Targeted delivery of CEBPA-saRNA for the treatment of pancreatic ductal adenocarcinoma by transferrin receptor aptamer decorated tetrahedral framework nucleic acid.

Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

GPC3-targeted CAR-M cells exhibit potent antitumor activity against hepatocellular carcinoma.

Chimeric antigen receptor (CAR)-modified macrophages are a promising treatment for solid tumor. So far the potential effects of CAR-M cell therapy have rarely been investigated in hepatocellular carcinoma (HCC). Glypican-3 (GPC3) is a biomarker for a variety of malignancies, including liver cancer, which is not expressed in most adult tissues. Thus, it is an ideal target for the treatment of HCC. In this study, we engineered mouse macrophage cells with CAR targeting GPC3 and explored its therapeutic potential in HCC. First, we generated a chimeric adenoviral vector (Ad5f35) delivering an anti-GPC3 CAR, Ad5f35-anti-GPC3-CAR, which using the CAR construct containing the scFv targeting GPC3 and CD3ζ intracellular domain. Phagocytosis and killing effect indicated that macrophages transduced with Ad5f35-anti-GPC3-CAR (GPC3 CAR-Ms) exhibited antigen-specific phagocytosis and tumor cell clearance in vitro, and GPC3 CAR-Ms showed significant tumor-killing effects and promoted expression of pro-inflammatory (M1) cytokines and chemokines. In 3D NACs-origami spheroid model of HCC, CAR-Ms were further demonstrated to have a significant tumor killing effect. Together, our study provides a new strategy for the treatment of HCC through CAR-M cells targeting GPC3, which provides a basis for the research and treatment of hepatocellular carcinoma.

Secretome of senescent hepatic stellate cells favors malignant transformation from nonalcoholic steatohepatitis-fibrotic progression to hepatocellular carcinoma.

Background: Hepatic fibrosis is a premalignant lesion, and how injured hepatocytes transform into malignancy in a fibrotic microenvironment is poorly understood. Senescence is one of major fates of activated hepatic stellate cells (HSCs). Paucity of literature is available regarding the influence of senescent HSCs on behavior of steatotic hepatocytes. Methods: Senescent HSCs were identified in a murine model of nonalcoholic steatohepatitis (NASH)-fibrosis-hepatocellular carcinoma (HCC) and human NASH-HCC specimens. Secretome of senescent HSCs was analyzed by label-free mass-spectrum (NanoRPLC-MS/MS) and verified quantitatively. Results: Senescent HSCs were increased along with the progression from nonalcoholic fatty liver (NAFL), NASH to NASH-fibrosis, and reached a peak at the stage of advanced fibrosis and then decreased when hepatocellular dysplasia or HCC was developed. Critical components affecting proliferation, epithelial-mesenchymal transition (EMT) or migration were identified from secretome of senescent HSCs, and may activate morphogenic hedgehog or oncogenic Wnt signaling pathways to accelerate malignant transformation of steatotic or dysplastic hepatocytes. Primary hepatocytes stimulated with conditioned medium from senescent HSCs, in co-culture or co-cultured in 3D spheroids with senescent HSCs exhibited an enhanced proliferating or EMT profile. Conclusion: Senescent HSCs secreted a characterized protein profile favoring malignant transformation of steatotic or dysplastic hepatocytes through activating morphogenic hedgehog or oncogenic Wnt signaling pathways in the progression from NASH to malignancy.

Tyrosine kinase receptor B attenuates liver fibrosis by inhibiting TGF-ß/SMAD signaling.

