HCC cellular proliferation, migration, and invasion, and reduced HCC cellular apoptosis. Furthermore, HOMER3-AS1 promoted macrophages recruitment and M2-like polarization. In vivo, HOMER3-AS1 significantly facilitated HCC progression. Mechanism investigations revealed that HOMER3-AS1 activated Wnt/β-catenin signaling via upregulating HOMER3. Functional rescue experiments revealed that HOMER3/Wnt/β-catenin axis mediated the roles of HOMER3-AS1 in promoting HCC cellular malignant phenotypes. Furthermore, colony stimulating factor-1 (CSF-1) was also identified as a critical downstream target of HOMER3-AS1. HOMER3-AS1 increased CSF-1 expression and secretion. Blocking CSF-1 reversed the roles of HOMER3-AS1 in inducing macrophages recruitment and M2 polarization. Furthermore, positive correlations between HOMER3-AS1 and HOMER3 expression, HOMER3-AS1 and CSF-1 expression, and HOMER3-AS1 expression and M2-like macrophages infiltration were found in human HCC tissues. In summary, our findings demonstrated that HOMER3-AS1 drives HCC progression via modulating the behaviors of both tumor cells and macrophages, which are dependent on the activation of HOMER3/Wnt/β-catenin axis and CSF-1, respectively. HOMER3-AS1 might be a promising prognostic and therapeutic target for HCC.


INTRODUCTION
Liver cancer is the sixth most commonly diagnosed malignancy and the third leading cause of malignancy-related death worldwide, with about 905,677 newly diagnosed cases and 830,180 liver cancer-caused deaths [1]. The main subtype of liver cancer is hepatocellular carcinoma (HCC), which accounts for 80%-90% of all liver cancer cases [2]. Although modern multidisciplinary therapies have been developed to fight against HCC, including surgical resection, liver transplantation, ablation, transcatheter arterial chemoembolization (TACE), molecular targeted therapies, and immunotherapy, only modest improvement in prognoses of HCC patients has been achieved [3]. Therefore, identifying molecular alterations in HCC that are responsible for HCC tumorigenesis and progression and developing novel more effective treatments are urgently needed [4][5][6].
The progression of HCC is not only regulated by malignant phenotypes of themselves, but also by tumor microenvironment (TME) [26][27][28]. Immune checkpoints inhibitors which enhance antitumor immune responses of T cells have been approved to treat HCC [29]. Apart from T cells, tumor-associated macrophages (TAMs) are another class of critical components of TME [8,30]. Increasing evidences revealed that TAMs have M2-like phenotypes and contribute to tumor progression [27,31]. Increased levels of M2-like TAMs in HCC were also revealed to be correlated with the poor prognosis of HCC patients [32]. Compared with the M2-like phenotype, macrophages can also be polarized to M1-like phenotypes, which frequently show antitumor activities [33,34]. Although accumulating studies have identified several factors inducing M1 or M2 polarization of macrophages, such as the induction of M2 phenotype by IL-4, IL-10, TGF-β, and colonystimulating factor 1 (CSF-1), the contributions of lncRNAs to M2 polarization of TAMs are still largely unknown [35,36].
In this study, we identified a poor prognosis-correlated lncRNA HOMER3-AS1 in HCC via analyzing the cancer genome atlas (TCGA) liver hepatocellular carcinoma (LIHC). The expression and clinical relevance of HOMER3-AS1 were further investigated in our HCC cohort. In vitro and in vivo functional experiments were undertaken to explore the biological roles of HOMER3-AS1 in HCC. The molecular mechanisms underlying the roles of HOMER3-AS1 in HCC were also investigated.

MATERIALS AND METHODS Bioinformatics analyses
The correlation between gene expression and overall survival of HCC patients based on the RNA sequencing expression data of HCC tissues from the cancer genome atlas (TCGA) liver hepatocellular carcinoma (LIHC) project was analyzed by the online in silico tool GEPIA (http://gepia.cancerpku.cn/). Gene expression correlation in HCC tissues based on the TCGA LIHC dataset was calculated by GEPIA (http://gepia.cancer-pku.cn/). The genes which have similar expression patterns with HOMER3-AS1 in HCC based on the TCGA LIHC dataset were searched by GEPIA (http://gepia. cancer-pku.cn/).

Clinical samples
Sixty-eight pairs of HCC tissues and adjacent noncancerous liver tissues were acquired at the Affiliated Hospital of Youjiang Medical University for Nationalities from HCC patients who received surgical resection with written informed consents. All tissues were diagnosed by two experienced pathologists. The clinicopathological characteristics of these 68 cases were presented in Table 1. This study was performed following the Declaration of Helsinki and approved by the Ethics Committee of Affiliated Hospital of Youjiang Medical University for Nationalities.
J. Pu et al.

