Endothelial cell dynamics in sepsis-induced acute lung injury and acute respiratory distress syndrome: pathogenesis and therapeutic implications.

Sepsis, a prevalent critical condition in clinics, continues to be the leading cause of death from infections and a global healthcare issue. Among the organs susceptible to the harmful effects of sepsis, the lungs are notably the most frequently affected. Consequently, patients with sepsis are predisposed to developing acute lung injury (ALI), and in severe cases, acute respiratory distress syndrome (ARDS). Nevertheless, the precise mechanisms associated with the onset of ALI/ARDS remain elusive. In recent years, there has been a growing emphasis on the role of endothelial cells (ECs), a cell type integral to lung barrier function, and their interactions with various stromal cells in sepsis-induced ALI/ARDS. In this comprehensive review, we summarize the involvement of endothelial cells and their intricate interplay with immune cells and stromal cells, including pulmonary epithelial cells and fibroblasts, in the pathogenesis of sepsis-induced ALI/ARDS, with particular emphasis placed on discussing the several pivotal pathways implicated in this process. Furthermore, we discuss the potential therapeutic interventions for modulating the functions of endothelial cells, their interactions with immune cells and stromal cells, and relevant pathways associated with ALI/ARDS to present a potential therapeutic strategy for managing sepsis and sepsis-induced ALI/ARDS.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment in vitro. Both conditioned media of miR-146a-5p-treated macrophages (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.

Autophagy Plays a Protective Role in Sodium Hydrosulfide-Induced Acute Lung Injury by Attenuating Oxidative Stress and Inflammation in Rats.

Sodium hydrosulfide (NaHS), as an exogenous hydrogen sulfide (H2S) donor, has been used in various pathological models. NaHS is usually considered to be primarily protective, however, the toxic effect of NaHS has not been well elucidated. The aim of this study was to investigate whether NaHS (1 mg/kg) can induce acute lung injury (ALI is a disease process characterized by diffuse inflammation of the lung parenchyma) and define the mechanism by which NaHS-induced ALI involves autophagy, oxidative stress, and inflammatory response. Wistar rats were randomly divided into three groups (control group, NaHS group, and 3-MA + NaHS group), and samples from each group were collected from 2, 6, 12, and 24 h. We found that intraperitoneal injection of NaHS (1 mg/kg) increased the pulmonary levels of H2S and oxidative stress-related indicators (reactive oxygen species, myeloperoxidase, and malondialdehyde) in a time-dependent manner. Intraperitoneal injection of NaHS (1 mg/kg) induced histopathological changes of ALI and inhibition of autophagy exacerbated the lung injury. This study demonstrates that administration of NaHS (1 mg/kg) induces ALI in rats and autophagy in response to ROS is protective in NaHS-induced ALI by attenuating oxidative stress and inflammation.

Toxic effects of hydrogen sulfide donor NaHS induced liver apoptosis is regulated by complex IV subunits and reactive oxygen species generation in rats.

In recent years, the protective effect of hydrogensulfide donor sodium hydrosulfide(NaHS) on multiple organs has been widely reported. The study aimed to explorethe effect of commonly used concentration of NaHS on theliver and its potential damage mechanism. Rats divided into 4 groups: control, NaHS I (1 mg/kg), II (3 mg/kg) and III(5 mg/kg) groups, and each group is divided into four-timepoints (2, 6, 12, and 24 hours). Results showed that H2S concentration increased, mitochondrial complex IV activity inhibited, the COX I and IV subunits and mitochondrial apoptosis pathway-related proteins expression increased in atime- and dose-dependent manner. We confirmed that 1 mg/kg NaHS had no injuryeffect on the liver, 3 and 5 mg/kg NaHS inhibitsthe activity of mitochondrial complex IV by promoting COX I and IV subunits expression, leading to the increase in ROS and ultimately inducing apoptosis and liver injury.

OsJAZ13 Negatively Regulates Jasmonate Signaling and Activates Hypersensitive Cell Death Response in Rice.