BACKGROUND AND AIMS: Liver fibrosis is a leading indicator for increased mortality and long-term comorbidity in NASH. Activation of HSCs and excessive extracellular matrix production are the hallmarks of liver fibrogenesis. Tyrosine kinase receptor (TrkB) is a multifunctional receptor that participates in neurodegenerative disorders. However, paucity of literature is available about TrkB function in liver fibrosis. Herein, the regulatory network and therapeutic potential of TrkB were explored in the progression of hepatic fibrosis. METHODS AND RESULTS: The protein level of TrkB was decreased in mouse models of CDAHFD feeding or carbon tetrachloride-induced hepatic fibrosis. TrkB suppressed TGF-ß-stimulated proliferation and activation of HSCs in 3-dimensional liver spheroids and significantly repressed TGF-ß/SMAD signaling pathway either in HSCs or in hepatocytes. The cytokine, TGF-ß, boosted Nedd4 family interacting protein-1 (Ndfip1) expression, promoting the ubiquitination and degradation of TrkB through E3 ligase Nedd4-2. Moreover, carbon tetrachloride intoxication-induced hepatic fibrosis in mouse models was reduced by adeno-associated virus vector serotype 6 (AAV6)-mediated TrkB overexpression in HSCs. In addition, in murine models of CDAHFD feeding and Gubra-Amylin NASH (GAN), fibrogenesis was reduced by adeno-associated virus vector serotype 8 (AAV8)-mediated TrkB overexpression in hepatocytes. CONCLUSION: TGF-ß stimulated TrkB degradation through E3 ligase Nedd4-2 in HSCs. TrkB overexpression inhibited the activation of TGF-ß/SMAD signaling and alleviated the hepatic fibrosis both in vitro and in vivo . These findings demonstrate that TrkB could be a significant suppressor of hepatic fibrosis and confer a potential therapeutic target in hepatic fibrosis.

Cyanidin chloride protects mice from methicillin-resistant Staphylococcus aureus-induced pneumonia by targeting Sortase A.

Methicillin-resistant Staphylococcus aureus (MRSA) has been developing rapidly in recent years. It poses a severe peril to global health care, and the new strategies to against the MRSA is urgently needed. Sortase A (SrtA) regulates the anchoring of many surface proteins. Compounds repress Staphylococcus aureus (S. aureus) cysteine transpeptidase SrtA are considered adequate potent virulence inhibitors. Then, we describe the identification of an effective SrtA inhibitor, cyanidin chloride, a bioflavonoid compound isolated from various plants. It has a reversible inhibitory effect on SrtA activity at an IC50 of 21.91 µg/mL. As a SrtA inhibitor, cyanidin chloride antagonizes SrtA-related virulence phenotypes due to its breadth and specificity, including fibrinogen adhesion, A549 cell invasion, biofilm formation, and surface protein (SpA) anchoring. Subsequently, molecular docking and fluorescence quenching revealed that SrtA and cyanidin chloride had robust mutual affinity. Further mechanistic studies revealed that Arg-197, Gly-167, and Sep-116 were the key-binding sites mediating the interaction between SrtA and cyanidin chloride. Notably, a significant therapeutic effect of cyanidin chloride in vivo was also observed on the mouse pneumonia model induced by MRSA. In conclusion, our study indicates that cyanidin chloride potentially represents a new candidate SrtA inhibitor for S. aureus and potentially be developed as a new antivirulence agent.

microRNA-106b-5p Promotes Cell Growth and Sensitizes Chemosensitivity to Sorafenib by Targeting the BTG3/Bcl-xL/p27 Signaling Pathway in Hepatocellular Carcinoma.

microRNAs (miRNAs) and miRNA-mediated regulatory networks are promising candidates in the prevention and treatment of cancer, but the role of specific miRNAs involved in hepatocellular carcinoma (HCC) remains to be elusive. Herein, we found that miR-106b-5p is upregulated in both HCC patients' tumor tissues and HCC cell lines. The miR-106b-5p expression level was positively correlated with α-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), and tumor size. Overexpression of miR-106b-5p promoted cell proliferation, migration, cell cycle G1/S transition, and tumor growth, while decreased miR-106b-5p expression had opposite effects. Mechanistic studies showed that B-cell translocation gene 3 (BTG3), a known antiproliferative protein, was a direct target of miR-106b-5p, whose expression level is inversely correlated with miR-106b-5p expression. Moreover, miR-106b-5p positively regulates cell proliferation in a BTG3-dependent manner, resulting in upregulation of Bcl-xL, cyclin E1, and CDK2, as well as downregulation of p27. More importantly, we also demonstrated that miR-106b-5p enhances the resistance to sorafenib treatment in a BTG3-dependent manner. The in vivo findings showed that mice treated with a miR-106b-5p sponge presented a smaller tumor burden than controls, while the mice injected cells treated with miR-106b-5p had more considerable tumor burden than controls. Altogether, these data suggest that miR-106b-5p promotes cell proliferation and cell cycle and increases HCC cells' resistance to sorafenib through the BTG3/Bcl-xL/p27 signaling pathway.