Stable cell lines construction
To construct HOMER3-AS1 stably overexpressed and control cells, HOMER3-AS1 overexpression plasmid or empty plasmid pcDNA3. paraformaldehyde for 30 min and permeabilized using 0.5% TritonX-100 for 10 min, the cells were stained with Apollo dye solution (RiboBio). The cell nucleus was further stained using DAPI. The number of EdU-positive and proliferative cells was detected using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Cell apoptosis was detected using the Caspase-3 Activity Assay Kit (Cell Signaling Technology, Danvers, MA, USA) strictly following the provided protocol. Cell migration and invasion were evaluated by transwell migration and invasion assays as we previously described [23].
Co-culture assay PMA-stimulated THP-1 cells were added into the upper chamber of 8 μm pore transwell inserts (Corning, NY, USA). SK-HEP-1 cells with HOMER3-AS1 overexpression or silencing were plated into the lower chamber. After culture for 48 h, THP-1 cells remaining in the upper chamber were removed. The migrated THP-1 cells were fixed, stained, and detected using a microscope. SK-HEP-1 cells with HOMER3-AS1 overexpression or silencing were plated into the upper chamber of 0.4 μm pore transwell inserts (Corning). PMA-stimulated THP-1 cells were plated into the lower chamber. After co-culture for 96 h, THP-1 cells were collected to extract RNA. Genes expression in THP-1 cells were detected by qRT-PCR. THP-1 cellular proliferation was detected by CCK-8 experiments.

Subcutaneous and orthotopic models
Five-week-old male BALB/C athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co. and fed in Specific Pathogen Free (SPF) conditions. Luciferase-labelled SK-HEP-1 cells with HOMER3-AS1 overexpression or silencing were subcutaneously injected into nude mice. When the subcutaneous xenografts grew to about 0.7 cm in diameter, they were removed and cut into small pieces, which were then transplanted into the liver of nude mice. At the 14 th day after transplantation, the tumors were detected by bioluminescence imaging using IVIS @ Lumina II system (Caliper Life Sciences, Hopkinton, MA, USA). No statistical method was used to determine the sample size. The experiments were not randomized. The investigators performed the bioluminescence imaging were blinded to mouse allocation. The animal experiments were undertaken with the approval of the Animal Ethics Committee of Affiliated Hospital of Youjiang Medical University for Nationalities.

RNA fluorescence in situ hybridization (FISH)
For in situ detection of HOMER3-AS1 in HCC tissues, the HOMER3-AS1 probes were designed and synthesized by Advanced Cell Diagnostics (Hayward, CA, USA). RNAscope Fluorescent Multiplex Detection Kit (Advanced Cell Diagnostics) was used to perform the hybridization and fluorescence detection.

Dual-luciferase reporter assays
Wild-type Wnt/β-catenin reporter TOPFlash (Addgene, Watertown, MA, USA) and pRL-TK (Promega, Madison, WI, USA) were co-transfected into SK-HEP-1 cells with HOMER3-AS1 stable overexpression or silencing. pRL-TK encodes Renilla luciferase and was used as endogenous control. After culture for another 48 h, the Firefly luciferase and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The results were calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity.

Statistical analysis
All statistical analyses were conducted using the GraphPad Prism 6.0 Software. The detailed statistical methods were shown in the figure and table legends. p < 0.05 was considered statistically significant.

HOMER3-AS1 is correlated with advanced stage and poor prognosis in HCC
The correlation between HOMER3-AS1 expression levels and overall survival in HCC was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index. html) based on the cancer genome atlas (TCGA) liver hepatocellular carcinoma (LIHC) dataset [38]. The results revealed that high HOMER3-AS1 (AC005932.1) expression was correlated with poor overall survival (Fig. 1a). To further test the correlation between HOMER3-AS1 expression levels and prognosis in HCC, we collected 68 HCC tissues and measured HOMER3-AS1 expression by qRT-PCR. Consistently, high HOMER3-AS1 expression was correlated with poor overall survival in our HCC cohort (Fig. 1b).
Analyses of the correlations between HOMER3-AS1 expression levels and clinicopathological characteristics revealed that high HOMER3-AS1 expression was correlated with advanced BCLC stage, high serum AFP concentration, and microvascular invasion ( Table 1). In these 68 HCC cases, the expression of HOMER3-AS1 in paired adjacent noncancerous liver tissues was also measured. The results revealed that HOMER3-AS1 was significantly upregulated in HCC tissues compared with noncancerous liver tissues (Fig. 1c). Furthermore, the expression of HOMER3-AS1 in immortalized human liver cell lines THLE-2 and THLE-3, and human HCC cell lines SK-HEP-1, SNU-398, and Huh7 was measured. The result revealed that HOMER3-AS1 was also significantly upregulated in HCC cell lines compared with immortalized liver cell lines (Fig. 1d). The high expression and clinical relevance of HOMER3-AS1 in HCC implied that HOMER3-AS1 might be an oncogenic lncRNA in HCC.