Jasmonate ZIM-domain (JAZ) proteins belong to the subgroup of TIFY family and act as key regulators of jasmonate (JA) responses in plants. To date, only a few JAZ proteins have been characterized in rice. Here, we report the identification and function of rice OsJAZ13 gene. The gene encodes three different splice variants: OsJAZ13a, OsJAZ13b, and OsJAZ13c. The expression of OsJAZ13 was mainly activated in vegetative tissues and transiently responded to JA and ethylene. Subcellular localization analysis indicated OsJAZ13a is a nuclear protein. Yeast two-hybrid assays revealed OsJAZ13a directly interacts with OsMYC2, and also with OsCOI1, in a COR-dependent manner. Furthermore, OsJAZ13a recruited a general co-repressor OsTPL via an adaptor protein OsNINJA. Remarkably, overexpression of OsJAZ13a resulted in the attenuation of root by methyl JA. Furthermore, OsJAZ13a-overexpressing plants developed lesion mimics in the sheath after approximately 30-45 days of growth. Tillers with necrosis died a few days later. Gene-expression analysis suggested the role of OsJAZ13 in modulating the expression of JA/ethylene response-related genes to regulate growth and activate hypersensitive cell death. Taken together, these observations describe a novel regulatory mechanism in rice and provide the basis for elucidating the function of OsJAZ13 in signal transduction and cell death in plants.

Dexmedetomidine improves acute stress-induced liver injury in rats by regulating MKP-1, inhibiting NF-κB pathway and cell apoptosis.

Acute stress is a frequent and unpredictable disease for many animals. Stress is widely considered to affect liver function. However, the underlying mechanism by which dexmedetomidine (DEX) attenuates acute stress-induced liver injury in rats remains unclear. In this study, we used forced swimming for 15 min and acute 3-hr restraint stress model. Behavioral tests and changes in norepinephrine levels confirmed the successful establishment of the acute stress model. Acute stress-induced liver injury, evidenced by hematoxylin and eosin-stained pathological sections and increased serum aminotransferase and aspartate aminotransferase levels, was reduced in DEX-treated livers. Reactive oxygen species and oxidative stress levels were dramatically decreased with DEX treatment compared with acute stress-induced liver injury. DEX significantly reduced acute stress-induced liver inflammation and apoptosis, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and inflammation and apoptosis-related protein levels. DEX treatment also effectively inhibited acute stress-induced c-Jun N-terminal kinase (JNK), P38, and BAD signaling pathway activation, and significantly induced MKP-1 activation. Thus, DEX has a protective effect on acute-stress-induced liver injury by reducing inflammation and apoptosis, which suggests a potential clinical application for DEX in stress syndrome.

Dexmedetomidine ameliorates lipopolysaccharide-induced acute kidney injury in rats by inhibiting inflammation and oxidative stress via the GSK-3ß/Nrf2 signaling pathway.

Acute kidney injury (AKI) is a frequent and serious complication of sepsis; however, there are currently no effective therapies. Inflammation and oxidative stress are the major mechanisms implicated in lipopolysaccharide (LPS)-induced AKI. Dexmedetomidine (DEX) has been reported to have remarkable anti-inflammatory and antioxidant effects. Here, we examined the renoprotective effects of DEX and potential underlying mechanisms in rats with LPS-induced AKI. We analyzed renal function and structure; serum inflammatory cytokine; renal oxidant and antioxidant levels; and renal expression of glycogen synthase kinase-3ß (GSK-3ß)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related proteins in rats 4 hr after administration of LPS. Pretreatment with DEX improved renal function and significantly reduced the levels of inflammatory cytokines and oxidative stress markers. Treatment with DEX and the GSK-3ß inhibitor SB216367 promoted phosphorylation of GSK-3ß, induced Nrf2 nuclear translocation, and increased transcription of the Nrf2 target genes heme oxygenase-1 and NAD(P)H quinone oxidoreductase-1, primarily in renal tubules. Alpha-2-adrenergic receptor (α2-AR) antagonist atipamezole and imidazoline I 2 receptor (I 2 R) antagonist idazoxan reversed the effects of DEX. These results suggest that the renoprotective effects of DEX are mediated via α2-AR and I 2 R-dependent pathways that reduce inflammation and oxidative stress through GSK-3ß/Nrf2 signaling.

Dexmedetomidine alleviates lipopolysaccharide-induced acute kidney injury by inhibiting the NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway.