Pre-mRNA processing factor 19 functions in DNA damage repair and radioresistance by modulating cyclin D1 in hepatocellular carcinoma.

Pre-mRNA processing factor 19 (PRP19) is elevated in hepatocellular carcinoma (HCC); however, little is known about its function in DNA damage repair in HCC. In this study, analysis of The Cancer Genome Atlas data and our tumor models after ionizing radiation (IR) treatment indicated that increased expression of PRP19 was positively correlated with DNA damage repair. Gain of PRP19 expression induced by plasmids resulted in decreases in apoptosis and double-strand breaks (DSBs), and an increase in cell survival after IR. Loss of PRP19 expression induced by small interfering RNAs resulted in the accumulation of apoptosis and DSBs, and a decrease in cell survival. Mechanistically, the effect of PRP19 on DNA damage repair was mediated by the modulation of cyclin D1 expression in HCC. PRP19 controlled the translation of cyclin D1 by modulating eukaryotic initiation factor 4E. PRP19 affected the DNA damage repair ability of cyclin D1 by interacting with the WD40 domain. The combination of PRP19 and cyclin D1 was more valuable than each single marker for predicting the prognosis of patients. Taken together, the present results demonstrate that PRP19 promotes DNA damage repair by modulating cyclin D1 expression and function, thereby contributing to the radioresistance in HCC.

Tetrahedral Framework Nucleic Acid Delivered RNA Therapeutics Significantly Attenuate Pancreatic Cancer Progression via Inhibition of CTR1-Dependent Copper Absorption.

Copper is vital for various life processes, whereas severely toxic at excess level. Intracellular copper homeostasis is strictly controlled by a set of transporters and chaperones encoded by the copper homeostasis genes. Increasing evidence has shown that copper is usually overloaded in multiple malignancies, including pancreatic cancer, which has an extremely poor prognosis. Recently, silencing the SLC31A1 gene, which encodes a major transmembrane copper transporter (CTR1), has been demonstrated to be an effective means for reducing the malignant degree of pancreatic cancer by downregulating the cellular copper levels. Herein, we utilized tetrahedral framework nucleic acids (tFNAs) as vehicles to overcome the biological barriers for delivering small molecular RNAs and efficiently transferred two kinds of CTR1 mRNA-targeted RNA therapeutics, siCTR1 or miR-124, into PANC-1 cells. Both therapeutic tFNAs, termed t-siCTR1 and t-miR-124, prevented copper intake more effective than the free RNA therapeutics via efficiently suppressing the expression of CTR1, thereby significantly attenuating the progression of PANC-1 cells. In this study, therapeutic tFNAs are constructed to target metal ion transporters for the first time, which may provide an effective strategy for future treatment of other metal metabolism disorders.

Tumor cell-imposed iron restriction drives immunosuppressive polarization of tumor-associated macrophages.