HOMER3-AS1 promoted HCC tumor progression in vivo
To further evaluate the roles of HOMER3-AS1 in HCC xenograft models, small pieces of subcutaneous tumors formed by luciferase labelled SK-HEP-1 cells with HOMER3-AS1 stable overexpression or silencing were orthotopically transplanted into the livers of nude mice. Bioluminescence imaging showed that the xenografts derived from HOMER3-AS1 overexpressed SK-HEP-1 cells were remarkably larger than those derived from control SK-HEP-1 cells (Fig. 3a). Conversely, the xenografts derived from HOMER3-AS1 silenced SK-HEP-1 cells were remarkably smaller than those derived from control SK-HEP-1 cells (Fig. 3b). Proliferation marker Ki67 IHC staining of orthotropic xenografts revealed that the xenografts derived from HOMER3-AS1 overexpressed SK-HEP-1 Fig. 2 Overexpression of HOMER3-AS1 promoted HCC cellular malignant phenotype. a HOMER3-AS1 expression in SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was measured by qRT-PCR. b Cell proliferation of SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was determined by CCK-8 experiments. c Cell proliferation of SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was determined by EdU incorporation experiments. Scale bars, 100 µm. The red color indicates EdU-positive nucleuses and blue color indicates all cellular nucleuses. Results are shown as the ratio of EdU-positive nucleuses to all cellular nucleuses. d Cell apoptosis of SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was determined by caspase-3 activity assays. e Cell migration of SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was determined by transwell migration assays. Scale bars, 100 µm. f Cell invasion of SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was determined by transwell invasion assays. Scale bars, 100 µm. Results are presented as mean ± SD based on three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 by Student's t-test.
HOMER3 expression is positively correlated with HOMER3-AS1 expression and poor prognosis in HCC HOMER3 expression in the same 68 pairs of HCC tissues and adjacent noncancerous liver tissues were measured. Similar with HOMER3-AS1, HOMER3 was significantly upregulated in HCC tissues compared with noncancerous liver tissues ( Supplementary  Fig. 3a). The expression of HOMER3 was significantly positively correlated with that of HOMER3-AS1 in these 68 HCC tissues ( Supplementary Fig. 3b). Similar with HOMER3-AS1, high HOMER3 expression was correlated with poor overall survival in this HCC cohort ( Supplementary Fig. 3c). The remarkably positive correlation between HOMER3 and HOMER3-AS1 expression in HCC tissues was also confirmed in the TCGA LIHC dataset, analyzed by GEPIA ( Supplementary Fig. 3d). Analyses of the TCGA LIHC dataset by GEPIA also revealed that high HOMER3 expression was correlated with poor overall survival in HCC (Supplementary Fig.  3e). Furthermore, the expressions of HOMER3-AS1 and HOMER3 in HCC tissues were detected by RNA FISH and IHC respectively. The results revealed that both HOMER3-AS1 and HOMER3 were mainly expressed in HCC cells, but not in stromal cells ( Supplementary  Fig. 3f), supporting clinical relevancy of HOMER3-AS1 and HOMER3 in HCC.

