{'fraction_non_alphanumeric': 0.03713812262403743, 'fraction_numerical': 0.02963251778925821, 'mean_word_length': 5.115946348733234, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Hsa‐microRNA‐370‐3p targeting Snail and Twist1 suppresses IL‐8/STAT3‐driven hepatocellular carcinoma metastasis

Abstract The pro‐inflammatory factor interleukin‐8 (IL‐8) is related to poor prognosis in hepatocellular carcinoma (HCC) patients. Interleukin‐8 enhanced HCC invasion by upregulating Snail and Twist1, whether this modulation relies on microRNAs (miR) is unclear. In this study, hsa‐miR‐370‐3p was screened as candidate miRNA targeting Snail and Twist1, and its expression was downregulated by IL‐8. Luciferase assays and RNA electrophoretic mobility shift assays were used to evaluate the interaction between miR‐370‐3p and targeted mRNAs. Coimmunoprecipitation, luciferase, and ChIP assays were undertaken to investigate the mechanisms underlying IL‐8‐mediated modification of miR‐370‐3p. Gain‐ and loss‐of‐function studies, Transwell assays, and a xenograft nude mouse model were used to investigate pro‐ and antitumor activities. Interleukin‐8 and miR‐370‐3p levels were analyzed for clinical relevance in HCC patients. Our results showed that HCC patients with high levels of IL‐8 experienced more metastasis and shorter survival. Interleukin‐8 induced epithelial–mesenchymal transition and promoted liver cancer cell migration, invasion, and metastasis both in vitro and in vivo. MicroRNA‐370‐3p interacted with its cognate mRNA within the 3′‐UTR regions of Twist1 and Snail mRNA directly and specifically and attenuated IL‐8 protumoral effects on liver cancer cells. Interleukin‐8 negatively modulated miR‐370‐3p through signal transducer and activator of transcription 3 (STAT3) activation by recruiting histone deacetylase 1 (HDAC1) to miR‐370‐3p promoter. The STAT3 and HDAC antagonists inhibited liver cancer cell migration and invasion. Patients with high miR‐370‐3p and low IL‐8 levels had longer overall survival. In conclusion, our study elucidated a novel axis IL‐8/STAT3/miR‐370‐3p/Twist1 and Snail relying on HDAC1 recruitment, which showed both diagnostic and therapeutic potentials of miR‐370‐3p in HCC metastasis.