BACKGROUND: Tumor-associated macrophages (TAM) are immunosuppressive cells that contribute to impaired anti-cancer immunity. Iron plays a critical role in regulating macrophage function. However, it is still elusive whether it can drive the functional polarization of macrophages in the context of cancer and how tumor cells affect the iron-handing properties of TAM. In this study, using hepatocellular carcinoma (HCC) as a study model, we aimed to explore the effect and mechanism of reduced ferrous iron in TAM. METHODS: TAM from HCC patients and mouse HCC tissues were collected to analyze the level of ferrous iron. Quantitative real-time PCR was used to assess M1 or M2 signature genes of macrophages treated with iron chelators. A co-culture system was established to explore the iron competition between macrophages and HCC cells. Flow cytometry analysis was performed to determine the holo-transferrin uptake of macrophages. HCC samples from The Cancer Genome Atlas (TCGA) were enrolled to evaluate the prognostic value of transferrin receptor (TFRC) and its relevance to tumor-infiltrating M2 macrophages. RESULTS: We revealed that ferrous iron in M2-like TAM is lower than that in M1-like TAM. In vitro analysis showed that loss of iron-induced immunosuppressive M2 polarization of mouse macrophages. Further experiments showed that TFRC, the primary receptor for transferrin-mediated iron uptake, was overexpressed on HCC cells but not TAM. Mechanistically, HCC cells competed with macrophages for iron to upregulate the expression of M2-related genes via induction of HIF-1α, thus contributing to M2-like TAM polarization. We further clarified the oncogenic role of TFRC in HCC patients by TCGA. TFRC is significantly increased in varieties of malignancies, including HCC, and HCC patients with high TFRC levels have considerably shortened overall survival. Also, TFRC is shown to be positively related to tumor-infiltrating M2 macrophages. CONCLUSIONS: Collectively, we identified iron starvation through TFRC-mediated iron competition drives functional immunosuppressive polarization of TAM, providing new insight into the interconnection between iron metabolism and tumor immunity.

Analysis of Long Noncoding RNAs in Aila-Induced Non-Small Cell Lung Cancer Inhibition.

Non-small cell lung cancer (NSCLC) has the highest morbidity and mortality among all carcinomas. However, it is difficult to diagnose in the early stage, and current therapeutic efficacy is not ideal. Although numerous studies have revealed that Ailanthone (Aila), a natural product, can inhibit multiple cancers by reducing cell proliferation and invasion and inducing apoptosis, the mechanism by which Aila represses NSCLC progression in a time-dependent manner remains unclear. In this study, we observed that most long noncoding RNAs (lncRNAs) were either notably up- or downregulated in NSCLC cells after treatment with Aila. Moreover, alterations in lncRNA expression induced by Aila were crucial for the initiation and metastasis of NSCLC. Furthermore, in our research, expression of DUXAP8 was significantly downregulated in NSCLC cells after treatment with Aila and regulated expression levels of EGR1. In conclusion, our findings demonstrate that Aila is a potent natural suppressor of NSCLC by modulating expression of DUXAP8 and EGR1.

Baicalein Induces Apoptosis of Pancreatic Cancer Cells by Regulating the Expression of miR-139-3p and miR-196b-5p.

Pancreatic cancer is a common malignant tumor with a high incidence and mortality rate. The prognosis of patients with pancreatic cancer is considerably poor due to the lack of effective treatment in clinically. Despite numerous studies have revealed that baicalein, a natural product, is responsible for suppressing multiple cancer cells proliferation, motility and invasion. The mechanism by which baicalein restraining pancreatic cancer progression remains unclear. In this study, we firstly verified that baicalein plays a critical role in inhibiting pancreatic tumorigenesis in vitro and in vivo. Then we analyzed the alteration of microRNAs (miRNAs) expression levels in Panc-1 cells incubated with DMSO, 50 and 100 µM baicalein by High-Throughput sequencing. Intriguingly, we observed that 20 and 39 miRNAs were accordingly up- and down-regulated through comparing Panc-1 cells exposed to 100 µM baicalein with the control group. Quantitative PCR analysis confirmed that miR-139-3p was the most up-regulated miRNA after baicalein treatment, while miR-196b-5p was the most down-regulated miRNA. Further studies showed that miR-139-3p induced, miR-196b-5p inhibited the apoptosis of Panc-1 cells via targeting NOB1 and ING5 respectively. In conclusion, we demonstrated that baicalein is a potent inhibitor against pancreatic cancer by modulating the expression of miR-139-3p or miR-196b-5p.

Fucoidan Inhibits the Progression of Hepatocellular Carcinoma via Causing lncRNA LINC00261 Overexpression.