CSF-1 upregulation is responsible for the recruitment and M2like polarization of macrophages by HOMER3-AS1
To elucidate the mechanisms mediating the roles of HOMER3-AS1 in macrophages recruitment and polarization, we searched the genes with similar expression pattern with HOMER3-AS1 in HCC based on the TCGA LIHC dataset analyzed by GEPIA. CSF-1 ranked the first with a Pearson correlation coefficient of 0.72 ( Supplementary Fig. 5a). Similar with HOMER3-AS1, high CSF-1 expression was correlated with poor overall survival in HCC based on the TCGA LIHC dataset analyzed by GEPIA (Supplementary Fig. 5b). CSF-1 expression in the same 68 HCC tissues of our cohort were measured. The expression of CSF-1 was significantly positively correlated with that of HOMER3-AS1 in our HCC cohort (Supplementary Fig. 5c). Similar with HOMER3-AS1, high CSF-1 expression was correlated with poor overall survival in this HCC cohort (Supplementary Fig. 5d). CSF-1 is frequently reported to be a critical regulator of macrophages generation, differentiation, and function [36,40]. Thus, we further detected the correlation between HOMER3-AS1, CSF-1, and macrophages recruitment in HCC tissues. CD163 IHC staining was performed in these 68 HCC tissues to indicate Fig. 4 HOMER3-AS1 increased HOMER3 expression and activated Wnt/β-catenin signaling. a HOMER3 mRNA levels in SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control was measured by qRT-PCR. b HOMER3 mRNA levels in SK-HEP-1 cells with HOMER3-AS1 stable silencing or control were measured by qRT-PCR. c HOMER3 protein levels in SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control was measured by western blot. d HOMER3 protein levels in SK-HEP-1 cells with HOMER3-AS1 stable silencing or control was measured by qRT-PCR. e Nuclear β-catenin protein levels in SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control was measured by western blot. f Nuclear β-catenin protein levels in SK-HEP-1 cells with HOMER3-AS1 stable silencing or control was measured by western blot. g β-catenin expression in the nucleus of SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control was detected by IF. h β-catenin expression in the nucleus of SK-HEP-1 cells with HOMER3-AS1 stable silencing or control was detected by IF. i β-catenin reporter TOPFlash and pRL-TK which encodes Renilla luciferase were transfected into SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. Dual-luciferase reporter assays were undertaken to determine β-catenin transcriptional activity. j β-catenin reporter TOPFlash and pRL-TK were transfected into SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. Dual-luciferase reporter assays were undertaken to determine β-catenin transcriptional activity. k Wnt/β-catenin downstream targets MYC and MMP7 expressions in SK-HEP-1 cells with HOMER3-AS1 stable overexpression or control. l Wnt/β-catenin downstream targets MYC and MMP7 expressions in SK-HEP-1 cells with HOMER3-AS1 stable silencing or control. Results are presented as mean ± SD based on three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t-test (a, g, i, k) or one-way ANOVA followed by Dunnett's multiple comparisons test (b, h, j, l).
macrophages infiltration and polarization. The results revealed that the HCC tissues with high levels of CD163 + macrophages also had higher HOMER3-AS1 and CSF-1 expression levels than those HCC tissues with low levels of CD163 + macrophages ( Supplementary Fig. 5e).

