METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

CBX2-mediated suppression of SIAH2 triggers WNK1 accumulations to promote glycolysis in hepatocellular carcinoma.

Previous studies have highlighted the poor prognosis of liver cancer, and treatment effects are overall limited. We aimed to confirm the biological roles of SIAH2 in liver cancer and provide potential therapeutic targets. Differential analysis was conducted based on public datasets and found that SIAH2 expressed lowly in HCC samples relative to normal tissues, which was demonstrated in tumor samples via immunohistochemistry (IHC). Besides, SIAH2 overexpression could significantly suppress HCC proliferation. SIAH2 deficiency induced cell proliferation, migration and self-renewal abilities in vitro and in vivo. Mechanistically, SIAH2 could interact with WNK1, and trigger the ubiquitination and degradation of WNK1 proteins. In addition, low SIAH2 depended on elevated WNK1 proteins to drive HCC malignant features, including proliferation, migration and stemness. Meanwhile, we further found that CBX2 could regulate SIAH2 expressions. CBX2 cooperated with EZH2 to mediate the H3K27me3 enrichment on the promoter region of SIAH2 to suppress its transcriptional levels. High CBX2/EZH2 levels in HCC correlated with poor prognosis of patients. Gene set enrichment analysis (GSEA) further implicated that WNK1 correlates tightly with glycolytic process in HCC samples. WNK1 overexpression was found to notably enhance glycolytic activity, whereas WNK1 deficiency could significantly suppress the HCC glycolysis activity. Lastly, the subcutaneous tumor model further demonstrated that targeting WNK1 was effective to inhibit the in vivo tumor growth of SIAH2low HCC. Collectively, down-regulated SIAH2 expressions induced by CBX2/EZH2 could drive progression and glycolysis via accumulating WNK1 proteins, indicating that CBX2/SIAH2/WNK1 axis is a potential prognostic biomarker and therapeutic vulnerability for human HCC.

N6 -methyladenosine-modified FAM111A-DT promotes hepatocellular carcinoma growth via epigenetically activating FAM111A.

As an epitranscriptomic modulation manner, N6 -methyladenosine (m6 A) modification plays important roles in various diseases, including hepatocellular carcinoma (HCC). m6 A modification affects the fate of RNAs. The potential contributions of m6 A to the functions of RNA still need further investigation. In this study, we identified long noncoding RNA FAM111A-DT as an m6 A-modified RNA and confirmed three m6 A sites on FAM111A-DT. The m6 A modification level of FAM111A-DT was increased in HCC tissues and cell lines, and increased m6 A level was correlated with poor survival of HCC patients. m6 A modification increased the stability of FAM111A-DT transcript, whose expression level showed similar clinical relevance to that of the m6 A level of FAM111A-DT. Functional assays found that only m6 A-modified FAM111A-DT promoted HCC cellular proliferation, DNA replication, and HCC tumor growth. Mutation of m6 A sites on FAM111A-DT abolished the roles of FAM111A-DT. Mechanistic investigations found that m6 A-modified FAM111A-DT bound to FAM111A promoter and also interacted with m6 A reader YTHDC1, which further bound and recruited histone demethylase KDM3B to FAM111A promoter, leading to the reduction of the repressive histone mark H3K9me2 and transcriptional activation of FAM111A. The expression of FAM111A was positively correlated with the m6 A level of FAM111A-DT, and the expression of methyltransferase complex, YTHDC1, and KDM3B in HCC tissues. Depletion of FAM111A largely attenuated the roles of m6 A-modified FAM111A-DT in HCC. In summary, the m6 A-modified FAM111A-DT/YTHDC1/KDM3B/FAM111A regulatory axis promoted HCC growth and represented a candidate therapeutic target for HCC.

The underlying pathophysiology association between the Type 2-diabetic and hepatocellular carcinoma.