| INTRODUC TI ON
Liver cancer, mainly HCC, is one of the most fatal cancers, especially in Asia. 1 The poor outcomes of HCC patients are largely due to metastatic spread, with 5-year recurrence rate exceeding 70%. 2 Intrahepatic metastasis frequently manifests as intrahepatic vascular microinvasion and micrometastasis, implying the highly aggressive nature of HCC cells. 1 Uncovering the mechanisms underlying metastasis could reveal previously unappreciated features of HCC.
The complex heterogeneity of epithelial cells was described as EMT, a transient and reversible dedifferentiation to a mesenchymallike or mesenchymal phenotype, which partly explains the phenotype of primary HCC accompanied by portal vein tumor thrombi and intrahepatic metastasis. 3 Epithelial-mesenchymal transition is executed in response to pleiotropic signaling factors that activate specific EMT-TFs and then trigger the dissociation of carcinoma cells from primary carcinomas. 4 Epithelial-mesenchymal transition-TFs are pivotal regulators, as they suppress E-cadherin, the prototypic adhesion molecule essential for mediating cell polarity, differentiation, migration, and stem cell-like properties, which leads to the transition from well-differentiated adenoma to invasive. 5 The most prominent EMT-TFs, such as Snail (SNAI1), Slug (SNAI2), and ZEB, bind to E-boxes present on the E-cadherin promoter and suppress its transcription directly. 6 Other EMT-TFs, such as bHLH proteins (Twist1 and Twist2), are commonly recognized as indirect repressors of E-cadherin. 7 It is now becoming clear that chronic inflammatory stimuli are indispensable in tumorigenesis and progression. 8 Various proinflammatory cytokines, including tumor necrosis factor, IL-8, IL-1β, IL-6, IL-10, IL-18, and IL-17, are important inflammatory mediators on neoplastic processes both in inflammation-induced cancer and in inflammation that follows tumor development. 9 Previous studies have identified IL-8 as a key factor involved in the progression of digestive tract tumors. 10 In liver cancer, increased IL-8 expression is positively related to portal vein invasion and distant metastasis and negatively related to PFS and OS. 11 Hepatocellular carcinoma patients with high levels of IL-8 also experience an impaired benefit from immunotherapy, following poor prognosis. 12 Our previous research also confirmed that IL-8 was associated with prognostic clinical factors, including lymph node metastasis, recurrence, and alanine aminotransferase levels. Interleukin-8 upregulates Twist1 and Snail expression and induces EMT in hepatic cancer cells, resulting in enhanced invasion. 13 However, the cellular and molecular mechanisms involved in this modulation are not completely understood.
In recent years, the expansion of knowledge of ncRNA biology has revealed critical roles in metastatic processes. 14 MicroRNAs are an emerging class of primarily ncRNAs that bind to the 3′-UTRs of specific mRNAs, which leads to either mRNA degradation or translational pausing. 15,16 Functionally, miRNAs are pivotal determinants during liver cancer progression. p53 upregulates miR-200 and miR-192 family members, then represses ZEB1/2 expression, which normally sustains the epithelial phenotype, thus promoting EMT and HCC invasion. 17 Cross-regulation between cytokine and miRNA pathways is beginning to be elucidated. 18 However, it is largely unknown whether miRNAs targeting EMT-TFs involve IL-8-activated EMT in HCC.
Enlightened by the abovementioned progress, in this study, we combined in silico, bioinformatic, in vitro, and in vivo approaches to systematically investigate the contribution of miRNAs targeting EMT-TFs induced by IL-8 in liver cancer cells. The molecular mechanism underlying IL-8-modulated miRNA expression was also investigated.  19 The miRNA/mRNA and miRNA/mRNA/protein hybridizations were detected with Odyssey CLx Infrared Imaging System (LI-COR Biosciences). Details are described in Appendix S1. The sequences of primers, siRNAs, miRNAs, and oligonucleotides for EMSA are listed in Table S1.

| Chromatin immunoprecipitation assay
This procedure was carried out as described by the ChIP assay kit (Beyotime). Briefly, 10-cm plates were seeded with cells and allowed to grow to approximately 90% confluence. A 1% final formaldehyde concentration was used to cross-link proteins to DNA, which was then stopped by adding glycine to the suspension. The cells were collected by centrifugation, and the nuclear pellet was resuspended in ChIP buffer. The cell lysate was subjected to sonication and then incubated with 5 μg of Abs overnight, followed by incubation with protein A/G agarose beads overnight at 4°C. Bound DNA-protein complexes were eluted, and cross-links were reversed after a series of washes. The purified DNA was resuspended in TE buffer for PCR.
The sequences of primers for ChIP are listed in Table S1, and the antibodies are listed in Table S2.

| Dataset information
The Cancer Genome Atlas public database (https://cance rgeno me.nih.gov/), which includes 40 pairs of liver cancer, was used to analyze differences in miR-370-3p expression between HCC and adjacent nontumor tissues. Another 424 pairs were used to analyze the correlation between IL-8 and STAT3.

| Statistical analysis
Statistical analysis was undertaken with SPSS software (SPSS Inc.).
Values are expressed as the mean ± SD. The difference was analyzed using ANOVA or χ 2 -test. Spearman's rank correlation coefficient was used to determine the correlation between two parameters.
The PFS and OS curves were assessed using Kaplan-Meier plots and compared with log-rank test. p < 0.05 was considered statistically significant.