DISCUSSION
Increasing understandings of the crosstalk between tumor cells and tumor microenvironment (TME) have driven advances in Fig. 6 HOMER3-AS1 promoted macrophages infiltration and M2-like polarization. a PMA-stimulated THP-1 cells were subjected to migration assays towards SK-HEP-1 cells with HOMER3-AS1 overexpression or control. b PMA-stimulated THP-1 cells were subjected to migration assays towards SK-HEP-1 cells with HOMER3-AS1 silencing or control. c Cell proliferation of PMA-stimulated THP-1 cells co-cultured with HOMER3-AS1 overexpressed or control SK-HEP-1 cells was determined by CCK-8 experiments. d Cell proliferation of PMA-stimulated THP-1 cells co-cultured with HOMER3-AS1 silenced or control SK-HEP-1 cells was determined by CCK-8 experiments. e Expression of markers associated with M1 and M2 polarization in PMA-stimulated THP-1 cells co-cultured with HOMER3-AS1 overexpressed or control SK-HEP-1 cells was detected by qRT-PCR. f Expression of markers associated with M1 and M2 polarization in PMA-stimulated THP-1 cells co-cultured with HOMER3-AS1 silenced or control SK-HEP-1 cells was detected by qRT-PCR. Results are presented as mean ± SD based on three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t-test (a, c, e) or one-way ANOVA followed by Dunnett's multiple comparisons test (b, d, f).
immunotherapy [41][42][43][44]. Immune checkpoints inhibitors, such as anti-PD-1 antibody nivolumab and pembrolizumab, have been approved for HCC treatment [45,46]. However, only about 14% of HCC patients are sensitive to anti-PD-1 antibody. Furthermore, frequent drug resistances to anti-PD-1 antibody were also found in clinical therapy [47]. Thus, further revealing the critical molecules driving HCC progression via mediating the crosstalk between HCC cells and TME would provide novel avenues for HCC therapy.
In this study, we identified a novel HCC-related lncRNA HOMER3-AS1. HOMER3-AS1 is located in chromosome 19p13.11. HOMER3-AS1 is transcribed in the anti-sense direction of HOMER3 and has 1041 nucleotides in length. Both RNA-sequencing data of the TCGA LIHC project and qRT-PCR detection of our HCC cohort revealed that high expression of HOMER3-AS1 is correlated with poor overall survival of HCC patients. Furthermore, our data also revealed that HOMER3-AS1 is upregulated in HCC tissues and correlated with advanced BCLC stage, high serum AFP Fig. 7 HOMER3-AS1 promoted macrophages infiltration and M2-like polarization via upregulating CSF-1. a CSF-1 expression in SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was measured by qRT-PCR. b CSF-1 expression in SK-HEP-1 and Huh7 cells with HOMER3-AS1 stable silencing was measured by qRT-PCR. c CSF-1 secretion from SK-HEP-1 and SNU-398 cells with HOMER3-AS1 stable overexpression was measured by ELISA. d CSF-1 secretion from SK-HEP-1 and Huh7 cells with HOMER3-AS1 stable silencing was measured by ELISA. e PMA-stimulated THP-1 cells were subjected to migration assays towards CM from HOMER3-AS1 overexpressed SK-HEP-1 cells treated with or without anti-CSF-1. f Cell proliferation of PMA-stimulated THP-1 cells after culture with CM from HOMER3-AS1 overexpressed SK-HEP-1 cells treated with or without anti-CSF-1. g Expression of markers associated with M1 and M2 polarization in PMA-stimulated THP-1 cells after culture with CM from HOMER3-AS1 overexpressed SK-HEP-1 cells treated with or without anti-CSF-1. h Schematic model of the roles of HOMER3-AS1 in regulating HCC cellular malignancy and TAMs infiltration and polarization. Results are presented as mean ± SD based on three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, by Student's t-test (a, c) or one-way ANOVA followed by Dunnett's multiple comparisons test (b, d-g).
concentration, and microvascular invasion. Therefore, these findings suggested HOMER3-AS1 as a potential prognostic biomarker for HCC. The positive correlation between HOMER3-AS1 and AFP suggested that HOMER3-AS1 may be a diagnostic biomarker for HCC, which needs further investigation.
Gain-and loss-of functions experiments revealed that HOMER3-AS1 has oncogenic roles in HCC. HOMER3-AS1 drives HCC progression in vitro and in vivo. HOMER3-AS1 enhances HCC cellular malignant phenotypes, including the promotion of HCC cellular proliferation, migration, and invasion, and the inhibition of HCC cellular apoptosis. Moreover, HOMER3-AS1 was also found to facilitate macrophages infiltration and M2-like polarization. Therefore, HOMER3-AS1 drives HCC progression not only by regulating the behaviors of HCC cells, but also by educating macrophages. Our findings also showed that depletion of HOMER3-AS1 significantly represses HCC progression in vitro and in vivo through reducing HCC cellular malignant phenotypes and macrophages recruitment and M2 polarization. HOMER3-AS1 represents a potential therapeutic target for HCC.
Except for the roles of HOMER3-AS1 in enhancing HCC cellular malignant phenotypes, HOMER3-AS1 was also found to induce macrophages recruitment and M2-like polarization. Several molecules were reported to mediate the crosstalk between cancer cells and macrophages [35,36,55]. Tumor cells secrete sonic hedgehog to drive TAMs M2 polarization [55]. OPN derived from HCC cells facilitate macrophages migration and M2 polarization through activating the CSF-1/CSF1R pathway [35]. CircASAP1 promotes macrophage infiltration via modulating the miR-326/miR-532-5p-CSF-1 pathway [36]. Combining bioinformatics analyses and experimental verification, we identified CSF-1 as the critical downstream target of HOMER3-AS1 and the mediator of the roles of HOMER3-AS1 in inducing macrophages recruitment and M2 polarization. The expression of CSF-1 is significantly positively correlated with that of HOMER3-AS1 in HCC tissues, verified in the TCGA LIHC dataset and our HCC cohort. The expression levels of CSF-1 and HOMER3-AS1 are both positively correlated with the number of CD163 positive macrophages. These clinical data supported the HOMER3-AS1/CSF-1/ macrophages regulatory axis. The detailed molecular mechanisms of how HOMER3-AS1 regulates CSF-1 need further investigation.
In summary, we identified lncRNA HOMER3-AS1 as a novel HCCrelated lncRNA, which is highly expressed in HCC tissues and correlated with poor survival of HCC patients. HOMER3-AS1 enhances HCC cellular malignant phenotypes via upregulating HOMER3 and activating Wnt/β-catenin signaling. HOMER3-AS1 facilitates macrophages infiltration and M2-like polarization via upregulating CSF-1 and inducing CSF-1 secretion (Fig. 7h). These findings suggested HOMER3-AS1 as a potential prognostic biomarker and therapeutic target for HCC.