USP40 promotes hepatocellular carcinoma progression through a YAP/USP40 positive feedback loop.

Yes-associated protein (YAP) is an essential driver of hepatocellular carcinoma (HCC) progression and the ubiquitin-proteasome system controls its abundance. However, the role of ubiquitin-specific protease 40 (USP40) in YAP stability remains unclear. Here, USP40 was first identified as a novel regulator of YAP abundance and its target genes in HCC cells. USP40 interacted with YAP to remove the lysine 48 (K48)-linked polyubiquitination of YAP at K252 and K315 sites, thereby maintaining YAP stability. USP40 facilitated the proliferation, colony formation, migration and spheroid formation of HCC cells in vitro and promoted HCC growth in vivo in a YAP-dependent manner. In turn, YAP transcriptionally activated USP40 expression in HCC cells. RNA sequencing analysis showed that about 37% of USP40-regulated genes overlapped with YAP-regulated genes. Interestingly, stiffness-induced USP40 upregulation was abolished by YAP knockdown, and USP40 knockdown attenuated stiffness-induced YAP accumulation in HCC cells. Clinical data demonstrated that USP40 was positively associated with YAP expression in HCC tissues and its high expression indicated a poor prognosis. In conclusion, the USP40/YAP positive feedback loop contributes to HCC progression, suggesting that USP40 may be a promising drug target for anti-HCC.

A novel nomogram predicting the early recurrence of hepatocellular carcinoma patients after R0 resection.

Background: Early tumor recurrence is one of the most significant poor prognostic factors for patients with HCC after R0 resection. The aim of this study is to identify risk factors of early recurrence, in addition, to develop a nomogram model predicting early recurrence of HCC patients. Methods: A total of 481 HCC patients after R0 resection were enrolled and divided into a training cohort (n = 337) and a validation cohort (n = 144). Risk factors for early recurrence were determined based on Cox regression analysis in the training cohort. A nomogram incorporating independent risk predictors was established and validated. Results: Early recurrence occurred in 37.8% of the 481 patients who underwent curative liver resection of HCC. AFP ≥ 400 ng/mL (HR: 1.662; P = 0.008), VEGF-A among 127.8 to 240.3 pg/mL (HR: 1.781, P = 0.012), VEGF-A > 240.3 pg/mL (HR: 2.552, P < 0.001), M1 subgroup of MVI (HR: 2.221, P = 0.002), M2 subgroup of MVI (HR: 3.120, P < 0.001), intratumor necrosis (HR: 1.666, P = 0.011), surgical margin among 5.0 to 10.0 mm (HR: 1.601, P = 0.043) and surgical margin < 5.0 mm (HR: 1.790, P = 0.012) were found to be independent risk factors for recurrence-free survival in the training cohort and were used for constructing the nomogram. The nomogram indicated good predictive performance with an AUC of 0.781 (95% CI: 0.729-0.832) and 0.808 (95% CI: 0.731-0.886) in the training and validation cohorts, respectively. Conclusions: Elevated serum concentrations of AFP and VEGF-A, microvascular invasion, intratumor necrosis, surgical margin were independent risk factors of early intrahepatic recurrence. A reliable nomogram model which incorporated blood biomarkers and pathological variables was established and validated. The nomogram achieved desirable effectiveness in predicting early recurrence in HCC patients.

Comprehensive analysis of the amino acid metabolism-related gene signature for prognosis, tumor immune microenvironment, and candidate drugs in hepatocellular carcinoma.