Dexmedetomidine (DEX) prevents kidney damage caused by sepsis, but the mechanism of this effect remains unclear. In this study, the protective molecular mechanism of DEX in lipopolysaccharide (LPS)-induced acute kidney injury was investigated and its potential pharmacological targets from the perspective of inhibiting oxidative stress damage and the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation. Intraperitoneal injection of DEX (30 µg/kg) significantly improved LPS (10 mg/kg) induced renal pathological damage and renal dysfunction. DEX also ameliorated oxidative stress damage by reducing the contents of reactive oxygen species, malondialdehyde and hydrogen peroxide, and increasing the level of glutathione, as well as the activity of superoxide dismutase and catalase. In addition, DEX prevented nuclear factor-kappa B (NF-κB) activation and I-kappa B (IκB) phosphorylation, as well as the expressions of NLRP3 inflammasome-associated protein and downstream IL-18 and IL-1ß. The messengerRNA (mRNA) and protein expressions of toll-like receptor 4 (TLR4), NADPH oxidase-4 (NOX4), NF-κB, and NLRP3 were also significantly reduced by DEX. Their expressions were further evaluated by immunohistochemistry, yielding results were consistent with the results of mRNA and protein detection. Interestingly, the protective effects of DEX were reversed by atipamezole-an alpha 2 adrenal receptor (α2 AR) inhibitor, whereas idazoxan-an imidazoline receptor (IR) inhibitor failed to reverse this change. In conclusion, DEX attenuated LPS-induced AKI by inhibiting oxidative stress damage and NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway, mainly acting on the α2 AR rather than IR.

Dexmedetomidine attenuates lipopolysaccharide-induced liver oxidative stress and cell apoptosis in rats by increasing GSK-3ß/MKP-1/Nrf2 pathway activity via the α2 adrenergic receptor.

Dexmedetomidine (DEX) protects against liver damage caused by sepsis. The purpose of this study was to confirm the regulatory effects of DEX on glycogen synthase kinase 3 beta (GSK-3ß) via the α2 adrenergic receptor (α2AR) and evaluate the role of GSK-3ß in lipopolysaccharide (LPS)-induced liver injury. Sprague-Dawley (SD) rats were administered an intraperitoneal injection of DEX (30 µg/kg) 30 min before an intraperitoneal injection of LPS (10 mg/kg). HE staining and serum biochemical test results indicated that DEX significantly improved liver histopathological damage and liver function indices. Furthermore, DEX increased super oxide dismutase (SOD) activity and L-glutathione (GSH) levels, and inhibited malondialdehyde (MDA) production. Western blot (WB) analysis demonstrated that treatment with the GSK-3ß inhibitor SB216763 increased antioxidant-related protein mitogen-activated protein kinase phosphatase 1 (MKP-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. In addition, anti-apoptosis-related proteins were up-regulated and pro-apoptosis-related proteins were down-regulated by SB21676 administration. WB analysis also showed that DEX increased anti-apoptosis-related protein levels and decreased pro-apoptotic protein levels in LPS-induced liver injury. Nrf2, p53, and activated caspase-3 levels were further evaluated using immunohistochemistry (IHC), producing results consistent with WB results. The α2AR antagonist atipamezole (AT) significantly reversed the protective effects of DEX, as shown by WB analysis. Our data suggested that α2AR plays an important role in reversing the effects of liver oxidative stress and apoptosis via DEX, and that DEX exerts antioxidant and anti-apoptosis effects through regulation of the GSK-3ß/MKP-1/Nrf2 pathway.

Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat ß-lactoglobulin (ßLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

Arabidopsis pentatricopeptide repeat protein SOAR1 plays a critical role in abscisic acid signalling.

A dominant suppressor of the ABAR overexpressor, soar1-1D, from CHLH/ABAR [coding for Mg-chelatase H subunit/putative abscisic acid (ABA) receptor (ABAR)] overexpression lines was screened to explore the mechanism of the ABAR-mediated ABA signalling. The SOAR1 gene encodes a pentatricopeptide repeat (PPR) protein which localizes to both the cytosol and nucleus. Down-regulation of SOAR1 strongly enhances, but up-regulation of SOAR1 almost completely impairs, ABA responses, revealing that SOAR1 is a critical, negative, regulator of ABA signalling. Further genetic evidence supports that SOAR1 functions downstream of ABAR and probably upstream of an ABA-responsive transcription factor ABI5. Changes in the SOAR1 expression alter expression of a subset of ABA-responsive genes including ABI5. These findings provide important information to elucidate further the functional mechanism of PPR proteins and the complicated ABA signalling network.

Coupling cellular localization and function of checkpoint kinase 1 (Chk1) in checkpoints and cell viability.

Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.

Cochaperonin CPN20 negatively regulates abscisic acid signaling in Arabidopsis.