Type 2-diabetic (T2D) disease has been reported to increase the incidence of liver cancer, however, the underlying pathophysiology is still not fully understood. Here, we aimed to reveal the underlying pathophysiology association between the T2D and hepatocellular carcinoma (HCC) and, therefore, to find the possible therapeutic targets in the occurrence and development of HCC. The methylation microarray data of T2D and HCC were extracted from the Gene Expression Omnibus and The Cancer Genome Atlas. A total of 504 differentially methylated genes (DMGs) between T2D samples and the controls were identified, whereas 6269 DMGs were identified between HCC samples and the control groups. There were 336 DMGs coexisting in diabetes and HCC, among which 86 genes were comethylated genes. These genes were mostly enriched in pathways as glycosaminoglycan biosynthesis, fatty acid, and metabolic pathway as glycosaminoglycan degradation and thiamine, fructose and mannose. There were 250 DMGs that had differential methylation direction between T2D DMGs and HCC DMGs, and these genes were enriched in the Sphingolipid metabolism pathway and immune pathways through natural killer cell-mediated cytotoxicity and ak-STAT signaling pathway. Eight genes were found related to the occurrence and development of diabetes and HCC. Moreover, the result of protein-protein interaction network showed that CDKN1A gene was related to the prognosis of HCC. In summary, eight genes were found to be associated with the development of HCC and CDKN1A may serve as the potential prognostic gene for HCC.

HIF­1α and RACGAP1 promote the progression of hepatocellular carcinoma in a mutually regulatory way.

Hypoxia, a condition characterized by low oxygen levels, serves an important role in the progression of hepatocellular carcinoma (HCC). However, the precise molecular mechanisms underlying hypoxia­induced HCC progression are yet to be fully elucidated. The present study assessed the involvement of two key factors, hypoxia­inducible factor­1α (HIF­1α) and Rac GTPase activating protein 1 (RACGAP1), in HCC development under hypoxic conditions. HIF­1α and RACGAP1 genes were overexpressed and knocked down in Hep3B and Huh7 cells using lentiviral transduction and the levels of HIF­1α and RACGAP1 in the cells were assessed using quantitative PCR, western blotting and immunofluorescence. Co­immunoprecipitation experiments were performed to evaluate the interaction between HIF­1α and RACGAP1. Subsequently, the proliferation, apoptosis, migration and invasion of Hep3B and Huh7 cells were assessed using the Cell Counting Kit­8 assay, flow cytometry, Transwell assay and migration experiments. The expression levels of HIF­1α and RACGAP1 in normal and HCC tumor samples were analyzed utilizing the Gene Expression Profiling Interactive Analysis database. Furthermore, correlations between HIF­1α/RACGAP1 gene expression levels and patient survival outcomes were evaluated using the Kaplan­Meier plotter. Knockdown of HIF­1α resulted in a significant decrease in RACGAP1 expression, whilst overexpression of HIF­1α resulted in a significant increase in RACGAP1 expression. Moreover, overexpression and knockdown of RACGAP1 had the same effect on HIF­1α expression. Additionally, it was demonstrated that HIF­1α and RACGAP1 interacted directly within a complex. Overexpression of HIF­1α or RACGAP1 significantly increased proliferation, invasion and migration, and significantly decreased the proportion of apoptotic Hep3B and Huh7 cells. Conversely, knockdown of HIF­1α or RACGAP1 significantly decreased proliferation, invasion and migration, and significantly increased the proportion of apoptotic Hep3B and Huh7 cells. In addition, the combined knockdown or overexpression of HIF­1α and RACGAP1 had a more pronounced effect on HCC cell migration compared with knockdown of HIF­1α alone. Furthermore, there was a significant positive correlation between the expression levels of HIF­1α and RACGAP1 in HCC tissues and patients with HCC and upregulation of both HIF­1α and RACGAP1 demonstrated a lower overall survival probability. In conclusion, HIF­1α and RACGAP1 may synergistically contribute to the development of HCC, highlighting their potential as valuable targets for HCC therapy.

N6-Methyladenosine-Modified LEAWBIH Drives Hepatocellular Carcinoma Progression through Epigenetically Activating Wnt/ß-Catenin Signaling.