Introduction: Metabolic rewiring satisfies increased nutritional demands and modulates many oncogenic processes in tumors. Amino acid metabolism is abnormal in many malignancies. Metabolic reprogramming of amino acids not only plays a crucial role in sustaining tumor cell proliferation but also influences the tumor immune microenvironment. Herein, the aim of our study was to elucidate the metabolic signature of amino acids in hepatocellular carcinoma (HCC). Methods: Transcriptome profiles of HCC were obtained from the TCGA and ICGC databases. Based on the expression of amino acid metabolism-related genes (AAMRGs), we clustered the HCC samples into two molecular subtypes using the non-negative matrix factorization algorithm. Then, we constructed the amino acid metabolism-related gene signature (AAMRGS) by Cox regression and LASSO regression. Afterward, the clinical significance of the AAMRGS was evaluated. Additionally, we comprehensively analyzed the differences in mutational profiles, immune cell infiltration, immune checkpoint expression, and drug sensitivity between different risk subgroups. Furthermore, we examined three key gene expressions in liver cancer cells by quantitative real-time PCR and conducted the CCK8 assay to evaluate the influence of two chemotherapy drugs on different liver cancer cells. Results: A total of 81 differentially expressed AAMRGs were screened between the two molecular subtypes, and these AAMRGs were involved in regulating amino acid metabolism. The AAMRGS containing GLS, IYD, and NQO1 had a high value for prognosis prediction in HCC patients. Besides this, the two AAMRGS subgroups had different genetic mutation probabilities. More importantly, the immunosuppressive cells were more enriched in the AAMRGS-high group. The expression level of inhibitory immune checkpoints was also higher in patients with high AAMRGS scores. Additionally, the two AAMRGS subgroups showed different susceptibility to chemotherapeutic and targeted drugs. In vitro experiments showed that gemcitabine significantly reduced the proliferative capacity of SNU449 cells, and rapamycin remarkedly inhibited Huh7 proliferation. The five HCC cells displayed different mRNA expression levels of GLS, IYD, and NQO1. Conclusions: Our study explored the features of amino acid metabolism in HCC and identified the novel AAMRGS to predict the prognosis, immune microenvironment, and drug sensitivity of HCC patients. These findings might help to guide personalized treatment and improve the clinical outcomes of HCC.

Matrix stiffness-induced upregulation of histone acetyltransferase KAT6A promotes hepatocellular carcinoma progression through regulating SOX2 expression.

BACKGROUND: Lysine acetyltransferase 6 A (KAT6A) is a MYST-type histone acetyltransferase (HAT) enzyme, which contributes to histone modification and cancer development. However, its biological functions and molecular mechanisms, which respect to hepatocellular carcinoma (HCC), are still largely unknown. METHODS: Immunohistochemical, western blot and qRT-PCR analysis of KAT6A were performed. A series of in vitro and in vivo experiments were conducted to reveal the role of KAT6A in the progression of HCC. RESULTS: We demonstrated that KAT6A expression was upregulated in HCC tissues and cell lines. Clinical analysis showed that increased KAT6A was significantly associated with malignant prognostic features and shorter survival. Gain- and loss-of-function experiments indicated that KAT6A promoted cell viability, proliferation and colony formation of HCC cells in vitro and in vivo. We confirmed that KAT6A acetylates lysine 23 of histone H3 (H3K23), and then enhances the association of the nuclear receptor binding protein TRIM24 and H3K23ac. Consequently, TRIM24 functions as a transcriptional activator to activate SOX2 transcription and expression, leading to HCC tumorigenesis. Restoration of SOX2 at least partially abolished the biological effects of KAT6A on HCC cells. Overexpression of KAT6A acetyltransferase activity-deficient mutants or TRIM24 mutants lacking H3K23ac binding sites did not affect SOX2 expression and HCC biological function. Moreover, matrix stiffness can upregulate the expression of KAT6A in HCC cells. CONCLUSIONS: Our data support the first evidence that KAT6A plays an oncogenic role in HCC through H3K23ac/TRIM24-SOX2 pathway, and represents a promising therapeutic strategy for HCC patients.