Previous study showed that the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) positively regulates abscisic acid (ABA) signaling. Here, we investigated the functions of a CHLH/ABAR interaction protein, the chloroplast co-chaperonin 20 (CPN20) in ABA signaling in Arabidopsis thaliana. We showed that down-expression of the CPN20 gene increases, but overexpression of the CPN20 gene reduces, ABA sensitivity in the major ABA responses including ABA-induced seed germination inhibition, postgermination growth arrest, promotion of stomatal closure and inhibition of stomatal opening. Genetic evidence supports that CPN20 functions downstream or at the same node of CHLH/ABAR, but upstream of the WRKY40 transcription factor. The other CPN20 interaction partners CPN10 and CPN60 are not involved in ABA signaling. Our findings show that CPN20 functions negatively in the ABAR-WRKY40 coupled ABA signaling independently of its co-chaperonin role, and provide a new insight into the role of co-chaperones in the regulation of plant responses to environmental cues.

Light-harvesting chlorophyll a/b-binding proteins, positively involved in abscisic acid signalling, require a transcription repressor, WRKY40, to balance their function.

The light-harvesting chlorophyll a/b-binding (LHCB) proteins are the apoproteins of the light-harvesting complex of photosystem II. In the present study, we observed that downregulation of any of the six LHCB genes resulted in abscisic acid (ABA)-insensitive phenotypes in seed germination and post-germination growth, demonstrating that LHCB proteins are positively involved in these developmental processes in response to ABA. ABA was required for full expression of different LHCB members and physiologically high levels of ABA enhanced LHCB expression. The LHCB members were shown to be targets of an ABA-responsive WRKY-domain transcription factor, WRKY40, which represses LHCB expression to balance the positive function of the LHCBs in ABA signalling. These findings revealed that ABA is an inducer that fine-tunes LHCB expression at least partly through repressing the WRKY40 transcription repressor in stressful conditions in co-operation with light, which allows plants to adapt to environmental challenges.

The great potential of flavonoids as candidate drugs for NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of approximately 25 % and is associated with high morbidity and high mortality. NAFLD is a leading cause of cirrhosis and hepatocellular carcinoma. Its pathophysiology is complex and still poorly understood, and there are no drugs used in the clinic to specifically treat NAFLD. Its pathogenesis involves the accumulation of excess lipids in the liver, leading to lipid metabolism disorders and inflammation. Phytochemicals with the potential to prevent or treat excess lipid accumulation have recently received increasing attention, as they are potentially more suitable for long-term use than are traditional therapeutic compounds. In this review, we summarize the classification, biochemical properties, and biological functions of flavonoids and how they are used in the treatment of NAFLD. Highlighting the roles and pharmacological uses of these compounds will be of importance for enhancing the prevention and treatment of NAFLD.

Inhibition of PPARγ by BZ26, a GW9662 derivate, attenuated obesity-related breast cancer progression by inhibiting the reprogramming of mature adipocytes into to cancer associate adipocyte-like cells.

Obesity has been associated with the development of 13 different types of cancers, including breast cancer. Evidence has indicated that cancer-associated adipocytes promote the proliferation, invasion, and metastasis of cancer. However, the mechanisms that link CAAs to the progression of obesity-related cancer are still unknown. Here, we found the mature adipocytes in the visceral fat of HFD-fed mice have a CAAs phenotype but the stromal vascular fraction of the visceral fat has not. Importantly, we found the derivate of the potent PPARγ antagonist GW9662, BZ26 inhibited the reprogramming of mature adipocytes in the visceral fat of HFD-fed mice into CAA-like cells and inhibited the proliferation and invasion of obesity-related breast cancer. Further study found that it mediated the browning of visceral, subcutaneous and perirenal fat and attenuated inflammation of adipose tissue and metabolic disorders. For the mechanism, we found that BZ26 bound and inhibited PPARγ by acting as a new modulator. Therefore, BZ26 serves as a novel modulator of PPARγ activity, that is, capable of inhibiting obesity-related breast cancer progression by inhibiting of CAA-like cell formation, suggesting that inhibiting the reprogramming of mature adipocytes into CAAs or CAA-like cells may be a potential therapeutic strategy for obesity-related cancer treatment.

The links between gut microbiota and obesity and obesity related diseases.

