Hexokinase1: A glucose sensor involved in drought stress response and sugar metabolism depending on its kinase activity in strawberry.

Hexokinase1 (HXK1) is a bifunctional enzyme that plays indispensable roles in plant growth, nitrogen utilization, and stress resistance. However, information on the HXK family members of strawberries and their functions in glucose sensing and metabolic regulation is scarce. In the present study, four HXKs were firstly identified in the genome of Fragaria vesca and F. pentaphylla. The conserved domains of the HXK1s were confirmed, and a site-directed mutation (S177A) was introduced into the FpHXK1. FpHXK1, which shares the highest identity with the AtHXK1 was able to restore the glucose sensitivity and developmental defects of the Arabidopsis gin2-1 mutant, but not its kinase-activity-impaired mutant (FpHXK1S177A ). The transcription of FpHXK1 was dramatically up-regulated under PEG-simulated drought stress conditions. The inhibition of the HXK kinase activity delayed the strawberry plant's responses to drought stress. Transient overexpression of the FpHXK1 and its kinase-impaired mutant differentially affected the level of glucose, sucrose, anthocyanins, and total phenols in strawberry fruits. All these results indicated that the FpHXK1, acting as a glucose sensor, was involved in drought stress response and sugar metabolism depending on its kinase activity.

Systematic Comparison of Structural Characterization of Polysaccharides from Ziziphus Jujuba cv. Muzao.

To investigate the structural information differences of Ziziphus Jujuba cv. Muzao polysaccharides, ten samples were successfully extracted from aqueous and alkaline solutions, prepared via DEAE-Sepharose Fast Flow through different eluents and Sephacryl S-300 columns, and systematically analyzed. Their characteristics were studied and then compared using chemical testing, high-performance gel permeation chromatography (HPGPC), gas chromatography (GC), methylation analysis, and NMR spectroscopy. The data achieved demonstrated that different jujube polysaccharide fractions possessed different structural characteristics, and most of them belonged to pectic polysaccharides. Overall, the structural information difference of jujube polysaccharides was preliminarily illuminated, which could not only promote the potential application of Z. Jujuba cv. Muzao polysaccharides but also provide an effective way to analyze the structures of polysaccharides from other genera jujube fruit.

A Novel Sodium Alginate-Carnauba Wax Film Containing Calcium Ascorbate: Structural Properties and Preservative Effect on Fresh-Cut Apples.

In order to improve the mechanical properties, nutritional value and fresh-keeping ability of conventional sodium alginate edible composite membranes, a new type of edible composite film was prepared by adding water-blocking agent carnauba wax, plasticizer glycerin, antioxidant and nutritional enhancer sodium ascorbate on a basis of traditional sodium alginate composite film. In this study, the physical, mechanical and structural properties of different film components were investigated. The results showed the components did not simply combine, but produced interaction forces which improved the stability and mechanical properties of composite film. When the amount of calcium ascorbate was 0.4%, the water vapor transmittance of the composite film reached a minimum of 0.65 g·mm/(cm2·d·kPa), and the tensile strength and elongation at break reached the maximum, which were 398.64 MPa and 17.93%, respectively. Additionally, the sodium alginate-carnauba wax film exhibited better performance on the preservation of fresh-cut apples. Compared with other composite films, the color and hardness of fresh-cut apples coated with this composite film were better maintained, and the losses of titration acid content and soluble solid content were reduced. Moreover, the weight loss rate, increase in polyphenol oxidase activity and total colony count were inhibited. All results determined that the edible film has good application value in the field of fresh-cut fruit preservation, which provides a theoretical basis for further research on edible film.

Genome-Wide Identification of Strawberry C2H2-ZFP C1-2i Subclass and the Potential Function of FaZAT10 in Abiotic Stress.

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsisthaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.

Transcriptomic Complexity in Strawberry Fruit Development and Maturation Revealed by Nanopore Sequencing.