| Interleukin-8 promotes metastasis of HCC and predicts poor outcomes in patients
Increased expression of the pro-angiogenic/pro-inflammatory chemokine IL-8 (CXCL8) and/or its receptors (CXCR1/CXCR2) has been characterized in various cells, such as cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, which suggests that IL-8 could function as a significant regulatory factor within tumor microenvironment. 20 In a cohort of freshfrozen serum samples collected from primary and metastatic HCC patients (n = 76 and 24, respectively), the metastatic group showed increased IL-8 expression ( Figure 1A, p = 0.05). Moreover, the OS in the IL-8-high group was significantly shorter than those in the IL-8-low group ( Figure 1B), implying that IL-8 affects patient survival by promoting metastasis. We examined the expression of several EMT-TFs, including Snail, Slug, Twist1, ZEB1, and ZEB2, and found that Snail and Twist1 apparently increased after IL-8 stimulation.
At the same time, the expression of epithelial marker proteins Ecadherin and ZO-1 decreased, and interstitial characteristic proteins N-cadherin, fibronectin, and vimentin increased ( Figure 1C).
A spike-like filopodia morphology change was also observed in IL-8-stimulated cells ( Figure 1D). Consequently, IL-8 promoted Huh-7 and HepG2 migration and invasion in vitro ( Figure 1E). An HCC metastasis model was established to mimic the blood spreading of cancer cells with Huh-7 cells injected into the mouse tail vein. As shown in Figure 1F, mice treated with IL-8 showed more severer liver metastasis than controls. Collectively, these results suggested that IL-8 promotes cancer cell metastasis and was correlated with poor prognosis.

| Identification of EMT-TFs targeting miRNA
We wanted to explore whether a factor regulates Snail and Twist1 simultaneously, perhaps a key link between IL-8 and EMT. Certain miRNAs target multiple genes simultaneously and exert synergistic effects on cellular biofunctions. 21 We undertook an in silico analysis using three algorithms (TargetScan, miRbase, and micro RNA.org) that predict targeting transcripts by miRNAs based on the 3′-UTR of Snail and Twist1.
Minimum free energy represents the hybridization ability between miRNAs and targeting sequences, and we have previously reported that MFE < −20 kcal/mol is a key parameter for efficient binding. 19 As shown in Figure S1, there are 11 miRNAs that potentially target Snail and Twist1; miR-370-3p is unique in that its MFE is lower than −20 kcal/mol. Mutated miR-370-3p was constructed as three nucleotides in the WT sequence, and MFE between miRNAs and mRNAs was significantly reduced to −18.6 kcal/mol and − 10.6 kcal/ mol, respectively ( Figure 2A). We then explored the differentially expressed miRNAs between HCC and normal samples in TCGA.
A Venn diagram of the screened differential genes and 11 candidate miRNAs was constructed, which identified miR-370 and miR-381 at the intersection. Our review of other studies found that the regulation of miR-370 on EMT-TFs has not been reported yet.
MicroRNA-381 was reported to influence IL-8 expression, indicating that miR-381 might be an upstream factor of IL-8. To further confirm the direct regulation of miR-370-3p on 3′-UTR of Snail and Twist1 mRNA, HEK293T cells were transfected with luciferase reporter vectors containing WT 3′-UTR of Snail or Twist1. As shown in Figure 2C, miR-370-3p mimic suppressed the luciferase activity of Snail 3′-UTR by approximately 20% and Twist1 3′-UTR by approximately 25%, whereas mutant miR-370-3p did not show any effect. We concluded that miR-370-3p targets Snail and Twist1 directly and specifically.

| Hsa-miR-370-3p inhibited hepatic cancer cell EMT and metastasis
The function of miR-370-3p in EMT is less clear, nor has it been studied in the context of HCC metastasis. We first transfected Huh-7 and HepG2 cells with synthetic miR-370-3p molecules (mimic, inhibitor, and corresponding negative controls) and verified that miR-370-3p downregulated Snail and Twist1 expression at both mRNA and protein levels ( Figure 3A,B). MicroRNA-370-3p mimic group showed increased E-cadherin and ZO-1, but decreased N-cadherin, vimentin, and fibronectin expression, and miR-370-3p inhibitor exerted the opposite effects ( Figure 3B).
Consistently, miR-370-3p mimics inhibited Huh-7 and HepG2 cell migration and invasion in vitro ( Figure 3C). The effects of miR-370-3p mimics were similar to those of siRNA Snail and siRNA Twist1 ( Figure S2 and S3), mice bearing Huh-7 miR-370 -3p showed much less liver metastasis than control. As we did not detect apparent changes in Huh-7 or HepG2 cell proliferation after miR-370-3p overexpression ( Figure S4A), we believed that the diminished invasion and metastasis were due to the anti-EMT effect of miR-370-3p.

