Decoding the transcriptome of muscular dystrophy due to Ptrf deficiency using single-nucleus RNA sequencing.

Lacking PTRF (polymerase I and transcript release factor), an essential caveolae component, causes a secondary deficiency of caveolins resulting in muscular dystrophy. The transcriptome responses of different types of muscle fibers and mononuclear cells in skeletal muscle to muscular dystrophy caused by Ptrf deletion have not been explored. Here, we created muscular dystrophy mice by Ptrf knockout and applied single-nucleus RNA sequencing (snRNA-seq) to unveil the transcriptional changes of the skeletal muscle at single-nucleus resolution. 11 613 muscle nuclei (WT, 5838; Ptrf KO, 5775) were classified into 12 clusters corresponding to 11 nuclear types. Trajectory analysis revealed the potential transition between type IIb_1 and IIb_2 myonuclei upon muscular dystrophy. Functional enrichment analysis indicated that apoptotic signaling and enzyme-linked receptor protein signaling pathway were significantly enriched in type IIb_1 and IIb_2 myonuclei of Ptrf KO, respectively. The muscle structure development and the PI3K-AKT signaling pathway were significantly enriched in type IIa and IIx myonuclei of Ptrf KO. Meanwhile, metabolic pathway analysis showed a decrease in overall metabolic pathway activity of myonuclei subtypes upon muscular dystrophy, with the most decrease in type IIb_1 myonuclei. Gene regulatory network analysis found that the activity of Mef2c, Mef2d, Myf5, and Pax3 regulons was enhanced in type II myonuclei of Ptrf KO, especially in type IIb_2 myonuclei. In addition, we investigated the transcriptome changes in adipocytes and found that muscular dystrophy enhanced the lipid metabolic capacity of adipocytes. Our findings provide a valuable resource for exploring the molecular mechanism of muscular dystrophy due to Ptrf deficiency.

Influence of lifestyle patterns on depression among adults with diabetes: a mediation effect of dietary inflammatory index.

BACKGROUND: Lifestyle has become a crucial modulator in the management of diabetes and is intimately linked with the development and exacerbation of comorbid depression. The study aimed to analyze lifestyle patterns and their impact on depression in individuals with diabetes and to explore the role of the Dietary Inflammatory Index (DII) in the relationship between lifestyle patterns and depression. METHODS: Data was attained from the National Health and Nutrition Examination Survey (NHANES) between 2009 and 2020. A latent class analysis (LCA) was performed on 3,009 diabetic adults based on lifestyle behaviors. A generalised linear model (GLM) was employed to analyse the effects of different lifestyle patterns on depression. The mediation effect model was utilised to examine the relationship between lifestyle patterns, DII and Patient Health Questionnaire-9 (PHQ-9) scores. RESULTS: The cohort was divided through LCA into unhealthy lifestyle (44.53%), unhealthy but non-alcohol use (48.06%) and healthy but smoking (7.41%) groups of lifestyle behaviors, the unhealthy but non-alcohol use group was identified as a risk factor for depression (OR = 1.379, 95%CI = 1.095 ~ 1.735, P = 0.006). The DII partially mediated the relationship between the unhealthy but non-alcohol use group and PHQ-9, and fully mediated the relationship between the healthy but smoking group and PHQ-9, with effect coefficients of - 0.018 (95%CI: -0.044 ~ - 0.001) and - 0.035 (95%CI: -0.083 ~ - 0.001). CONCLUSIONS: Lifestyle patterns significantly influence the occurrence of depression among diabetes patients. The dietary inflammation plays a varying mediating role between different lifestyle patterns and depression. Restricting pro-inflammatory diets or encouraging anti-inflammatory diets, combined with the promotion of healthy lifestyle practices, may be an effective method for preventing and alleviating symptoms of depression among patients with diabetes.

Exploring high-resolution chromatin interaction changes and functional enhancers of myogenic marker genes during myogenic differentiation.