Purpose: N6-methyladenosine (m6A) modification plays an important role in regulating RNA maturation, stability, and translation. Thus, m6A modification is involved in various pathophysiological processes including hepatocellular carcinoma (HCC). However, the direct contribution of m6A modifications to RNA function in HCC remains unclear. Here, we identified LEAWBIH (long non-coding RNA epigenetically activating Wnt/ß-catenin signalling in HCC) as an m6A-modified long non-coding RNA (lncRNA) and investigated the effects of m6A on the function of LEAWBIH in HCC. Methods: Quantitative polymerase chain reaction was performed to measure the gene expression in tissues and cells. The level of m6A modification was detected using a methylated RNA immunoprecipitation assay and single-base elongation- and ligation-based qPCR amplification method. Cell proliferation was evaluated using the Glo cell viability and CCK-8 assays. Cell migration and invasion were evaluated using Transwell migration and invasion assays. The mechanisms of m6A modified LEAWBIH were investigated using chromatin isolation by RNA purification, chromatin immunoprecipitation, and dual-luciferase reporter assays. Results: LEAWBIH was highly expressed and correlated with poor survival in HCC patients. LEAWBIH was identified as a m6A-modified transcript. m6A modification increased LEAWBIH transcript stability. The m6A modification level of LEAWBIH was increased in HCC, and a high m6A modification level of LEAWBIH predicted poor survival. LEAWBIH promotes HCC cell proliferation, migration, and invasion in an m6A modification-dependent manner. Mechanistic investigations revealed that m6A-modified LEAWBIH activated Wnt/ß-catenin signaling. m6A-modified LEAWBIH binds to the m6A reader YTHDC1, which further interacts with and recruits H3K9me2 demethylase KDM3B to CTNNB1 promoter, leading to H3K9me2 demethylation and CTNNB1 transcription activation. Functional rescue assays showed that blocking Wnt/ß-catenin signaling abolished the role of LEAWBIH in HCC. Conclusion: m6A-modified LEAWBIH exerts oncogenic effects in HCC by epigenetically activating Wnt/ß-catenin signaling, highlighting m6A-modified LEAWBIH as a promising therapeutic target for HCC.

m6A modification of AC026356.1 facilitates hepatocellular carcinoma progression by regulating the IGF2BP1-IL11 axis.

N6-methyladenosine (m6A) is the most common RNA modification in eukaryotic RNAs. Although the important roles of m6A in RNA fate have been revealed, the potential contribution of m6A to RNA function in various diseases, including hepatocellular carcinoma (HCC), is still unclear. In this study, we identified a novel m6A-modified RNA AC026356.1. We found that AC026356.1 was increased in HCC tissues and cell lines. High expression of AC026356.1 was correlated with poor survival of HCC patients. m6A modification level of AC026356.1 was also increased in HCC and more significantly correlated with poor survival of HCC patients. Functional assays showed that m6A-modified AC026356.1 promoted HCC cellular proliferation, migration, and liver metastasis. Gene set enrichment analysis showed that AC026356.1 activated IL11/STAT3 signaling. Mechanistic investigation showed that m6A-modified AC026356.1 bound to IGF2BP1. The interaction between m6A-modified AC026356.1 and IGF2BP1 promoted the binding of IL11 mRNA to IGF2BP1, leading to increased IL11 mRNA stability and IL11 secretion. Functional rescue assays showed that depletion of IL11 reversed the oncogenic roles of AC026356.1. These findings revealed the potential influences of m6A modification on RNA biological functions and suggested that targeting m6A modification may be a novel strategy for HCC treatment.

Long non-coding RNA PAARH promotes hepatocellular carcinoma progression and angiogenesis via upregulating HOTTIP and activating HIF-1α/VEGF signaling.

Hepatocellular carcinoma (HCC) is one of the leading lethal malignancies and a hypervascular tumor. Although some long non-coding RNAs (lncRNAs) have been revealed to be involved in HCC. The contributions of lncRNAs to HCC progression and angiogenesis are still largely unknown. In this study, we identified a HCC-related lncRNA, CMB9-22P13.1, which was highly expressed and correlated with advanced stage, vascular invasion, and poor survival in HCC. We named this lncRNA Progression and Angiogenesis Associated RNA in HCC (PAARH). Gain- and loss-of function assays revealed that PAARH facilitated HCC cellular growth, migration, and invasion, repressed HCC cellular apoptosis, and promoted HCC tumor growth and angiogenesis in vivo. PAARH functioned as a competing endogenous RNA to upregulate HOTTIP via sponging miR-6760-5p, miR-6512-3p, miR-1298-5p, miR-6720-5p, miR-4516, and miR-6782-5p. The expression of PAARH was significantly positively associated with HOTTIP in HCC tissues. Functional rescue assays verified that HOTTIP was a critical mediator of the roles of PAARH in modulating HCC cellular growth, apoptosis, migration, and invasion. Furthermore, PAARH was found to physically bind hypoxia inducible factor-1 subunit alpha (HIF-1α), facilitate the recruitment of HIF-1α to VEGF promoter, and activate VEGF expression under hypoxia, which was responsible for the roles of PAARH in promoting angiogenesis. The expression of PAARH was positively associated with VEGF expression and microvessel density in HCC tissues. In conclusion, these findings demonstrated that PAARH promoted HCC progression and angiogenesis via upregulating HOTTIP and activating HIF-1α/VEGF signaling. PAARH represents a potential prognostic biomarker and therapeutic target for HCC.