The use of alternative transcription start or termination sites (aTSS or aTTS) as well as alternative splicing (AS) produce diverse transcript isoforms, playing indispensable roles in the plant development and environmental adaptations. Despite the advances in the finding of the genome-wide alternatively spliced genes in strawberry, it remains unexplored how AS responds to the developmental cues and what relevance do these outcomes have to the gene function. In this study, we have systematically investigated the transcriptome complexity using long-read Oxford Nanopore Technologies along the four successive developmental stages. The full-length cDNA sequencing results unraveled thousands of previously unexplored transcript isoforms raised from aTSS, aTTS, and AS. The relative contributions of these three processes to the complexity of strawberry fruit transcripts were compared. The aTSS and aTTS were more abundant than the AS. Differentially expressed transcripts unraveled the key transitional role of the white fruit stage. Isoform switches of transcripts from 757 genes were observed. They were associated with protein-coding potential change and domain gain or loss as the main consequences. Those genes with switched isoforms take part in the key processes of maturation in the late stages. A case study using yeast two hybrid analysis supported the functional divergence of the two isoforms of the B-box protein 22. Our results provided a new comprehensive overview of the dynamic transcriptomic landscape during strawberry fruit development and maturation.

Metabolomic Analysis Revealed Distinct Physiological Responses of Leaves and Roots to Huanglongbing in a Citrus Rootstock.

Huanglongbing (HLB) is an obstinate disease in the citrus industry. No resistant citrus resources were currently available, but various degrees of Huanglongbing tolerance exist in different germplasm. Citrus junos is emerging as one of the popular rootstocks widely used in the citrus production. However, its responses to the HLB causal agent, Candidatus Liberibacter asiaticus (CLas), were still elusive. In the current study, we investigated the physiological, anatomical, and metabolomic responses of a C. junos rootstock 'Pujiang Xiangcheng' by a controlled CLas grafting inoculation. The summer flushes and roots were impaired at 15 weeks after inoculation, although typical leaf symptomatic phenotypes were not obvious. The chlorophyll pigments and the photosynthetic rate were compromised. The phloem sieve tubes were still working, despite the fact that the callose was deposited and the starch granules were accumulated in the phloem cells. A wide, targeted metabolomic analysis was carried out to explore the systematic alterations of the metabolites at this early stage of infection in the leaves and root system. The differentially accumulated metabolites in the CLas-affected leaves and roots compared with the mock-inoculation control tissues revealed that distinct responses were obvious. Besides the commonly observed alteration of sugar and amino acids, the active break down of starch in the roots was discovered. The different types of fatty acids were altered in the two tissues, with a more pronounced content decline in the roots. Our results not only provided fundamental knowledge about the response of the C. junos rootstock to the HLB disease, but also presented new insights into the host-pathogen interaction in the early stages.

Regulatory Effect of Sea-Buckthorn Procyanidins on Oxidative Injury HUVECs.

As society develops and aging populations increase, the incidence of arteriosclerosis, a seriously harmful cardiovascular disease (CVD) which mostly results from endothelial cellular oxidative damage, has continuously risen. Procyanidins from sea-buckthorn is a powerful antioxidant, although its protective effect on the cardiovascular system is not yet clearly understand. In this study, oxidative damaged HUVECs induced by palmitate acid (PA) were used as a model and the regulatory effect of procyanidins from sea-buckthorn (SBP) on HUVECs were investigated. The results showed SBP can be used for 12 h by HUVECs and had no detective cytotoxicity to them under 400 µg/L. Also, different concentrations of SBP can increase mitochondrial membrane potential and NO level and decrease LDH leakage in a dose-effect relationship, indicating SBP can improve oxidative damage. In addition, western blots and qPCR results showed SBP regulation on oxidative injured HUVECs is probably through p38MAPK/NF-κB signal pathway. This study revealed the molecular mechanism of procyanidins in decreasing endothelial oxidative damage, providing a theoretical foundation for further research on natural bioactive compounds to exert antioxidant activity in the body and prevent and improve cardiovascular diseases.

Immunomodulatory effect of intracellular polysaccharide from mycelia of Agaricus bitorquis (QuéL.) Sacc. Chaidam by TLR4-mediated MyD88 dependent signaling pathway.

Agaricus bitorquis (QuéL.) Sacc. Chaidam is a valuable edible fungus in Qinghai-Tibet plateau and ABSP is a novel intracellular polysaccharide from its mycelia. GC and NMR analysis determined ABSP is galactoglucomannan-like polysaccharide that may have immunomodulatory effect. This study used RAW264.7 as model cell to determine immunomodulatory effect of ABSP. After ABSP treatment, viability and phagocytic ability promoted, and NO, ROS, TNF-α levels also raised which proved ABSP had immune regulation to RAW264.7. WB and qRT-PCR determined the key proteins and genes expression of TLR4, MyD88, TRAF-6 and NF-κB significantly increased while protein and gene expression of TRAM had no significant increase. Also, TNF-α level extremely decreased by adding inhibitors of TLR4 and MyD88 which confirmed ABSP could immunologically regulate RAW264.7 byTLR4-MyD88 dependent pathway. This study would provide theoretical basis for further study on ABSP and be helpful for development of beneficial functionally foods and exploitation of this resource.