HIF-1α-activated long non-coding RNA KDM4A-AS1 promotes hepatocellular carcinoma progression via the miR-411-5p/KPNA2/AKT pathway.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer with poor clinical outcomes. Long non-coding RNAs (lncRNAs) are extensively involved in the tumorigenesis and progression of HCC. However, more investigations should be carried out on novel lncRNAs and their effects on HCC. Here we identified a novel lncRNA KDM4A-AS1, which was aberrantly overexpressed in HCC tissues, associated with unfavorable clinical features and poor prognosis of patients. KDM4A-AS1 promoted HCC cell proliferation, migration, and invasion in vitro and contributed to HCC growth and lung metastasis in vivo. Mechanistically, KDM4A-AS1 was inversely modulated by miR-411-5p at the post-transcriptional level and facilitated Karyopherin α2 (KPNA2) expression by competitively binding miR-411-5p, thereby activating the AKT pathway. KPNA2 silencing, miR-411-5p overexpression, and AKT inhibitor (MK2206) consistently reversed KDM4A-AS1-enhanced proliferation, mobility, and EMT of HCC cells. KDM4A-AS1 was identified as a novel hypoxia-responsive gene and transactivated by hypoxia-inducible factor 1α (HIF-1α) in HCC cells. In turn, KDM4A-AS1 regulated HIF-1α expression through the KPNA2/AKT signaling pathway. Hence, this study revealed a novel hypoxia-responsive lncRNA, KDM4A-AS1, which contributed to HCC growth and metastasis via the KDM4A-AS1/KPNA2/HIF-1α signaling loop. Our findings provide a promising prognostic and therapeutic target for HCC.

Matrix stiffness modulates hepatic stellate cell activation into tumor-promoting myofibroblasts via E2F3-dependent signaling and regulates malignant progression.

The hepatic stellate cells (HSCs) activation by myofibroblastic differentiation is critical for liver fibrosis. Crosstalk between stromal cells and tumor cells in the microenvironment alters the properties and facilitates the growth and metastasis of tumor cells. How mechanical stimuli originally stiffness of extracellular matrix (ECM) contribute to tumor development remains poorly understood. Here, we demonstrated that stiffness contributes to mechanosignal transduction in HSCs, which promotes hepatocellular carcinoma (HCC) cells growth and metastasis through secretion of FGF2. On stiffness matrix, HSCs activation was confirmed by immunofluorescence (IF) and Western blot (WB) for α-smooth muscle actin (SMA). Increasing matrix stiffness promoted HSCs activation by CD36-AKT-E2F3 mechanosignaling through shRNA-mediated E2F3 knockdown, AKT inhibitors, and CD36 shRNA. Moreover, ChIP-qPCR. Confirmed that E2F3 combined the promoter of FGF2, and stiffness promoted FGF2 expression. On a stiff matrix, HCC cells cultured with conditioned media (CM) from HSCs increased HCC cells growth and metastasis by binding FGFR1 to activate PI3K/AKT and MEK/ERK signaling pathways. Moreover, conditional E2F3 knockout mice were subjected to CCl4 treatment to assess the role of E2F3 in HSC activation. Additionally, the DEN-induced HCC model was also used to evaluate the role of E2F3 in liver fibrosis and HCC growth. In conclusion, we demonstrated that stiffness-induced HSC activation by E2F3 dependent. Stiffness activated CD36-AKT-E2F3 signaling and targeted FGF2 transcription, subsequently, activated HCC growth and metastasis by FGFR1-mediated PI3K/AKT and MEK/ERK signaling.

Deubiquitinase PSMD7 promotes the proliferation, invasion, and cisplatin resistance of gastric cancer cells by stabilizing RAD23B.