ADORA2A-AS1 Restricts Hepatocellular Carcinoma Progression via Binding HuR and Repressing FSCN1/AKT Axis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies. Increasing evidence revealed that long noncoding RNAs (lncRNAs) were frequently involved in various malignancies. Here, we explored the clinical significances, roles, and mechanisms of lncRNA ADORA2A antisense RNA 1 (ADORA2A-AS1) in HCC. METHODS: The clinical significances of ADORA2A-AS1 in HCC were analyzed using RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) project. The expressions of ADORA2A-AS1, Fascin Actin-Bundling Protein 1 (FSCN1), Matrix Metallopeptidase 2 (MMP2), and Baculoviral IAP Repeat Containing 7 (BIRC7) in HCC tissues and cells were measured by qRT-PCR. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), caspase-3 activity assay, transwell migration and invasion assays, and xenograft growth and metastasis experiments were performed to evaluate the roles of ADORA2A-AS1 in HCC. RNA pull-down, RNA immunoprecipitation, qRT-PCR, Western blot, and RNA stability assay were performed to elucidate the mechanisms of ADORA2A-AS1 in HCC. RESULTS: ADORA2A-AS1 was identified as an HCC-related lncRNA, whose low expression was correlated with advanced stage and poor outcome in HCC. Gain- and loss-of functional experiments demonstrated that ADORA2A-AS1 inhibited HCC cell proliferation, induced cell apoptosis, repressed cell migration and invasion, and repressed xenograft growth and metastasis in vivo. Mechanistically, ADORA2A-AS1 competitively bound HuR (Hu Antigen R), repressed the binding of HuR to FSCN1 transcript, decreased FSCN1 transcript stability, and downregulated FSCN1 expression. The expression of FSCN1 was negatively correlated with ADORA2A-AS1 in HCC tissues. Through downregulating FSCN1, ADORA2A-AS1 repressed AKT pathway activation. Functional rescue assays showed that blocking of FSCN1/AKT axis abrogated the roles of ADORA2A-AS1 in HCC. CONCLUSION: Low-expression ADORA2A-AS1 is correlated with poor survival of HCC patients. ADORA2A-AS1 exerts tumor-suppressive roles in HCC via binding HuR and repressing FSCN1/AKT axis.

Hepatoma Cell-Derived Extracellular Vesicles Promote Liver Cancer Metastasis by Inducing the Differentiation of Bone Marrow Stem Cells Through microRNA-181d-5p and the FAK/Src Pathway.

Bone marrow mesenchymal stem cells (BMSCs) are beneficial to repair the damaged liver. Tumor-derived extracellular vesicles (EV) are notorious in tumor metastasis. But the mechanism underlying hepatoma cell-derived EVs in BMSCs and liver cancer remains unclear. We hypothesize that hepatoma cell-derived EVs compromise the effects of BMSCs on the metastasis of liver cancer. The differentially expressed microRNAs (miRNAs) were screened. HepG2 cells were transfected with miR-181d-5p mimic or inhibitor, and the EVs were isolated and incubated with BMSCs to evaluate the differentiation of BMSCs into fibroblasts. Hepatoma cells were cultured with BMSCs conditioned medium (CM) treated with HepG2-EVs to assess the malignant behaviors of hepatoma cells. The downstream genes and pathways of miR-181d-5p were analyzed and their involvement in the effect of EVs on BMSC differentiation was verified through functional rescue experiments. The nude mice were transplanted with BMSCs-CM or BMSCs-CM treated with HepG2-EVs, and then tumor growth and metastasis in vivo were assessed. HepG2-EVs promoted fibroblastic differentiation of BMSCs, and elevated levels of α-SMA, vimentin, and collagen in BMSCs. BMSCs-CM treated with HepG2-EVs stimulated the proliferation, migration, invasion and epithelial-mesenchymal-transition (EMT) of hepatoma cells. miR-181d-5p was the most upregulated in HepG2-EVs-treated BMSCs. miR-181d-5p targeted SOCS3 to activate the FAK/Src pathway and SOCS3 overexpression inactivated the FAK/Src pathway. Reduction of miR-181d-5p in HepG2-EVs or SOCS3 overexpression reduced the differentiation of BMSCs into fibroblasts, and compromised the promoting effect of HepG2-EVs-treated BMSCs-CM on hepatoma cells. In vivo, HepG2-EVs-treated BMSCs facilitated liver cancer growth and metastasis. In conclusion, HepG2-EVs promote the differentiation of BMSCs, and promote liver cancer metastasis through the delivery of miR-181d-5p and the SOCS3/FAK/Src pathway.