| Interleukin-8 suppressed miR-370-3p expression by STAT3 occupying the promoter
A previous study showed that miR-370 expression was influenced by IL-6, 23 which prompted us to further explore whether IL-8 regulates the expression of miR-370-3p. Interestingly, miR-370-3p expression was dramatically decreased after IL-8 treatment ( Figure 5A). We previously detected apparent phosphorylation of the Tyr 705 residue of STAT3 in IL-8-treated liver cancer cells. 13 Using HCC data from TCGA, we found that the expression of STAT3 was positively correlated with IL-8 expression ( Figure 5B).
As a transcription factor, STAT3 frequently binds to the promoter of genes and mediates their epigenetic modification. 24 Some miRNAs interact with the JAK-STAT3 pathway to regulate inflammation and oncogenesis, which could be potentially applied as clinical inhibitors. 25 We hypothesized that STAT3 might display transcription-modulatory properties between IL-8 and miR-370-3p through chromatin topological modulation. We confirmed this hypothesis by investigating UCSC and MEME websites, which identified the potential STAT3 binding motif on miR-370-3p promoter ( Figure 5E). A dual-luciferase reporter assay verified that miR-370-3p promoter activity was significantly decreased upon STAT3 overexpression ( Figure 5F). The direct interaction between STAT3 and miR-370-3p promoter was further confirmed by ChIP assays ( Figure 5G). Taken together, these results showed that IL-8 regulates miR-370-3p expression through STAT3 directly binding to miR-370-3p promoter.