Ubiquitination, a crucial post-translational modification, controls substrate degradation and can be reversed by deubiquitinases (DUBs). An increasing number of studies are showing that DUBs regulate the malignant behavior and chemotherapy resistance of gastric cancer (GC) by stabilizing various proteins. However, the expression level and biological function of the DUB, proteasome 26S subunit, non-ATPase 7 (PSMD7), in GC remains unknown. Herein, we report for the first time that PSMD7 is frequently overexpressed in GC tissues. Elevated levels of PSMD7 were also detected in GC cell lines. Notably, the upregulation of PSMD7 closely correlated with malignant clinical parameters and reduced the survival of GC patients. Functionally, we found that PSMD7 knockdown consistently suppressed the proliferation, migration, and invasion of AGS and SGC-7901 cells. Ectopic expression of PSMD7 facilitated GC cell proliferation and mobility. Based on protein-protein interaction prediction, RAD23 homolog B (RAD23B) protein was identified as a candidate substrate of PSMD7. PSMD7 positively regulated the abundance of RAD23B and xeroderma pigmentosum, complementation group C (XPC) protein in GC cells. The interaction between PSMD7 and RAD23B was confirmed using protein immunoprecipitation. PSMD7 knockdown enhanced the ubiquitination and degradation of RAD23B protein in GC cells. PSMD7 promoted cell viability, apoptosis resistance, and DNA damage repair in GC cells upon cisplatin (DDP) treatment. Moreover, PSMD7 silencing inhibited tumor growth and enhanced the sensitivity of GC cells to DDP treatment in mice. In summary, PSMD7 was highly expressed in GC and contributed to the malignant behavior and DDP resistance of tumor cells by stabilizing RAD23B.

Repression of the miR-627-5p by histone deacetylase 3 contributes to hypoxia-induced hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is one of the most common solid tumors globally. Our previous studies revealed that miR-627-5p suppresses HCC progression via targeting BCL3/CCND1 pathway. However, the molecular mechanism by which miR-627-5p was downregulated in HCC remains to be further elucidated. As a hallmark of solid tumors, hypoxia results in the rapid growth, strongly potential invasion and high frequent metastasis of cancer cells. Hypoxia-inducible factors (HIFs), mainly including HIF-1 and HIF-2, are the classical transcription factors which mediate hypoxia-related gene transcription. Here, we demonstrated that miR-627-5p was repressed by hypoxia in a HIF-1-dependent manner in HCC cells. But HIF-1 regulated miR-627-5p expression not directly through the hypoxia-response element (HRE) sites of MIR627 gene. In contrast, histone deacetylase 3 (HDAC3) was identified as a HIF-1 target gene, and the occupancy of HIF-1 to HRE site was essential for hypoxia-mediated HDAC3 induction. And upregulated HDAC3 was closely related to the malignant clinical and pathological characteristics and worse prognosis of HCC. Furthermore, HDAC3-mediated histone deacetylation in promoter region of MIR627 was critical for hypoxia-mediated miR-627-5p repression. And miR-627-5p mediated the effects of hypoxic condition on HCC progression. Thus, this study has revealed that miR-627-5p was repressed by hypoxia under the mediation of HDAC3 in HCC, and there existed a HIF-1α/HDAC3/miR-627-5p/BCL3/CCND1 signal pathway in HCC.

Long noncoding RNA FIRRE contributes to the proliferation and glycolysis of hepatocellular carcinoma cells by enhancing PFKFB4 expression.

Recent reports show that long noncoding RNA (lncRNA) FIRRE contributes to the proliferation, apoptosis resistance, and invasion of colorectal cancer and diffuse large B-cell lymphoma. However, the biological function of FIRRE in hepatocellular carcinoma (HCC) remains unknown. Here, we disclosed that the FIRRE level was frequently increased in HCC compared to nontumor tissues. Compared with normal liver cells, we also confirmed the upregulated level of FIRRE in HCC cells. Notably, the FIRRE high expression was related to malignant clinical features, including advanced TNM stage and tumor size ≥5 cm, and conferred to worse survival of HCC. Functionally, FIRRE knockdown repressed the proliferation and glycolysis of HCCLM3 cells. Overexpression of FIRRE strengthened Huh7 cell proliferation and glycolysis. Notably, FIRRE positively regulated the glycolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) expression in HCC cells. PFKFB4 was highly expressed and positively associated with FIRRE level in HCC tissues. The upregulated expression of PFKFB4 was associated with high tumor grade and advanced TNM stage. TCGA data revealed that the PFKFB4 high expression indicated a poor prognosis of HCC. Mechanistically, modulating FIRRE level did not affect the stability of PFKFB4 mRNA. FIRRE was mainly distributed in HCC cells' nucleus and promoted PFKFB4 transcription and expression via cAMP-responsive element-binding protein (CREB). PFKFB4 could abolish the effects of FIRRE knockdown on HCC cell proliferation and glycolysis. To conclude, the highly expressed FIRRE facilitated HCC cell proliferation and glycolysis by enhancing CREB-mediated PFKFB4 transcription and expression.