M2 macrophage-derived extracellular vesicles facilitate CD8+T cell exhaustion in hepatocellular carcinoma via the miR-21-5p/YOD1/YAP/ß-catenin pathway.

Hepatocellular carcinoma (HCC) is a common malignancy. CD8+ T cell-mediated immune response is critical for the inhibition of HCC progression. M2 macrophages participate in HCC progression. This study set out to investigate the effect of M2 macrophage-derived extracellular vesicles (EVs) on CD8+ T cell exhaustion in HCC. M2 macrophage-derived EVs were isolated and identified. The murine model of primary HCC was established through DEN/CCl4 induction, and model mice were injected with EVs. Peripheral blood mononuclear cells (PBMCs) were isolated from the mouse liver and CD8+ T cells were sorted. The expressions of immune checkpoint inhibitory receptors and effector cytokines on CD8+ T cells were detected, followed by the evaluation of CD8+ T cell proliferation and killing function. miR-21-5p expression in M2 macrophage-derived EVs was detected. The binding relationship between miR-21-5p and YOD1 was verified. The activation of the YAP/ß-catenin pathway was detected. Consequently, M2 macrophage-derived EVs promoted CD8+ T cell exhaustion in HCC mice. miR-21-5p expression was upregulated in M2 macrophage-derived EVs, and EVs carried miR-21-5p into HCC tissues. miR-21-5p targeted YOD1. Inhibition of miR-21-5p or overexpression of YOD1 annulled the promoting effect of EVs on CD8+ T cell exhaustion. YOD1 inactivated the YAP/ß-catenin pathway. In conclusion, M2 macrophage-derived EVs facilitated CD8+ T cell exhaustion via the miR-21-5p/YOD1/YAP/ß-catenin axis. This study may confer novel insights into the immunotherapy of HCC.

Hypoxia-induced Fascin-1 upregulation is regulated by Akt/Rac1 axis and enhances malignant properties of liver cancer cells via mediating actin cytoskeleton rearrangement and Hippo/YAP activation.

In solid tumors, hypoxia facilitates malignant progression of cancer cells by triggering epithelial-mesenchymal transition (EMT) and cancer stemness. Fascin-1, an actin-bundling protein, takes part in the formation of many actin-based cellular structures. In the present study, we explored the potential functions of hypoxia-induced upregulation of Fascin-1 in liver cancer. Transcriptome RNA-sequencing was conducted to identify hypoxia-related genes. The potential functions of Fascin-1 were evaluated by western blot, transwell migration and invasion assays, sphere-formation assay, tumor xenograft growth, gelatin zymography analysis, immunofluorescence, cell viability assay, soft agar assay, and flow cytometry. We found that Fascin-1 was upregulated by hypoxia in liver cancer cell lines, elevated in liver cancer patients and correlated with larger tumor size, lymph node metastasis, distant metastasis, and shorter overall survival. Knockdown of Fascin-1 suppressed migration, invasion, EMT, stemness, and tumor xenograft growth of liver cancer cells under both normoxia and hypoxia conditions, while forced Fascin-1 expression showed opposite effects. Moreover, hypoxia-induced upregulation of Fascin-1 was regulated by the Akt/Rac1 signaling, and inhibition of Akt/Rac1 signaling by EHop-016 and MK-2206 restrained migration, invasion, EMT, and stemness of liver cancer cells under hypoxia. Furthermore, Fascin-1 knockdown suppressed MMP-2 and MMP-9 expression, impaired actin cytoskeleton rearrangement, inactivated Hippo/YAP signaling, and increased Sorafenib sensitivity in liver cancer cells. Our study provided a novel insight of Fascin-1 in regulating migration, invasion, EMT, and stemness of liver cancer cells under normoxia and hypoxia conditions.

