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Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real‐time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor β (TGF‐β) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR‐202‐5p, which served as a tumor‐suppressing microRNA via the TGF‐β pathway. We address that NORAD has a tumor‐promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF‐β pathway as a ceRNA of Homo sapiens (hsa)‐miR‐202‐5p. Here, we reported a novel axis that NORAD could act as a competitive endogenous RNA to modulate miR‐202‐5p expression, which targets the 3′‐untranslated regions of transforming growth factor β receptors to regulate the transforming growth factor β pathway. It indicated an oncogenic role of NORAD in hepatocellular carcinoma (HCC) and may contribute to HCC research and treatment. [ABSTRACT FROM AUTHOR]

Evading immune destruction is an emerging hallmark of cancer and a potential key step in tumorigenesis. Immune checkpoint blocker (ICB)-based combination therapies revolutionize the landscape of systemic therapy for HCC. However, the molecular underpinnings governing immune evasion and responses remain unclear. Our study aims to find new regulatory molecules that drive HCC immune escape and tumorigenesis and find new promising immunotherapeutic approaches for HCC. In our study, laser capture microdissection (LCM) and miRNA sequencing combined with in vitro and in vivo experiment s identified miR-93-5p as a crucial initiating oncogene during liver progenitor cell (LPC) malignant transformation and immune escape. Mechanistically, miR-93-5p could directly target canonical tumour suppressors such as APC to promote LPC malignant transformation and hepatocarcinogenesis. More importantly, miR-93-5p could induce deviant GAL-9 augmentation to inactivate infiltrated CD8(+) T cells and induce immune evasion by targeting several epigenetic regulators, such as AEBP2, and then regulating H3K4me3/H3K27me3 bivalency. Experiments in C57BL/6 mice demonstrated that blockade of Gal-9 abrogated miR-93-5p-induced HCC progression and improved their prognosis. Clinically, we identified a unique subtype of HCC closely associated with high GAL-9 expression and anti-PD1 treatment resistance. Our study highlights the pivotal role of the miR-93-5p/Gal-9 axis in driving HCC immune escape and tumorigenesis. Blocking GAL-9 is an effective and promising immunotherapeutic approach for HCC. • miR-93-5p was demonstrated to be a crucial initiating oncogene during liver progenitor cell (LPC) malignant transformation. • miR-93-5p could directly target several tumour suppressors to promote LPC malignant transformation and hepatocarcinogenesis. • miR-93-5p induced GAL-9 augmentation to inactivate infiltrated CD8(+) T cells and induce immune evasion. • Blockade of Gal-9 abrogated miR-93-5p-induced HCC progression and improved animals prognosis. • A unique subtype of HCC was identified to be closely associated with high GAL-9 expression and anti-PD1 treatment resistance. [ABSTRACT FROM AUTHOR]

Background: Laparoscopic liver resection (LLR) has now been established as a safe and minimally invasive technique that is deemed feasible for treating hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). However, the role of LLR in treating combined hepatocellular-cholangiocarcinoma (cHCC-CC) patients has been rarely reported. This study aimed to assess the efficacy of LLR when compared with open liver resection (OLR) procedure for patients with cHCC-CC. Methods: A total of 229 cHCC-CC patients who underwent hepatic resection (34 LLR and 195 OLR patients) from January 2014 to December 2018 in Zhongshan Hospital, Fudan University were enrolled and underwent a 1:2 propensity score matching (PSM) analysis between the LLR and OLR groups to compare perioperative and oncologic outcomes. Overall survival (OS) and recurrence-free survival (RFS) parameters were assessed by the log-rank test and the sensitivity analysis. Results: A total of 34 LLR and 68 OLR patients were included after PSM analysis. The LLR group displayed a shorter postoperative hospital stay (6.61 vs. 8.26 days; p value < 0.001) when compared with the OLR group. No significant differences were observed in the postoperative complications' incidence or a negative surgical margin rate between the two groups (p value = 0.409 and p value = 1.000, respectively). The aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory indicators in the LLR group were significantly lower than those in the OLR group on the first and third postoperative days. Additionally, OS and RFS were comparable in both the LLR and OLR groups (p value = 0.700 and p value = 0.780, respectively), and similar results were obtained by conducting a sensitivity analysis. Conclusion: LLR can impart less liver function damage, better inflammatory response attenuation contributing to a faster recovery, and parallel oncologic outcomes when compared with OLR. Therefore, LLR can be recommended as a safe and effective therapeutic modality for treating selected cHCC-CC patients, especially for those with small tumors in favorable location. [ABSTRACT FROM AUTHOR]