Development and Validation of a Prognostic Score for Hepatocellular Carcinoma Patients in Immune Checkpoint Inhibitors Therapies: The Hepatocellular Carcinoma Modified Gustave Roussy Immune Score.

Background: There is not yet an effective marker in predicting the efficacy of immune checkpoint inhibitors (ICIs) in treating hepatocellular carcinoma (HCC) patients. The Gustave Roussy Immune Score (GRIm-Score) based on three objective variables, namely, neutrophil-to-lymphocyte ratio (NLR), serum albumin level (ALB), and lactate dehydrogenase (LDH), was developed as feasible prognostic indication in lung cancer patients receiving ICIs therapies. Our study aimed to adapt the GRIm-Score (HCC-GRIm-Score) in HCC patients who received ICIs therapies and thus improving the predictive ability. Methods: From January 2018 to September 2020, 261 patients who received ICIs therapy were retrospectively included and divided into training and validation groups. After determining the factors for HCC-GRIm-Score by multivariable analysis from training group, the optimized HCC-GRIm-Score was validated and compared to the original GRIm-Score and the Barcelona clinic liver cancer (BCLC) staging system. Results: One hundred sixty-one and 80 patients were assigned into the training and validation groups, respectively. Two more factors, aspartate transaminase-to-alanine transaminase ratio [hazard ratio (HR), 1.51; 95% confidence interval (CI), 0.94-2.42] and total bilirubin (HR, 1.76; 95% CI, 1.07-2.88), were identified as independent prognostic factors for overall survival (OS) and integrated in the HCC-GRIm-Score system according to the multivariable analysis. A risk score based on the HCC-GRIm-Score indicated that patients presenting high score (>2) suffered from significantly shorter median OS of 10.3 months compared to those with a low score (not reached; HR, 2.99; 95% CI, 1.89-4.75; p < 0.001). In the validation group of 80 patients, the patients presenting a high score showed an inferior OS (HR 5.62, 95% CI, 1.25-25.24; p = 0.024). HCC-GRIm-Score had the highest area under curve of 0.719 (95% CI, 0.661-0.773) compared to original GRIm-Score and BCLC staging system. Conclusion: The present study confirmed that the modified HCC-GRIm-Score system provided superior predictive ability in identifying the HCC patients potentially benefit from ICIs therapies, compared to the original GRIm-Score and the BCLC staging system.

Targeted sequencing reveals the mutational landscape responsible for sorafenib therapy in advanced hepatocellular carcinoma.

Rationale: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance. Methods: Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of TP53 in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant OCT4 were applied to explore the function of OCT4 in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the KITLG promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes. Results: Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in OCT4 (OCT4mut) was strongly associated with good response to sorafenib, whereas the stop-gain mutation in TP53 showed the opposite outcome both in vitro and in vivo. OCT4wt-induced stem cell factor (encoded by KITLG gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of KITLG promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of KITLG and sorafenib resistance. Conclusion: Our study reported a novel somatic mutation in OCT4 (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.

A Novel Pectic Polysaccharide of Jujube Pomace: Structural Analysis and Intracellular Antioxidant Activities.

After extraction from jujube pomace and purification by two columns (DEAE-Sepharose Fast Flow and Sepharcyl S-300), the structure of SAZMP4 was investigated by HPGPC, GC, FI-IR, GC-MS, NMR, SEM, and AFM. Analysis determined that SAZMP4 (Mw = 28.94 kDa) was a pectic polysaccharide mainly containing 1,4-linked GalA (93.48%) with side chains of 1,2,4-linked Rha and 1,3,5-linked Ara and terminals of 1-linked Rha and 1-linked Ara, which might be the homogalacturonan (HG) type with side chains of the RG-I type, corresponding to the results of NMR. In AFM and SEM images, self-assembly and aggregation of SAZMP4 were respectively observed indicating its structural features. The antioxidant activity of SAZMP4 against H2O2-induced oxidative stress in Caco-2 cells was determined by activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as malondialdehyde (MDA) and reactive oxygen species (ROS) levels, indicating SAZMP4 can be a natural antioxidant. Also, a better water retention capacity and thermal stability of SAZMP4 was observed based on DSC analysis, which could be applied in food industry as an additive.