Identification and validation of redox-immune based prognostic signature for hepatocellular carcinoma.

The intimate interaction between redox signaling and immunity has been widely revealed. However, the clinical application of relevant therapeutic is unavailable due to the absence of validated markers that stratify patients. Here, we identified novel biomarkers for prognosis prediction in hepatocellular carcinoma (HCC). Prognostic redox-immune-related genes for predicting overall survival (OS) of HCC were identified using datasets from TCGA, LIRI-JP, and GSE14520. LASSO Cox regression was employed to construct the signature model and generate a risk score in the TCGA cohort. The signature contained CDO1, G6PD, LDHA, GPD1L, PPARG, FABP4, CCL20, SPP1, RORC, HDAC1, STC2, HDGF, EPO, and IL18RAP. Patients in the high-risk group had a poor prognosis compared to the low-risk group. Univariate and multivariate Cox regressions identified this signature as an independent factor for predicting OS. Nomogram constructed by multiple clinical parameters showed good performance for predicting OS indicated by the c-index, the calibration curve, and AUC. GSEA showed that oxidoreductase activity and peroxisome-related metabolic pathways were enriched in the low-risk group, while glycolysis activity and hypoxia were higher in the high-risk group. Furthermore, immune profiles analysis showed that the immune score and stromal score were significantly decreased in the high-risk group in the TCGA cohort. There was a considerably lower infiltration of anti-tumor immune cells while a higher proportion of pro-tumor immune cells in silico. Immune markers were distinctly expressed between the subgroups, and redox-sensitive immunoregulatory biomarkers were at higher levels in the high-risk group. Altogether, we identified a redox-immune prognostic signature. A more severe redox perturbation-driven immunosuppressive environment in the high-risk group stratified by the signature may account for poor survival. This may provide a clue to the combined therapy targeting redox and immune in HCC.

Long non-coding RNA MAPKAPK5-AS1/PLAGL2/HIF-1α signaling loop promotes hepatocellular carcinoma progression.

BACKGROUND: Long non-coding RNAs (lncRNAs) are widely involved in human cancers' progression by regulating tumor cells' various malignant behaviors. MAPKAPK5-AS1 has been recognized as an oncogene in colorectal cancer. However, the biological role of MAPKAPK5-AS1 in hepatocellular carcinoma (HCC) has not been explored. METHODS: Quantitative real-time PCR was performed to detect the level of MAPKAPK5-AS1 in HCC tissues and cell lines. The effects of MAPKAPK5-AS1 on tumor growth and metastasis were assessed via in vitro experiments, including MTT, colony formation, EdU, flow cytometry, transwell assays, and nude mice models. The western blotting analysis was carried out to determine epithelial-mesenchymal transition (EMT) markers and AKT signaling. The interaction between MAPKAPK5-AS1, miR-154-5p, and PLAGL2 were explored by luciferase reporter assay and RNA immunoprecipitation. The regulatory effect of HIF-1α on MAPKAPK5-AS1 was evaluated by chromatin immunoprecipitation. RESULTS: MAPKAPK5-AS1 expression was significantly elevated in HCC, and its overexpression associated with malignant clinical features and reduced survival. Functionally, MAPKAPK5-AS1 knockdown repressed the proliferation, mobility, and EMT of HCC cells and induced apoptosis. Ectopic expression of MAPKAPK5-AS1 contributed to HCC cell proliferation and invasion in vitro. Furthermore, MAPKAPK5-AS1 silencing suppressed, while MAPKAPK5-AS1 overexpression enhanced HCC growth and lung metastasis in vivo. Mechanistically, MAPKAPK5-AS1 upregulated PLAG1 like zinc finger 2 (PLAGL2) expression by acting as an endogenous competing RNA (ceRNA) to sponge miR-154-5p, thereby activating EGFR/AKT signaling. Importantly, rescue experiments demonstrated that the miR-154-5p/PLAGL2 axis mediated the function of MAPKAPK5-AS1 in HCC cells. Interestingly, we found that hypoxia-inducible factor 1α (HIF-1α), a transcript factor, could directly bind to the promoter to activate MAPKAPK5-AS1 transcription. MAPKAPK5-AS1 regulated HIF-1α expression through PLAGL2 to form a hypoxia-mediated MAPKAPK5-AS1/PLAGL2/HIF-1α signaling loop in HCC. CONCLUSIONS: Our results reveal a MAPKAPK5-AS1/PLAGL2/HIF-1α signaling loop in HCC progression and suggest that MAPKAPK5-AS1 could be a potential novel therapeutic target of HCC.