Corrigendum: Hepatoma Cell-Derived Extracellular Vesicles Promote Liver Cancer Metastasis by Inducing the Differentiation of Bone Marrow Stem Cells Through microRNA-181d-5p and the FAK/Src Pathway.

[This corrects the article DOI: 10.3389/fcell.2021.607001.].

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/ß-catenin signaling via upregulating HOMER3. Functional rescue experiments revealed that HOMER3/Wnt/ß-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/ß-catenin axis and CSF-1, respectively. HOMER3-AS1 might be a promising prognostic and therapeutic target for HCC.

lncRNA MAGI2-AS3 Exerts Antioncogenic Roles in Hepatocellular Carcinoma via Regulating the miR-519c-3p/TXNIP Axis.

INTRODUCTION: Our work was aimed to explore the mechanisms of MAGI2 antisense RNA 3 (MAGI2-AS3) in regulating hepatocellular carcinoma (HCC) carcinogenesis. METHODS: MAGI2-AS3, microRNA-519c-3p (miR-519c-3p), and thioredoxin interacting protein (TXNIP) levels in HCC were detected by the RT-qPCR method. Cell proliferation and apoptosis rate were measured using Cell Counting Kit-8 assay and flow cytometry assay. Relationship between MAGI2-AS3, TXNIP, and miR-519c-3p were analyzed via luciferase activity assay, RNA pull-down assay, and RNA immunoprecipitation assay. Mouse xenograft models of HCC were conducted to explore the roles of MAGI2-AS3 in vivo. RESULTS: MAGI2-AS3 levels were elevated, and miR-519c-3p decreased in HCC. MAGI2-AS3 overexpression inhibits while its knockdown stimulates HCC cell growth through miR-519c-3p. Moreover, miR-519c-3p overexpression stimulates HCC cell growth. MAGI2-AS3 serves as competing endogenous RNA (ceRNA) of miR-519c-3p to regulate TXNIP in HCC. And, TXNIP upregulation weakened the influence of MAGI2-AS3 knockdown on HCC cell behaviors. Additionally, MAGI2-AS3 overexpression suppressed HCC tumor growth in vivo. CONCLUSION: MAGI2-AS3 inhibits HCC tumorigenesis through miR-519c-3p/TXNIP axis in vitro and in vivo, indicating MAGI2-AS3 plays a crucial role in HCC development.

Long Noncoding RNA PART1 Promotes Hepatocellular Carcinoma Progression via Targeting miR-590-3p/HMGB2 Axis.

INTRODUCTION: In East Asia, hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancer types. Long noncoding RNA (lncRNA) prostate androgen-regulated transcript 1 (PART1) was reported to play crucial roles in regulating cancer progression. However, roles and mechanisms of action of PART1 in hepatocellular carcinoma (HCC) still remain unknown. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) method was used to detect the PART1 expression level in HCC cells. Cell proliferation, colony formation, and transwell invasion assays were performed to investigate the biological roles of PART1 on HCC cell behaviors. Bioinformatic analysis methods were performed to analyze connections of microRNA-590-3p (miR-590-3p) with PART1 or high mobility group box 2 (HMGB2) in HCC. Moreover, expression levels of PART1, miR-590-3p, and HMGB2 in HCC tissues and normal tissues were analyzed at ENCORI. RESULTS: PART1 expression was found to be significantly upregulated in HCC tissues and cells. Functionally, silencing of PART1 significantly suppressed HCC cell proliferation, colony formation and invasion in vitro, while forcing PART1 exerts opposite biological effects. Mechanically, miR-590-3p/HMGB2 axis was downstream target of PART1, and silencing of miR-590-3p or forcing of HMGB2 could rescue the stimulation effects of PART1 overexpression on HCC cell behaviors. DISCUSSION: Our results provided evidence that PART1 serves as oncogenic lncRNA through sponging miR-590-3p to upregulate HMGB2 expression in HCC.

SLC6A8 Knockdown Suppresses the Invasion and Migration of Human Hepatocellular Carcinoma Huh-7 and Hep3B Cells.