A case of group infections with Paraginimus species in Henan, Central China.

In July of 2012, mass infections with Paragonimus species were detected in the Henan province sickening 11 of 51 people. In May 2011, these individuals had participated in an excursion during which freshwater crabs were caught and served after being toasted. Before the group infections with Paraginimus species was confirmed, 5 of the 11 patients had been misdiagnosed as tuberculosis (TB) and treated with an anti-TB drug regimen for six months. The most common and typical manifestations were eosinophilia (11/11, 100%) and pulmonary manifestations including, among others, stethalgia and cough (7/11 63.6%). Sero-examination revealed that all 11 patients were seropositive for Paragonimus species. Surprisingly, in our case, one patient presented with hemoptysis and eggs in respiratory secretions, and this is the first time P. skrjabini eggs are detected in the sputum of a patient from the Henan province. Paragonimus metacercariae were collected from 6 of 11 (54.5%) crabs caught at the infection site and were identified as Paraginiumus skrjabini by morphological and molecular examinations. Epidemiological and laboratory evidence confirmed that this is a case of group infection with P. skrjabini. As one of the most neglected tropical diseases (NTD), paragonimiasis should be differentiated diagnosed from TB to avoid the delay of treatment. To our knowledge, this is the second report of a case of group infections with Paraginimus species in Henan, Central China. The first case was reported in 1995. As a kind of food-borne parasitic disease, paragonimiasis should be included in the public health education agenda.

Circular RNAs negatively regulate cancer stem cells by physically binding FMRP against CCAR1 complex in hepatocellular carcinoma.

Circular RNA (circRNA) possesses great pre-clinical diagnostic and therapeutic potentials in multiple cancers. It has been reported playing roles in multiple malignant behaviors including proliferation, migration, metastasis and chemoresistance. However, the underlying correlation between circRNAs and cancer stem cells (CSCs) has not been reported yet. Methods: circZKSCAN1 level was detected in HCC tissue microarrays to clarify its prognostic values. Gain and loss function experiments were applied to investigate the role of circZKSCAN1 in HCC stemness. Bioinformatic analysis was used to predict the possible downstream RNA binding protein and further RNA immunoprecipitation sequencing was carried out to identify the RBP-regulated genes. Results: The absence of circZKSCAN1 endowed several malignant properties including cancer stemness and tightly correlated with worse overall and recurrence-free survival rate in HCC. Bioinformatics analysis and RNA immunoprecipitation-sequencing (RIP-seq) results revealed that circZKSCAN1 exerted its inhibitive role by competitively binding FMRP, therefore, block the binding between FMRP and ß-catenin-binding protein-cell cycle and apoptosis regulator 1 (CCAR1) mRNA, and subsequently restrain the transcriptional activity of Wnt signaling. In addition, RNA-splicing protein Quaking 5 was found downregulated in HCC tissues and responsible for the reduction of circZKSCAN1. Conclusion: Collectively, this study revealed the mechanisms underlying the regulatory role of circZKSCAN1 in HCC CSCs and identified the newly discovered Qki5-circZKSCAN1-FMRP-CCAR1-Wnt signaling axis as a potentially important therapeutic target for HCC treatment.

An alkali-extracted polysaccharide from Zizyphus jujuba cv. Muzao: Structural characterizations and antioxidant activities.

An acidic polysaccharide (SAZMP3) was isolated from jujube fruit residues by alkaline extraction and purified with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. The structural characterizations of SAZMP3 were determined by high-performance gelpermeation chromatography, gas chromatography, Fourier transform infrared spectroscopy, methylation analysis, NMR spectroscopy, scanning electron microscopy, and atomic force microscopy. The results determined SAZMP3 was an acidic polysaccharide mainly contained rhamnose, galactose, and galacturonic acid with a molecular weight of 9.37 kDa and its backbone might mainly be composed of 1,4-α-D-GalAp with side chains of 1,3-ß-D-Galp, 1,3,5-linked Araf, 1,2,4-α-L-Rhap and terminals of 1-linked Araf, 1-linked Rhap, 1-linked Galp by methylation and NMR analysis. Besides, SAZMP3 has shown potential antioxidant activity in scavenging free radicals suggesting that it is an antioxidant agent, which might contribute to the functional food qualities of jujube fruit.