Hepatocellular carcinoma (HCC) as a main type of primary liver cancers has become one of the most deadly tumors because of its high morbidity and poor prognosis. Fucoidan is a family of natural, heparin-like sulfated polysaccharides extracted from brown algae. It is not only a widely used dietary supplement, but also participates in many biological activities, such as anti-oxidation, anti-inflammation and anti-tumor. However, the mechanism of fucoidan induced inhibition of HCC is elusive. In our study, we demonstrated that fucoidan contributes to inhibiting cell proliferation in vivo and in vitro, restraining cell motility and invasion and inducing cell cycle arrest and apoptosis. According to High-Throughput sequencing of long-non-coding RNA (lncRNA) in MHCC-97H cells treated with 0.5 mg/mL fucoidan, we found that 56 and 49 lncRNAs were correspondingly up- and down-regulated. LINC00261, which was related to the progression of tumor, was highly expressed in fucoidan treated MHCC-97H cells. Moreover, knocking down LINC00261 promoted cell proliferation by promoting the expression level of miR-522-3p, which further decreased the expression level of downstream SFRP2. Taken together, our results verified that fucoidan effectively inhibits the progression of HCC via causing lncRNA LINC00261 overexpression.

Hepatic microenvironment underlies fibrosis in chronic hepatitis B patients.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a leading cause of liver morbidity and mortality worldwide. Liver fibrosis resulting from viral infection-associated inflammation and direct liver damage plays an important role in disease management and prognostication. The mechanisms underlying the contribution of the liver microenvironment to fibrosis in HBV patients are not fully understood. There is an absence of effective clinical treatments for liver fibrosis progression; thus, establishing a suitable in vitro microenvironment in order to design novel therapeutics and identify molecular biomarkers to stratify patients is urgently required. AIM: To examine a subset of pre-selected microenvironment factors of chronic HBV patients that may underlie fibrosis, with a focus on fibroblast activation. METHODS: We examined the gene expression of key microenvironment factors in liver samples from patients with more advanced fibrosis compared with those with less severe fibrosis. We also used the human stellate cell line LX-2 in the in vitro study. Using different recombinant cytokines and growth factors or their combination, we studied how these factors interacted with LX-2 cells and pinpointed the cross-talk between the aforementioned factors and screened the most important factors. RESULTS: Of the secreted factors examined, transforming growth factor (TGF)-ß1, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were increased in patients with advanced fibrosis. We found that besides TGF-ß1, IL-1ß can also induce a profibrotic cascade by stimulating the expression of connective tissue growth factor and platelet-derived growth factor (PDGF) in LX-2 cells. Furthermore, the proinflammatory response can be elicited in LX-2 cells following treatment with IL-1ß and TNF-α, suggesting that stellate cells can respond to proinflammatory stimuli. By combining IL-1ß and TGF-ß1, we observed not only fibroblast activation as shown by αlpha-smooth muscle actin and PDGF induction, but also the inflammatory response as shown by increased expression of IL-1ß. CONCLUSION: Collectively, our data from HBV patients and in vitro studies demonstrate that the hepatic microenvironment plays an important role in mediating the crosstalk between profibrotic and proinflammatory responses and modulating fibrosis in chronic HBV patients. For the establishment of a suitable in vitro microenvironment for HBV-induced liver fibrosis, not only TGF-ß1 but also IL-1ß should be considered as a necessary environmental factor.

microRNA-93-5p promotes hepatocellular carcinoma progression via a microRNA-93-5p/MAP3K2/c-Jun positive feedback circuit.