Liver cancer is considered the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths worldwide. Currently, there is no specific and effective therapy for hepatocellular carcinoma. Therefore, developing novel diagnostic and therapeutic strategies against hepatocellular carcinoma is of paramount importance. Solute carrier family 6 member 8 (SLC6A8) encodes the solute carrier family 6-8 to transport creatine into cells in a Na+ and Cl-- dependent manner. SLC6A8 deficiency is characterized by intellectual disabilities, loss of speech, and behavioral abnormalities. Of concern, the association of SLC6A8 with hepatocellular carcinoma remains elusive. In this study, we revealed that SLC6A8 knockdown significantly induced apoptosis and suppressed the migration and invasion of Hep3B and Huh-7 cells. These findings depicted the vital role of SLC6A8 in the initiation and progression of human hepatocellular carcinoma.

IGF2BP2 Promotes Liver Cancer Growth Through an m6A-FEN1-Dependent Mechanism.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. N6-methyladenosine (m6A) plays an important role in posttranscriptional gene regulation. METTL3 and IGF2BP2 are key genes in the m6A signal pathway and have recently been shown to play important roles in cancer development and progression. In our work, higher METTL3 and IGF2BP2 expression were found in HCC tissues and were associated with a poor prognosis. In addition, IGF2BP2 overexpression promoted HCC proliferation in vitro and in vivo. Mechanistically, IGF2BP2 directly recognized and bound to the m6A site on FEN1 mRNA and enhanced FEN1 mRNA stability. Overall, our study revealed that METTL3 and IGF2BP2, acting as an oncogene, maintained FEN1 expression through an m6A-IGF2BP2-dependent mechanism in HCC cells, and indicated a potential biomarker panel for prognostic prediction in liver cancer.

miR-632 Functions as Oncogene in Hepatocellular Carcinoma via Targeting MYCT1.

microRNAs (miRNAs) have been widely recognized as crucial regulators for tumorigenesis. However, the role of miR-632 in hepatocellular carcinoma (HCC) remains largely unknown. miR-632 expression in HCC cell lines was determined by quantitative real-time polymerase chain reaction. The role of miR-632 expression on overall survival of HCC patients was examined on the Kaplan-Meier plotter Web site. The dual luciferase reporter method was performed to investigate whether myc target 1 (MYCT1) was a target of miR-632. Cell counting kit-8 assay, colony formation assay, and Transwell invasion assay were performed to examine cell proliferation, colony formation, and cell invasion of HCC cells. The results showed miR-632 expression was elevated in HCC cell lines compared to normal cell lines. Loss-of-function experiments demonstrated that miR-632 downregulation was able to inhibit HCC cell proliferation, colony formation, and cell invasion. Moreover, miR-632 could negatively regulate the expression of MYCT1 in HCC cells. Importantly, the study showed miR-632 and MYCT1 were negatively correlated by analyzing the public data sets obtained from the Gene Expression Omnibus. Knockdown of MYCT1 by small interfering RNA partially reversed the effects of miR-632 on HCC cell events. The present study suggests that miR-632 regulates growth and invasion of HCC cells through targeting MYCT1.

miR-584-5p regulates hepatocellular carcinoma cell migration and invasion through targeting KCNE2.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancer type. This study was aimed to investigate the role of microRNA-584-5p (miR-584-5p) in regulating HCC progression. METHODS: The expression of miR-584-5p in HCC cell lines was analyzed by quantitative real-time polymerase chain reaction. Effects of miR-584-5p depletion on HCC cell proliferation, migration, and invasion in vitro were analyzed by cell counting kit-8 assay, wound-healing assay, and transwell invasion assay. miR-584-5p targeting potassium voltage-gated channel subfamily E regulatory subunit 2 (KCNE2) was identified using bioinformatics algorithm and dual-luciferase activity reporter assay. Kaplan-Meier Plotter website was used to investigate the effect of miR-584-5p or KCNE2 expression on the overall survival of HCC patients. RESULTS: In vitro functional assays showed miR-584-5p depletion decreased HCC cell proliferation, cell migration, and cell invasion. Moreover, miR-584-5p functions by directly targeting KCNE2, and it in turn, mediates the effects of miR-584-5p on HCC cell behaviors. CONCLUSIONS: These results demonstrated that miR-584-5p functions as an oncogenic miRNA in HCC.