ZMYND8 promotes the growth and metastasis of hepatocellular carcinoma by promoting HK2-mediated glycolysis.

The bromodomain protein zinc finger MYND-type containing 8 (ZMYND8) plays a critical role in human breast cancer. However, the expression and biological function of ZMYND8 in hepatocellular carcinoma (HCC) are poorly understood. In this study, ZMYND8 expression was found to be elevated in HCC based on the cancer genome atlas (TCGA) and gene expression omnibus (GEO) databases. Next, we confirmed that ZMYND8 was frequently overexpressed in HCC tissues compared with adjacent non-tumor tissues. The up-regulated level of ZMYND8 was also observed in HCC cell lines. Elevated ZMYND8 expression was correlated with unfavorable clinicopathological features and poor prognosis of HCC patients. Functionally, ectopic expression of ZMYND8 potentiated the proliferation, migration, and invasion of Hep3B cells. Conversely, ZMYND8 knockdown led to the reduced proliferation and invasiveness of HCCLM3 cells. ZMYND8 silencing restrained the growth of HCCLM3 cells in vivo. Mechanistically, ZMYND8 enhanced glucose consumption, lactate production, and ATP level in HCC cells. Pharmacological inhibition of glycolysis using 2-DG blocked the promoting effects of ZMYND8 on HCC cell proliferation and mobility. Furthermore, hexokinase 2 (HK2), a key enzyme of glycolysis, was identified as the downstream target of ZMYND8 in HCC cells. ZMYND8 promoted HK2 transcription by recruiting bromodomain containing 4 (BRD4) to its promoter. Knockdown of HK2 abrogated the oncogenic functions of ZMYND8 in HCC. Altogether, these data indicated that ZMYND8 promoted the growth and metastasis of HCC by promoting HK2-mediated glycolysis and might serve as a promising biomarker and therapeutic target for HCC.

A Novel Lactate Metabolism-Related Gene Signature for Predicting Clinical Outcome and Tumor Microenvironment in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the main subtype of primary liver cancer with high malignancy and poor prognosis. Metabolic reprogramming is a hallmark of cancer and has great importance on the tumor microenvironment (TME). As an abundant metabolite, lactate plays a crucial role in cancer progression and the immunosuppressive TME. Nonetheless, the potential roles of lactate in HCC remain unclear. In this study, we downloaded transcriptomic data of HCC patients with corresponding clinical information from the TCGA and ICGC portals. The TCGA-HCC dataset used as the training cohort, while the ICGC-LIRI-JP dataset was served as an external validation cohort. Cox regression analysis and the LASSO regression model were combined to construct the lactate metabolism-related gene signature (LMRGS). Then, we assessed the clinical significance of LMRGS in HCC. Besides, enriched molecular functions, tumor mutation burden (TMB), infiltrating immune cells, and immune checkpoint were comprehensively analyzed in different LMRGS subgroups. In total, 66 differentially expressed lactate metabolism-related genes (LMRGs) were screened. The functions of LMRGs were mainly enriched in mitochondrial activity and metabolic processes. The LMRGS comprised of six key LMRGs (FKTN, PDSS1, PET117, PUS1, RARS1, and RNASEH1) had significant clinical value for independently predicting the prognosis of HCC patients. The overall survival and median survival of patients in the LMRGS-high group were significantly shorter than in the LMRGS-low group. In addition, there were differences in TMB between the two LMRGS subgroups. The probability of genetic mutations was higher in the LMRGS-high group. Most importantly, the LMRGS reflected the TME characteristics. In the LMRGS-high group, the immune microenvironment presented a suppressed state, accompanied by more inhibitory immune cell infiltration, including follicular helper T cells and regulatory T cells. Additionally, the expression of inhibitory checkpoint molecules was much higher in the LMRGS-high group. Our study suggested that the LMRGS was a robust biomarker to predict the clinical outcomes and evaluate the TME of patients with HCC.