Background: The aim of this study is to investigate the expression profile of multiple epithelial mesenchymal transition (EMT)-related molecules in intrahepatic cholangiocarcinoma (ICC) and the related prognostic significance. Methods: Immunohistochemistry was performed to determine the expression of E-cadherin, Vimentin, Snail, slug and β-catenin in a tissue microarray consisting of tumor tissues of 140 ICC patients undergoing curative resection. The correlation between the expression of these molecules and the clinicopathological characteristics of ICC patients was analyzed, and their prognostic implication was evaluated. Results: Reduced E-cadherin and increased Vimentin expression, the characteristic changes of EMT, identified in 55.0% and 55.7% of primary ICCs, respectively, were correlated with lymphatic metastasis and poorer overall survival (OS) and disease-free survival (DFS) of ICCs. The overexpression of snail and nonmembranous β-catenin, which are the major regulators of the EMT, were identified in 49.2% and 45.7% of primary ICCs, while little slug expression was detected in ICCs. Cytoplasmic/nuclear β-catenin did not significantly predict worse DFS and was not related with E-cadherin loss. The overexpression of snail predicted worse OS and DFS. Snail overexpression correlated with the down-regulation of E-cadherin and the up-regulation of Vimentin. Inhibition of snail in an ICC cell line decreased the expression of E-cadherin, enhanced the expression of Vimentin and impaired the invasion and migration ability of ICC cells. Conclusions: These data support the hypothesis that EMT plays vital roles in ICC progression and suggest that snail but not slug and β-catenin plays a crucial role in the EMT induction of ICC. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. Method: The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Results: Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. Conclusion: In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are a class of regulatory RNAs with complex roles in healthy and diseased tissues. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) remains poorly understood, including the mechanisms by which the circular ubiquitin-binding associated protein 2 (circUBAP2) contributes to tumorigenesis. We analyzed the expression of circUBAP2 in 20 paired samples of HCC and healthy tissue as well as in seven HCC cell lines via quantitative real-time polymerase chain reaction. Functional experiments, such as CCK8 viability assays, colony formation assays, wound healing, transwell assays and flow cytometry, were conducted to assess the effects of circUBAP2 in vitro. To further elucidate the mechanisms by which circUBAP2 acts, we conducted dual-luciferase assays, western blots, RNA pull-down assays and rescue experiments. CircUBAP2 was highly upregulated in most HCC tissues and was associated with poor prognosis. HCC patients with high circUBAP2 expression had greater vascular invasion and worse differentiation. Functionally, circUBAP2 overexpression enhanced HCC cell proliferation, migration and invasion and inhibited apoptosis. Furthermore, we found that circUBAP2 upregulated c-Myc expression by sponging miR-1294, thus contributing to hepatocarcinogenesis. Inhibiting circUBAP2 expression in HCC attenuated the oncogenic effects of c-Myc. These findings suggest that circUBAP2 promotes HCC growth and metastasis. CircUBAP2 may have value as an independent prognostic biomarker or as a new target for the treatment of HCC. [ABSTRACT FROM AUTHOR]