Skeletal muscle differentiation (myogenesis) is a complex and highly coordinated biological process regulated by a series of myogenic marker genes. Chromatin interactions between gene's promoters and their enhancers have an important role in transcriptional control. However, the high-resolution chromatin interactions of myogenic genes and their functional enhancers during myogenesis remain largely unclear. Here, we used circularized chromosome conformation capture coupled with next generation sequencing (4C-seq) to investigate eight myogenic marker genes in C2C12 myoblasts (C2C12-MBs) and C2C12 myotubes (C2C12-MTs). We revealed dynamic chromatin interactions of these marker genes during differentiation and identified 163 and 314 significant interaction sites (SISs) in C2C12-MBs and C2C12-MTs, respectively. The interacting genes of SISs in C2C12-MTs were mainly involved in muscle development, and histone modifications of the SISs changed during differentiation. Through functional genomic screening, we also identified 25 and 41 putative active enhancers in C2C12-MBs and C2C12-MTs, respectively. Using luciferase reporter assays for putative enhancers of Myog and Myh3, we identified eight activating enhancers. Furthermore, dCas9-KRAB epigenome editing and RNA-Seq revealed a role for Myog enhancers in the regulation of Myog expression and myogenic differentiation in the native genomic context. Taken together, this study lays the groundwork for understanding 3D chromatin interaction changes of myogenic genes during myogenesis and provides insights that contribute to our understanding of the role of enhancers in regulating myogenesis.

Mir-629 Repressed LATS2 Expression and Promoted the Proliferation of Prostate Cancer Cells.

MicroRNAs (miRNAs) have been discovered to play critical role in regulating prostate cancer (PC) progression. The function role of miR-629 in tumor progression of PC has not been studied. Here, we found that miR-629 was markedly upregulated in PC as determined using the cancer genome atlas (TCGA) dataset, clinical tissues, and cell lines. Functional analysis (MTT assays, colony formation assays, soft agar growth assay and BrdU incorporation assay) indicated that overexpression of miR-629 was drastically promoted, while miR-629-in significantly suppressed cell proliferation. LATS2 was predicted as a direct target of miR-629 and was confirmed by western blot and dual luciferase assay. Through downregulation of large tumor suppressor 2 (LATS2) by overexpression of miR-629, the p21 mRNA and protein were decreased while the Cyclin D3 mRNA and protein were enhanced, suggesting promoting of cell proliferation process. Additionally, knockdown of LATS2 reversed the inhibitory effect by miR-629-in in PC. Our study indicated that miR-629 might serve as a new promising target for PC treatment.

Identification of enhancers responsible for the coordinated expression of myosin heavy chain isoforms in skeletal muscle.

BACKGROUND: Skeletal muscles consist of fibers of differing contractility and metabolic properties, which are primarily determined by the content of myosin heavy chain (MYH) isoforms (MYH7, MYH2, MYH1, and MYH4). The regulation of Myh genes transcription depends on three-dimensional chromatin conformation interaction, but the mechanistic details remain to be determined. RESULTS: In this study, we characterized the interaction profiles of Myh genes using 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing). The interaction profile of Myh genes changed between fast quadriceps and slow soleus muscles. Combining chromatin immunoprecipitation-sequencing (ChIP-seq) and transposase accessible chromatin with high-throughput sequencing (ATAC-seq), we found that a 38 kb intergenic region interacting simultaneously with fast Myh genes promoters controlled the coordinated expression of fast Myh genes. We also identified four active enhancers of Myh7, and revealed that binding of MYOG and MYOD increased the activity of Myh7 enhancers. CONCLUSIONS: This study provides new insight into the chromatin interactions that regulate Myh genes expression.

Glutathione Is Involved in the Reduction of Methylarsenate to Generate Antibiotic Methylarsenite in Enterobacter sp. Strain CZ-1.