MicroRNA­875­5p inhibits tumor growth and metastasis of hepatocellular carcinoma by targeting eukaryotic translation initiation factor 3 subunit a.

Accumulating evidence has demonstrated that aberrant microRNA (miRNA) expression is involved in hepatocellular carcinoma (HCC) progression. Previous findings suggested that miRNA (miR)­875­5p participates in the development of various types of cancer. However, the expression and function of miR­875­5p in HCC remains largely unclear. The analysis of clinical samples in the present study demonstrated that miR­875­5p expression was downregulated in HCC tissues compared to adjacent non­tumor tissues, which was associated with a large tumor size, venous infiltration, advanced tumor­node­metastasis stage and unfavorable overall survival. In vitro experiments revealed that ectopic expression of miR­875­5p suppressed, whereas inhibition of miR­875­5p promoted HCC cell proliferation, migration, invasion and epithelial­to­mesenchymal transition (EMT) progression. Overexpression of miR­875­5p restrained HCC tumor growth and metastasis in vivo. Mechanistically, eukaryotic translation initiation factor 3 subunit a (eIF3a) was identified as the downstream target of miR­875­5p in HCC. Further experiments demonstrated that the expression of eIF3a was upregulated and negatively correlated with that of miR­875­5p in HCC tissues. In addition, miR­875­5p negatively regulated the luciferase activity of wild­type, but not mutant 3'­untranslated region (3'UTR) of eIF3a mRNA. miR­875­5p suppressed eIF3a expression at the mRNA and protein level in HCC cells. Additionally, eIF3a exerted an oncogenic role, and knockdown of eIF3a inhibited the proliferation, motility and EMT of HCC cells. In addition, eIF3a overexpression abolished the inhibitory effects of miR­875­5p on the proliferation, motility and EMT in HCC cells. In conclusion, miR­875­5p, which was downregulated in HCC, may inhibit tumor growth and metastasis by eIF3a downregulation via targeting its 3'UTR and may be a promising prognostic and therapeutic strategy in HCC.

Hypoxia-inducible long noncoding RNA NPSR1-AS1 promotes the proliferation and glycolysis of hepatocellular carcinoma cells by regulating the MAPK/ERK pathway.

Hepatocellular carcinoma (HCC), which accounts for approximately 90% of primary liver cancer, is commonly treated with surgical resection. However, most patients lose the opportunity to receive this therapeutic strategy due to delayed diagnosis and rapid tumor progression. Long noncoding RNAs (lncRNAs) have been demonstrated to play essential roles in the initiation and progression of HCC. However, the function of the novel lncRNA neuropeptide S receptor 1 antisense RNA 1 (NPSR1-AS1) in HCC and its potential mechanism, is unclear. Here, our microarray data revealed NPSR1-AS1 as a novel hypoxia-responsive lncRNA in HCC cells. Interestingly, hypoxia-inducible factor-1α (HIF-1α) knockdown abolished hypoxia-induced NPSR1-AS1 expression in HCC cells. NPSR1-AS1 expression was upregulated in HCC tissues and cell lines. Next, the ectopic expression of NPSR1-AS1 facilitated the proliferation and glycolysis of HCC cells. In contrast, NPSR1-AS1 silencing repressed HCC cell proliferation and glycolysis. Mechanistically, NPSR1-AS1 overexpression increased the levels of p-ERK1/2 and pyruvate kinase M2 (PKM2) in HCC cells. NPSR1-AS1 knockdown abrogated hypoxia-induced the activation of the MAPK/ERK pathway in HCC cells. Importantly, NPSR1-AS1 depletion partially reversed hypoxia-induced proliferation and glycolysis of HCC cells in vitro. In conclusion, hypoxia-inducible NPSR1-AS1 promotes the proliferation and glycolysis of HCC cells, possibly by regulating the MAPK/ERK pathway, suggesting an underlying therapeutic strategy for HCC.