Objective: Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. Methods: The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues. Results: Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients. Conclusions: Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is an aggressive malignancy with few effective treatment options. Lenvatinib is the first-line therapy for HCC but has only limited clinical benefit. Here, we explored the role and mechanism of the WD repeat domain 4 (WDR4) in lenvatinib resistance to improve clinical benefit. We found that lenvatinib-resistant HCC tissues/cells exhibited increased the N7-methylguanosine (m7G) modification and WDR4 expression. By a gain/loss of function experiment, we showed that WDR4 promoted HCC lenvatinib resistance and tumor progress both in vitro and in vivo. By proteomics analysis and RNA immunoprecipitation PCR, we found that tripartite motif protein 28 (trim28) was an important WDR4 target gene. WDR4 promoted TRIM28 expression, further affected target genes expression, and thus increased cell-acquired stemness and lenvatinib resistance. Clinical tissue data showed that TRIM28 expression was correlated with WDR4 levels, and the expression of both was positively correlated with poor prognosis. Our study provides new insight into the role of WDR4, suggesting a potential therapeutic target to enhance the lenvatinib sensitivity of HCC. • WDR4 is high expressed in lenvatinib-resistant HCC cells and tissues. • WDR4 help cell acquire lenvatinib resistant by promote TRIM28 expression, further affect stem ness, and their expression were positively correlated with poor prognoses. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (ICC) is an aggressive type of cancer, and its incidence and mortality rates are increasing worldwide. Mortalin is a highly conserved chaperone protein involved in multiple pathological and physiological processes, including anti-apoptosis, carcinogenesis and metastasis. The Bcl-2 family of proteins can be divided into pro-survival and pro-apoptotic members, including B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax). The aim of the present study was to investigate the association between mortalin, Bcl-2 and Bax, as well as the prognostic significance of the combined expression of mortalin, Bcl-2 and Bax in ICC. Immunohistochemistry was used to determine the expression of mortalin, Bcl-2 and Bax in 116 ICC samples and to assess the association between expression of 3 markers and clinicopathological features of ICC patients. This revealed that ICC tumor tissues overexpressed mortalin and Bcl-2 and exhibited low expression of Bax in ICC tumor tissues compared with that in corresponding peritumoral samples. According to Pearson's correlation coefficient analysis, high expression of mortalin in ICC was positively correlated with Bcl-2 expression and negatively correlated with Bax expression. Furthermore, multiple linear regression analysis demonstrated that mortalin was positively associated with Bcl-2, but not with Bax, in patients with ICC. Patients with ICC exhibiting high expression of mortalin/Bcl-2 or low expression of Bax were closely associated with a malignant ICC phenotype, a relatively low overall survival rate and a high recurrence rate. Multivariate analysis indicated that mortalin and Bcl-2 were independent prognostic indicators for ICC patients. Meanwhile, the concomitant overexpression of mortalin and Bcl-2 and the low expression of Bax were independent markers for predicting a relatively poor prognosis of ICC. The overexpression of mortalin and Bcl-2 and/or the low expression of Bax are implicated in the anti-apoptotic effect and tumor progression of ICC. Mortalin or Bcl-2, or a combination of mortalin, Bcl-2 and Bax may be used to predict the prognosis of ICC, as well as potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Previous studies have indicated that resistin-like molecule beta (RELM beta), an intestinal goblet cell-specific protein, is markedly increased in the intestinal tumors of min mice and over-expressed in a human colon cancer cell line. We hypothesized that RELM beta might be enhanced in human colon cancer. The aim of this study was to examine the clinical importance of RELM beta expression in colon cancer patients and to correlate its expression with various clinicopathological parameters, upstream regulatory molecule expression, tumor proliferative capacity, and patients' survival. Of the 80 colon cancer patients studied, 65 (81.25%) tested positive for RELM beta, mainly in the cytoplasm of colon mucosa. Contrasting sharply with the strongly RELM beta-positive tumors, normal colon mucous membrane was negative or weakly positive. RELM beta positivity in colon cancer was correlated with histological grade of differentiation and lymph node metastasis, but not with age, gender, tumor location and size, tumor infiltration, Dukes' stage, liver metastasis, and venous invasion. RELM beta expression was significantly correlated with the expression of transcription factor CDX-2 (P < 0.01) but not with that of proliferative index Ki-67 (P > 0.05). The mean postoperative survival time (2.76 years) of RELM beta-positive patients was significantly longer than that (1.26 years) of RELM beta-negative patients (P = 0.032). These findings support evidence of the enhanced RELM beta expression in colon cancer patients and suggest that further investigation is warranted to explore the role of RELM beta in colon cancer. [ABSTRACT FROM AUTHOR]