| Interleukin-8 and miR-370-3p levels correlate with prognosis of HCC patients
All the above results in vitro and in vivo verified that miR-370-3p plays critical roles in the IL-8-dependent EMT regulatory circuit, and we consequently explored the clinical value in patients.
Transcatheter arterial chemoembolization, chemotherapy, and TKIs are the main treatments for liver cancer, but TACE trauma aggravates hypoxia and inflammatory reactions. It has been reported that chemotherapy and radiotherapy also increase inflammation. 27 We measured serum IL-8 levels in 17 untreated HCC patients, 9 of whom subsequently underwent TACE. We found that IL-8 levels increased after TACE treatment ( Figure 7A). The level of IL-8 also increased in the culture supernatant of HepG2 treated with several common chemotherapeutic drugs, such as Doxorubicin and Oxaliplatin, but TKI Sorafenib had no significant effect ( Figure 7B).
As miR-370-3p targets Snail and Twist1, it is rational to suppose  28 The miR-7/IGF1R/Snail axis increases E-cadherin expression and partially reverses EMT in gastric cancer. 33 However, it has not been reported whether miR-NAs regulate multiple EMT-TFs simultaneously.
According to the library screening results, we undertook comprehensive studies to verify that miR-370-3p modulates Snail as well as Twist1 directly and specifically. Although miR-370-3p suppressed the expression of Snail and Twist1 by approximately 20% and 25%, respectively, they significantly reduced liver cancer cell invasion and metastasis by approximately 60%, due to the synergistic inhibition effects on two targets.
Epithelial-mesenchymal transition-TFs play pleiotropic roles in cancer progression and are involved in cellular processes both in classical EMT (the loss of cell-to-cell adhesion) and other cellular processes (cell apoptosis, cell proliferation, stemness, and immunosuppression). 1 However, the synergistic effect of other target genes of miR-370-3p might also contribute to inhibition of invasion. In fact, miR-370-3p acts as a tumor suppressor in different types of cancer by targeting multiple protumorigenic genes. 34,35 For example, miRNA-370 suppresses the progression and proliferation of human astrocytoma and glioblastoma by negatively regulating β-catenin and causing activation of FOXO3a. 36 Upregulation of miRNA-370 promotes cell apoptosis and inhibits proliferation by targeting PTEN in human gastric cancer. 37 Other indirect molecular mechanisms underlying miR-370-3p antitumor activities have also been reported, such as regulating phosphatidylinositol, MAPK, and B-cell receptor signaling. 38 MicroRNA-370-3p alleviated ulcerative colitis-associated colorectal cancer by inhibiting the inflammatory response and EMT. 39 The androgen receptor-mediated ADAR2/circFNTA/miR-370-3p/FNTA pathway activates KRAS signaling to alter bladder cancer cell invasion and chemosensitivity. 40 In addition, several interactions among miRNAs, EMT inducers, and EMT-TFs facilitate cancer progression. Tight interconnected and complex regulatory loops between miRNAs and EMT-TFs control epithelial cell plasticity, providing self-enhancing signals and robustness to maintain epithelial or mesenchymal status. 41 The ZEB/miR-200 and Snail/miR-34 networks are significant feedback loops during EMT. 42,43 Twist1 induces the production of IL-8, which activates MMPs and promotes invasion of breast epithelial and cancer cells. 44 Additionally, miRNAs modulate genes related to inflammation; for example, the CXCL12/miR-370-3p/HMGA2 pathway is involved in tumor growth and invasiveness of nonfunctional pituitary adenomas. 45 Reduced Let-7 directly activates the IL-6 cascade, which involves STAT3, increasing nuclear factor-κB transcriptional activity and transformation from normal to cancer. 46 A previous study showed that hypermethylation of CpG was involved in IL-6-reduced miR-370 suppression. 23 In the present study, we reported a novel mechanism by which IL-8 downregulated miR-370-3p expression through the STAT3 pathway and HDAC recruitment. Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting. 47 Our results confirmed that SAHA and chidamide significantly reduced the migration and invasion of cancer cells, which implicates potential therapy for HCC. We noticed that many ongoing clinical trials are exploring the therapeutic value of HDAC inhibitors in HCC (NCT01075113 and NCT00321594). From a new perspective, our study showed that miR-370-3p inhibits metastasis by regulating EMT-TFs both Snail and Twist1; we also elucidated that the IL-8/STAT3/miR-370-3p pathway relies on HDAC1 recruitment, which characterizes a crucial mechanism underlying the metastasis of HCC. This has opened up a new avenue for further pharmacological research and suggests that HDAC inhibitors might be an adjuvant therapy to prevent HCC metastasis.
Nevertheless, our study is limited and cannot explain the full mechanism by which IL-8 promotes or miRNA-370-3p inhibits HCC invasion and metastasis. First, other EMT-TFs might also be involved in the process of IL-8-induced HCC invasion. Second, IL-8 can active many signal pathways. such as the PI3K/Akt pathway, to exert protumor effects. In addition to inducing EMT, IL-8 promotes tumor metastasis as well as inducing angiogenesis or recruiting larger numbers of immunosuppressive cells into the tumor microenvironment.
Third, miR-370-3p could modulate other targeting genes to attenuate the protumor functions of IL-8. Finally, other miRNAs that regulate multiple EMT-TFs also possibly play important roles underlying IL-8-induced HCC invasion and metastasis. What we have reported so far is only one of the potential mechanisms by which IL-8 promotes the metastasis of HCC and miRNA-370-3p interference, and more possible mechanisms need to be explored deeply.

ACK N OWLED G M ENTS
The Flag-HDAC constructs were generously provided by Professor Tiebang Kang (Sun Yat-sen University Cancer Center). We thank Professor Rongping Guo (Sun Yat-sen University Cancer Center) for providing MHCC97H, Hep3B, SNU-368, PLC and CRL-8024 cell lines.

D I SCLOS U R E
The authors have no conflicts of interest to declare.

E TH I C S S TATEM ENT
Approval of the research protocol: The study was approved by the research medical ethics committee of Sun Yat-sen University.