CTNNB1 mutation suppresses infiltration of immune cells in hepatocellular carcinoma through miRNA-mediated regulation of chemokine expression.

Mutations in the CTNNB1 gene in hepatocellular carcinoma (HCC) are related to immune exclusion, and HCC patients with CTNNB1 mutations tend to be primarily resistant to anti-PD1 therapy. However, systemic evaluation of immune cell infiltration in HCC with mutant CTNNB1 is lacking, and the mechanism of immune exclusion resulting from CTNNB1 mutations remains unclear. Based on CTNNB1 mutation status in HCC, we investigated RNA and miRNA expression and infiltration of immune cells. Data downloaded from TCGA showed that HCC with CTNNB1 mutation had an increased expression of CTNNB1. HCC with CTNNB1 mutation showed a reduction in infiltration score as well as in abundance of certain kinds of immune cells, including CD4 naïve T cells, CD4+ T cells, Tex cells, Th2 cells, Tfh cells, B cells, macrophages, and NK cells. Furthermore, there were 13 chemokines downregulated among all the 14 differentially expressed chemokines (DE-CKs) in CTNNB1 mutants compared to those in the wild type. A positive correlation was found between the expression of DE-CKs and infiltration score, as well as infiltration level of 6 types of immune cells, namely B cells, CD8+ cells, CD4+ cells, macrophages, neutrophils, and dendritic cells. Additionally, 302 differentially expressed immune-related genes (DE-IRGs) were involved mainly in the human immune response and cytokine-cytokine receptor interaction. The target DE-IRGs of differentially expressed miRNAs (DE-miRNAs) were identified and used to construct a network with DE-miRNAs and DE-CKs. Overall, CTNNB1 mutation in HCC led to a decrease in chemokine expression and subsequent suppression of immune cell infiltration partly through regulating specific miRNA-IRG-CK axes, pointing to a potential combination of interference of Wnt/ß-catenin signaling with immunotherapy in HCC with CTNNB1 mutation.

Long noncoding RNA PICSAR/miR-588/EIF6 axis regulates tumorigenesis of hepatocellular carcinoma by activating PI3K/AKT/mTOR signaling pathway.

Accumulating evidence has identified long noncoding RNAs (lncRNAs) as regulators in tumor progression and development. Here, we elucidated the function and possible molecular mechanisms of the effect of lncRNA-PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA) on the biological behaviors of HCC. In the present study, we found that PICSAR was upregulated in HCC tissues and cells and correlated with progression and poor prognosis in HCC patients. Gain- and loss-of-function experiments indicated that PICSAR enhanced cell proliferation, colony formation, and cell cycle progression and inhibited apoptosis of HCC cells. PICSAR could function as a competing endogenous RNA by sponging microRNA (miR)-588 in HCC cells. Mechanically, miR-588 inhibited HCC progression and alternation of miR-588 reversed the promotive effects of PICSAR on HCC cells. In addition, we confirmed that eukaryotic initiation factor 6 (EIF6) was a direct target of miR-588 in HCC and mediated the biological effects of miR-588 and PICSAR in HCC, resulting in PI3K/AKT/mTOR pathway activation. Our data identified PICSAR as a novel oncogenic lncRNA associated with malignant clinical outcomes in HCC patients. PICSAR played an oncogenic role by targeting miR-588 and subsequently promoted EIF6 expression and PI3K/AKT/mTOR activation in HCC. Our results revealed that PICSAR could be a potential prognostic biomarker and therapeutic target for HCC.