Cumulative evidence suggests that microRNAs (miRNAs) promote gene expression in cancers. However, the pathophysiologic relevance of miRNA-mediated RNA activation in hepatocellular carcinoma (HCC) remains to be established. Our previous miRNA expression profiling in seven-paired HCC specimens revealed miR-93-5p as an HCC-related miRNA. In this study, miR-93-5p expression was assessed in HCC tissues and cell lines by quantitative real-time PCR and fluorescence in situ hybridization. The correlation of miR-93-5p expression with survival and clinicopathological features of HCC was determined by statistical analysis. The function and potential mechanism of miR-93-5p in HCC were further investigated by a series of gain- or loss-of-function experiments in vitro and in vivo. We identified that miR-93-5p, overexpressed in HCC specimens and cell lines, leads to poor outcomes in HCC cases and promotes proliferation, migration, and invasion in HCC cell lines. Mechanistically, rather than decreasing target mRNA levels as expected, miR-93-5p binds to the 3'-untranslated region (UTR) of mitogen-activated protein kinase kinase kinase 2 (MAP3K2) to directly upregulate its expression and downstream p38 and c-Jun N-terminal kinase (JNK) pathway, thereby leading to cell cycle progression in HCC. Notably, we also demonstrated that c-Jun, a downstream effector of the JNK pathway, enhances miR-93-5p transcription by targeting its promoter region. Besides, downregulation of miR-93-5p significantly retarded tumor growth, while overexpression of miR-93-5p accelerated tumor growth in the HCC xenograft mouse model. Altogether, we revealed a miR-93-5p/MAP3K2/c-Jun positive feedback loop to promote HCC progression in vivo and in vitro, representing an RNA-activating role of miR-93-5p in HCC development.

Enhanced mLST8 Expression Correlates with Tumor Progression in Hepatocellular Carcinoma.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway, containing mTOR complex 1 (mTORC1) and mTORC2, is dysregulated in multiple cancers, including hepatocellular carcinoma (HCC). Mammalian lethal with sec-13 protein 8 (mLST8) is a shared constituent of both mTORC1 and mTORC2, yet little is known regarding its role in HCC development. METHODS: mLST8 expression was detected in a total of 186 pairs of HCC and adjacent non-tumor specimens. The correlation between mLST8 level and clinicopathological features or prognostic significance were analyzed. The role of mLST8 on biological functions was also preliminarily studied. RESULTS: The study revealed that the mLST8 level was dramatically higher in HCC specimens than in adjacent non-tumor specimens. mLST8 overexpression positively correlated with tumor size, differentiation, and vessel invasion. Cases with elevated mLST8 level had more unfavorable overall survival (OS) and disease-free survival (DFS) than those with downregulated mLST8 level. Multivariate analysis demonstrated that mLST8 upregulation was an independent predictive marker for OS and DFS. Calibration curves from nomogram models indicated an excellent coherence between nomogram prediction and actual situation. Decision curve analysis proved that mLST8-based nomograms presented much higher predictive accuracy when compared with conventional clinical staging systems. Mechanistically, mLST8 enhanced cell proliferation and invasion through the AKT (protein kinase B) pathway. CONCLUSIONS: Our study demonstrates that mLST8 exerts an oncogenic role in HCC and may become a promising prognostic biomarker and therapeutic target for HCC patients.

OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. METHODS: We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. RESULTS: Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681 postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated with reduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the α-ketoglutarate (αKG):citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived αKG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in an αKG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. CONCLUSION: Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. LAY SUMMARY: Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer.

A-to-I RNA Editing in Cancer: From Evaluating the Editing Level to Exploring the Editing Effects.

As an important regulatory mechanism at the posttranscriptional level in metazoans, adenosine deaminase acting on RNA (ADAR)-induced A-to-I RNA editing modification of double-stranded RNA has been widely detected and reported. Editing may lead to non-synonymous amino acid mutations, RNA secondary structure alterations, pre-mRNA processing changes, and microRNA-mRNA redirection, thereby affecting multiple cellular processes and functions. In recent years, researchers have successfully developed several bioinformatics software tools and pipelines to identify RNA editing sites. However, there are still no widely accepted editing site standards due to the variety of parallel optimization and RNA high-seq protocols and programs. It is also challenging to identify RNA editing by normal protocols in tumor samples due to the high DNA mutation rate. Numerous RNA editing sites have been reported to be located in non-coding regions and can affect the biosynthesis of ncRNAs, including miRNAs and circular RNAs. Predicting the function of RNA editing sites located in non-coding regions and ncRNAs is significantly difficult. In this review, we aim to provide a better understanding of bioinformatics strategies for human cancer A-to-I RNA editing identification and briefly discuss recent advances in related areas, such as the oncogenic and tumor suppressive effects of RNA editing.