Background: Recently, the dysregulation of circular RNA (circRNA) have been shown to have important regulatory roles in cancer development and progression, including hepatocellular carcinoma (HCC). However, the roles of most circRNAs in HCC are still unknown. Methods: The expression of circular tripartite motif containing 33–12 (circTRIM33–12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33–12 in HCC progression was assessed by western blotting, CCK-8, flow cytometry, transwell and a subcutaneous tumor mouse assays both in vitro and in vivo. In vivo circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33–12 and miR-191. Results: Here, we found that circTRIM33–12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33–12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33–12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33–12 upregulated TET1 expression by sponging miR-191, resulting in significantly reduced 5-hydroxymethylcytosine (5hmC) levels in HCC cells. Conclusions: These results reveal the important role of circTRIM33–12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression. [ABSTRACT FROM AUTHOR]

Background: RING finger protein 38 (RNF38), a member of the RNF protein family, has just emerged as a vital driver of cancer progression. However, the oncogenic mechanisms of RNF38 remain unexplored. Methods: Using frozen tumor tissue and tissue microarray from hepatocellular carcinoma (HCC) patients, we tried to probe the expression of RNF38 in HCC and its clinical value. Then the biological functions of RNF38 were analyzed in vivo and vitro. Stable isotope labeling with amino acids (SILAC) in cell culture and co-immunoprecipitation proteomic analyses were combined to reveal the potential mechanism of RNF38 in HCC progression. Results: We report that RNF38 expression was markedly higher in HCC tissues than in peritumor tissues. Correspondingly, RNF38 overexpression promoted the HCC cell migration and invasion and inhibited apoptosis both in vitro and in vivo. And elevated RNF38 expression induced HCC cell epithelial-mesenchymal transition by facilitating transforming growth factor-β (TGF-β) signaling via ubiquitinating and degrading neuroblast differentiation-associated protein (AHNAK), a well-established inhibitor of TGF-β signaling. Furthermore, AHNAK interference restored the HCC cell invasion and metastasis deprived by RNF38 downregulation. Clinically, elevated RNF38 and transforming growth factor beta receptor 1 (TGFBR1) expression was related to short overall survival (OS) and high cumulative recurrence rates in HCC patients. Conclusions: High levels of RNF38 promote HCC by facilitating TGF-β signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC. [ABSTRACT FROM AUTHOR]

Background: This study was performed to investigate the role of nucleotide-binding oligomerization domain (NOD)-like receptor X1 (NLRX1) in regulating hepatocellular carcinoma (HCC) progression. Methods: Expression levels of NLRX1 in clinical specimens and cell lines were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Transwell assays were conducted to evaluate the effect of NLRX1 on cell invasion, and flow cytometry was used to assess apoptosis. Expression patterns of key molecules in the phosphoinositide 3-kinase (PI3K)-AKT pathways were determined via WB. The effect of NLRX1 on cell senescence was evaluated with β-galactosidase assays. Kaplan-Meier analyses and Cox regression models were used for prognostic evaluation. Results: NLRX1 was downregulated in tumor tissue compared with adjacent normal liver tissue. Low tumor NLRX1 expression was identified as an independent indicator for HCC prognosis (recurrence: hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26–2.76, overall survival [OS] 2.26, 95% CI 1.44–3.56). NLRX1 over-expression (OE) significantly inhibited invasiveness ability and induced apoptosis in HCC cells. In vivo experiments showed that NLRX1 knock-down (KD) significantly promoted HCC growth. Mechanistically, NLRX1 exhibited a suppressor function by decreasing phosphorylation of AKT and thus downregulating Snail1 expression, which inhibited epithelial-mesenchymal-transition (EMT) in HCC cells. Moreover, NLRX1 OE could induce cell senescence via an AKT-P21-dependent manner. Conclusions: NLRX1 acted as a tumor suppressor in HCC by inducing apoptosis, promoting senescence, and decreasing invasiveness by repressing PI3K-AKT signaling pathway. Future investigations will focus on restoring expression of NLRX1 to provide new insights into HCC treatment. [ABSTRACT FROM AUTHOR]