Distinct role of nuclear receptor corepressor 1 regulated de novo fatty acids synthesis in liver regeneration and hepatocarcinogenesis in mice.

It is urgent that the means to improve liver regeneration (LR) be found, while mitigating the concurrent risk of hepatocarcinogenesis (HCG). Nuclear receptor corepressor 1 (NCoR1) is a co-repressor of nuclear receptors, which regulates the expression level of metabolic genes; however, little is known about its potential contribution for LR and HCG. Here, we found that liver-specific NCoR1 knockout in mice (NCoR1Δhep ) dramatically enhances LR after partial hepatectomy and, surprisingly, blocks the process of diethylnitrosamine (DEN)-induced HCG. Both RNA-sequencing and metabolic assay results revealed improved expression of Fasn and Acc2 in NCoR1Δhep mice, suggesting the critical role of de novo fatty acid synthesis (FAS) in LR. Continual enhanced de novo FAS in NCoR1Δhep mice resulted in overwhelmed adenosine triphosphate ATP and nicotinamide adenine dinucleotide phosphate (NADPH) consumption and increased mitochondrial reactive oxygen species production, which subsequently attenuated HCG through inducing apoptosis of hepatocytes at an early stage after DEN administration. CONCLUSION: NCoR1 functions as a negative modulator for hepatic de novo FAS and mitochondria energy adaptation, playing distinct roles in regeneration or carcinogenesis. (Hepatology 2018;67:1071-1087).

A neglected risk for sparganosis: eating live tadpoles in central China.

A 29-year-old farmer from central China was sent into the Emergency Department of the Affiliated Hospital of Zhengzhou University. He had a 15-day history of persistent high fever, abdominal distention and pain. The patient was clinically diagnosed as appendicitis and peritonitis, and treated with antibiotics in a local hospital, did not improve. On exploratory laparotomy, the appendicular perforation and peritonitis were seen; appendicectomy were performed, and antibiotics were given. However, high fever and abdominal pain still persisted; intestinal adhesion and obstruction, ascites appeared. He was given the "critically ill notice". He had eosinophilia (12.95%) and the history of eating live frog tadpoles for treating his cutaneous pruritus 3 days before onset of the disease. Serum anti-sparganum antibodies assayed by ELISA were positive. This patient has hospitalized for one and half months and spend more than US$ 12 000. This patient was primarily diagnosed as visceral sparganosis, and cured with praziquantel.Sparganosis is one neglected but important parasitic zoonosis of poverty. Human infections were mainly acquired by eating raw or uncooked meat of frogs and snakes infected with plerocercoids, using frog or snake flesh as poultices, or drinking raw water contaminated with infected copepods. However, sparganosis caused by ingestion of live tadpoles are emerging in central China. Our surveys showed that 11.93% of tadpoles in Henan province are infected with plerocercoids. Eating live tadpoles is a high risk for sparganum infection. The comprehensive public health education should be carried out for people in endemic areas and the bad habit of eating live tadpoles must be discouraged.

Choline Kinase α Mediates Interactions Between the Epidermal Growth Factor Receptor and Mechanistic Target of Rapamycin Complex 2 in Hepatocellular Carcinoma Cells to Promote Drug Resistance and Xenograft Tumor Progression.