The obesity epidemic has become a global public health crisis in recent years and is continuing to worsen at an alarming rate. However, the pathophysiological mechanisms involved in the development of obesity and obesity-related diseases are still being unraveled. In the past ten years, the gut microbiota has been identified as a crucial player affecting the onset and progression of obesity and obesity-related diseases, especially with respect to changes in its composition and metabolites during obesity progression. Herein, we summarize the roles and mechanisms of gut microbiota's composition and metabolite changes in the gut play in obesity and obesity related diseases. Furthermore, we discuss potential therapeutic treatments that can be used to modulate the gut microbiome composition and target the relevant metabolic pathways of obesity and obesity-related metabolic diseases.

An overview of ferroptosis in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a public health problem associated with high mortality and high morbidity rates worldwide. Presently, its complex pathophysiology is still unclear, and there is no specific drug to reverse NAFLD. Ferroptosis is an iron-dependent and non-apoptotic form of cell death characterized by the iron-induced accumulation of lipid reactive oxygen species (ROS), which damage nucleic acids, proteins, and lipids; generate intracellular oxidative stress; and ultimately cause cell death. Emerging evidence indicates that ferroptosis is involved in the progression of NAFLD, although the mechanism of action of ferroptosis in NAFLD is still poorly understood. Herein, we summarize the mechanism of action of ferroptosis in certain diseases, especially in the pathogenesis of NAFLD, and discuss the potential therapeutic approaches currently used to treat NAFLD. This review also highlights further directions for the treatment and prevention of NAFLD and related diseases.

Isolating High Antimicrobial Ability Lignin From Bamboo Kraft Lignin by Organosolv Fractionation.

Lignin from different biomasses possess biological antioxidation and antimicrobial activities, which depend on the number of functional groups and the molecular weight of lignin. In this work, organosolv fractionation was carried out to prepare the lignin fraction with a suitable structure to tailor excellent biological activities. Gel permeation chromatography (GPC) analysis showed that decreased molecular weight lignin fractions were obtained by sequentially organosolv fractionation with anhydrous acetone, 50% acetone and 37.5% hexanes. Nuclear magnetic resonance (NMR) results indicated that the lignin fractions with lower molecular weight had fewer substructures and a higher phenolic hydroxyl content, which was positively correlated with their antioxidation ability. Both of the original lignin and fractionated lignins possessed the ability to inhibit the growth of Gram-negative bacteria (Escherichia coli and Salmonella) and Gram-positive bacteria (Streptococcus and Staphylococcus aureus) by destroying the cell wall of bacteria in vitro, in which the lignin fraction with the lowest molecular weight and highest phenolic hydroxyl content (L3) showed the best performance. Besides, the L3 lignin showed the ability to ameliorate Escherichia coli-induced diarrhea damages of mice to improve the formation of intestinal contents in vivo. These results imply that a lignin fraction with a tailored structure from bamboo lignin can be used as a novel antimicrobial agent in the biomedical field.

Aberrant splicing of Hugl-1 is associated with hepatocellular carcinoma progression.

PURPOSE: Lethal giant larvae functions as a cell polarity regulator and a tumor suppressor in Drosophila. Its evolutionary conservation implies a tumor suppressor role for its human homologue, Hugl-1. The aims of this study were to characterize Hugl-1 and to determine the clinical significance of Hugl-1 alterations in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Sequence alterations of Hugl-1 from 80 HCC specimens and 5 HCC cell lines were characterized by reverse transcription-PCR and sequence analysis. Western blot was used for determining Hugl-1 expression. The biological activities of Hugl-1 and its aberrant variants were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound healing assay, Boyden chamber assay, and tumorigenicity assay. RESULTS: In 32.5% (26 of 80) of the specimens and 20.0% (one of five) of HCC cell lines, 23 unique aberrant Hugl-1 transcripts were identified, most of which resulted from skipping part of and/or entire exon or insertion of intron sequences. The majority of these aberrant Hugl-1 transcripts encoded truncated proteins lacking one or more conserved WD-40 repeat motifs. Two truncated Hugl-1 proteins were found exclusively in HCC tissues. Aberrant Hugl-1 transcripts (78.3%, 20 of 23) had a short "direct repeat" sequence flanking their deleted regions. The abnormal Hugl-1 was significantly correlated with poor differentiation and large tumor size of HCC. Overexpression of two representative HCC-derived aberrant Hugl-1 variants promoted HCC cell migration, invasion, and tumorigenicity in nude mice. CONCLUSIONS: We provide the first evidence that Hugl-1 mRNA is frequently mutated by aberrant splicing exclusively in HCC, which may be involved in HCC progression.