Upregulated calcium-binding tyrosine phosphorylation-regulated protein-a/b regulates cell proliferation and apoptosis and predicts poor prognosis in hepatocellular carcinoma.

BACKGROUND: Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is a group of isoforms produced by alternative splicing and is overexpressed in human malignancies including hepatocellular carcinoma (HCC). However, the prognostic value and biological functions of its major protein isoforms, named CABYR-a/b (combined CABYR-a and CABYR-b), in HCC remain to be established. METHODS: CABYR-a/b expression was detected in HCC tissues and cell lines by quantitative real-time polymerase chain reaction and Western blot analysis. The correlation of CABYR-a/b expression with clinical characteristics and its prognosis impact were determined by statistical analysis. Finally, the biological functions and molecular mechanism of CABYR-a/b were also investigated using molecular biology approaches. RESULTS: The present research found that CABYR-a/b was markedly elevated in HCC specimens and cell lines. Upregulated CABYR-a/b level had positive association with tumor size and differentiation in patients. Moreover, cases with elevated CABYR-a/b level had poorer overall survival (OS) and disease-free survival (DFS) than those with reduced CABYR-a/b level. Multivariate analysis and prognostic nomograms demonstrated that CABYR-a/b overexpression was an independent predictive indicator for OS and DFS. The calibration curve for the odds of OS and DFS demonstrated that the prediction by nomograms was in excellent accordance with actual situation. CABYR-a/b downregulation suppressed cell proliferation and induced G1-phase arrest via decreasing cyclin D1 and cyclin dependent kinase 4, while promoted apoptosis by reducing B-cell lymphoma 2 (Bcl-2) and increasing Bcl-2-associated death promoter. CONCLUSION: Our research indicates that CABYR-a/b exerts an oncogenic effect on HCC development and may become a new prognostic indicator for patients with HCC.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells.

OBJECTIVE: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases. DESIGN: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter. RESULTS: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver. CONCLUSION: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.

Comprehensive analysis of long non­coding RNA­messenger RNA­microRNA co­expression network identifies cell cycle­related lncRNA in hepatocellular carcinoma.

Long non­coding RNAs (lncRNAs) have been shown to contribute to progression and prognosis of hepatocellular carcinoma (HCC). However, expression profiling and interaction of lncRNAs with messenger RNAs (mRNAs) and microRNAs (miRNAs) remain largely unknown in HCC. The expression profiling of lncRNAs, mRNA and miRNAs was obtained using microarray. The Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to characterize potential functions of differentially expressed mRNAs. Cytoscape was applied to construct an lncRNA­miRNA­mRNA co­expression network and candidate lncRNAs were validated via quantitative PCR in 30 pairs of HCC and adjacent tumor­free tissues. In this study, 1,056 upregulated and 1,288 downregulated lncRNAs were identified, while 2,687 mRNAs and 6 miRNAs were aberrantly expressed in HCC compared with adjacent tumor­free tissues. Potential functions of differentially expressed mRNAs were demonstrated to significantly participate in modulating critical genes in the cell cycle, such as cyclin E1 and cyclin B2. After screening, 95 lncRNAs, 5 miRNAs and 36 mRNAs were recruited for construction of lncRNA­mRNA­miRNA co­expression network in the cell cycle pathway. Subsequently, the top 5 lncRNAs that potentially modulate critical genes in the cell cycle were selected as the candidates for further verification. Kaplan­Meier curves using the Cancer Genome Atlas database showed that 13 targeted mRNAs were associated with overall survival of HCC patients. Finally, three lncRNAs, including ENST00000522221, lnc­HACE1­6:1 and lnc­ICOSLG­11:1, are significantly upregulated in HCC tissues compared with adjacent tumor­free tissues. These findings suggest that lncRNAs play essential roles in the pathogenesis of HCC via regulating coding genes and miRNAs, and may be important targets for diagnosis and treatment of this disease.