BACKGROUND & AIMS: Choline kinase α (CHKA) catalyzes conversion of choline to phosphocholine and can contribute to carcinogenesis. Little is known about the role of CHKA in the pathogenesis of hepatocellular carcinoma (HCC). METHODS: We performed whole-exome and transcriptome sequence analyses of 9 paired HCC and non-tumor-adjacent tissues. We performed tissue chip analyses of 120 primary HCC and non-tumor-adjacent tissues from patients who received surgery in Shanghai, China from January 2006 through December 2009; 48 sets of specimens (HCC and non-tumor-adjacent tissues) were also analyzed. CHKA gene copy number was quantified and findings were validated by quantitative reverse transcription polymerase chain reaction analysis. CHKA messenger RNA and protein levels were determined by polymerase chain reaction, immunohistochemical, and immunoblot analyses. CHKA was examined in 2 hepatocyte cell lines and 7 HCC-derived cell lines, and knocked down with small interfering RNAs in 3 HCC cell lines. Cells were analyzed in proliferation, wound healing, migration, and invasion assays. Cells were injected into tail veins of mice and tumor growth and metastasis were quantified. Immunoprecipitation and immunofluorescence assays were conducted to determine interactions between CHKA and the epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin complex 2. RESULTS: Levels of CHKA messenger RNA were frequently increased in HCC tissues compared with nontumor tissues; increased expression was associated with amplification at the CHKA loci. Tumors that expressed high levels of CHKA had more aggressive phenotypes, and patients with these tumors had shorter survival times after surgery compared to patients whose tumors expressed low levels of CHKA. HCC cell lines that stably overexpressed CHKA had higher levels of migration and invasion than control HCC cells, and formed larger xenograft tumors with more metastases in mice compared to HCC cells that did not overexpress CHKA. CHKA was required for physical interaction between EGFR and mechanistic target of rapamycin complex 2. This complex was required for HCC cells to form metastatic xenograft tumors in mice and to become resistant to EGFR inhibitors. CONCLUSIONS: We found levels of CHKA to be increased in human HCCs compared to nontumor tissues, and increased expression to be associated with tumor aggressiveness and reduced survival times of patients. Overexpression of CHKA in HCC cell lines increased their invasiveness, resistance to EGFR inhibitors, and ability to form metastatic tumors in mice by promoting interaction of EGFR with mechanistic target of rapamycin complex 2.

Aldehyde dehydrogenase-2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP-activated protein kinase signaling in mice.

Potential biomarkers that can be used to determine prognosis and perform targeted therapies are urgently needed to treat patients with hepatocellular carcinoma (HCC). To meet this need, we performed a screen to identify functional genes associated with hepatocellular carcinogenesis and its progression at the transcriptome and proteome levels. We identified aldehyde dedydrogenase-2 (ALDH2) as a gene of interest for further study. ALDH2 levels were significantly lower at the mRNA and protein level in tumor tissues than in normal tissues, and they were even lower in tissues that exhibited increased migratory capacity. A study of clinical associations showed that ALDH2 is correlated with survival and multiple migration-associated clinicopathological traits, including the presence of metastasis and portal vein tumor thrombus. The result of overexpressing or knocking down ALDH2 showed that this gene inhibited migration and invasion both in vivo and in vitro. We also found that ALDH2 altered the redox status of cells by regulating acetaldehyde levels and that it further activated the AMP-activated protein kinase (AMPK) signaling pathway. CONCLUSION: Decreased levels of ALDH2 may indicate a poor prognosis in HCC patients, while forcing the expression of ALDH2 in HCC cells inhibited their aggressive behavior in vitro and in mice largely by modulating the activity of the ALDH2-acetaldehyde-redox-AMPK axis. Therefore, identifying ALDH2 expression levels in HCC might be a useful strategy for classifying HCC patients and for developing potential therapeutic strategies that specifically target metastatic HCC. (Hepatology 2017;65:1628-1644).

Interferon-microRNA signalling drives liver precancerous lesion formation and hepatocarcinogenesis.

OBJECTIVE: Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance. DESIGN: Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484(-/-), Ifnar1(-/-) and Tgfbr2(△hep) mice were employed to determine the critical role of the interferon (IFN)-microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms. RESULTS: miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484(-/-) mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-ß/Gli and nuclear factor-κB/type I IFN pathways. Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2(△hep) and Ifnar1(-/-) mice. CONCLUSIONS: These findings demonstrate a new protumourigenic axis involving type I IFN-microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.

Serum miRNAs as predictive and preventive biomarker for pre-clinical hepatocellular carcinoma.

The extremely poor prognosis of patients with symptomatic hepatocellular carcinoma (HCC) diagnosed clinically at advanced stages suggests an urgent need for biomarkers that can be used for prospective surveillance and pre-clinical screening for early presence of pre-malignant lesions and tumors. In a retrospective longitudinal phase 3 biomarker study in seven medical centers of China, time-series and 6 months interval-serum samples were collected from chronic hepatitis B virus infected (CHB) patient cohorts at the pre-malignant or pre-clinical stages (average 6 months prior to clinical diagnosis) and CHB patients that did not develop cancer, and circulating miRNAs measured. A set of serum miRNAs including miR-193a-3p, miR-369-5p, miR-672, miR-429 and let-7i* were identified in pre-clinical HCC patients and have the potential to screen for CHB patients at high risk to develop HCC 6-12 months after miRNAs measurement. These circulating miRNAs combined with the conventional screening tools using α-fetoprotein and ultrasound, may have great promise for the prediction and prevention of HCC in high-risk populations.