Methylarsenate (MAs(V)) is a product of microbial arsenic (As) biomethylation and has also been widely used as an herbicide. Some microbes are able to reduce nontoxic MAs(V) to highly toxic methylarsenite (MAs(III)) possibly as an antibiotic. The mechanism of MAs(V) reduction in microbes has not been elucidated. Here, we found that the bacterium Enterobacter sp. CZ-1 isolated from an As-contaminated paddy soil has a strong ability to reduce MAs(V) to MAs(III). Using a MAs(III)-responsive biosensor to detect MAs(V) reduction in E. coli Trans5α transformants of a genomic library of Enterobacter sp. CZ-1, we identified gshA, encoding a glutamate-cysteine ligase, as a key gene involved in MAs(V) reduction. Heterologous expression of gshA increased the biosynthesis of glutathione (GSH) and MAs(V) reduction in E. coli Trans5α. Deletion of gshA in Enterobacter sp. CZ-1 abolished its ability to synthesize GSH and decreased its MAs(V) reduction ability markedly, which could be restored by supplementation of exogenous GSH. In the presence of MAs(V), Enterobacter sp. CZ-1 was able to inhibit the growth of Bacillus subtilis 168; this ability was lost in the gshA-deleted mutant. In addition, deletion of gshA greatly decreased the reduction of arsenate to arsenite. These results indicate that GSH plays an important role in MAs(V) reduction to generate MAs(III) as an antibiotic. IMPORTANCE Arsenic is a ubiquitous environmental toxin. Some microbes detoxify inorganic arsenic through biomethylation, generating relatively nontoxic pentavalent methylated arsenicals, such as methylarsenate. Methylarsenate has also been widely used as an herbicide. Surprisingly, some microbes reduce methylarsenate to highly toxic methylarsenite possibly to use the latter as an antibiotic. How microbes reduce methylarsenate to methylarsenite is unknown. Here, we show that gshA encoding a glutamate-cysteine ligase in the glutathione biosynthesis pathway is involved in methylarsenate reduction in Enterobacter sp. CZ-1. Our study provides new insights into the crucial role of glutathione in the transformation of a common arsenic compound to a natural antibiotic.

Ursolic acid inhibits the activation of smoothened-independent non-canonical hedgehog pathway in colorectal cancer by suppressing AKT signaling cascade.

It is being brought to light that smoothened (SMO)-independent non-canonical Hedgehog signaling is associated with the pathogenesis of various cancers. Ursolic acid (UA), a pentacyclic triterpenoid present in many medicinal herbs, manifests potent effectiveness against multiple malignancies including colorectal cancer (CRC). In our previous study, UA was found to protect against CRC in vitro by suppression of canonical Hedgehog signaling cascade. Here, the influence of UA on SMO-independent non-canonical Hedgehog signaling in CRC was investigated in the present study, which demonstrated that UA hampered the proliferation and migration, induced the apoptosis of HCT-116hSMO- cells with SMO gene knockdown, accompanied by the augmented expression of the suppressor of fused (SUFU), and lessened levels of MYC (c-Myc), glioma-associated oncogene (GLI1) and Sonic Hedgehog (SHH), and lowered phosphorylation of protein kinase B (PKB, AKT), suggesting that UA diminished non-canonical Hedgehog signal transduction in CRC. In HCT-116hSMO- xenograft tumor, UA ameliorated the symptoms, impeded the growth and caused the apoptosis of CRC, with heightened SUFU expression, and abated levels of MYC, GLI1, and SHH, and mitigated phosphorylation of AKT, indicating that UA down-regulated non-canonical Hedgehog signaling cascade in CRC. Taken together, UA may alleviate CRC by suppressing AKT signaling-dependent activation of SMO-independent non-canonical Hedgehog pathway.

[Mechanism of ursolic acid in regulating colorectal cancer cell HCT116 autophagy through hedgehog signaling pathway].

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.

miRNA-10a-5p Alleviates Insulin Resistance and Maintains Diurnal Patterns of Triglycerides and Gut Microbiota in High-Fat Diet-Fed Mice.

miRNA-10a is rhythmically expressed and regulates genes involved in lipid and glucose metabolism. However, the effects of miRNA-10a on obesity and glucose intolerance, as well as on the diurnal pattern of expression of circadian clock genes, remain unknown. We explored the effects of miRNA-10a-5p on insulin resistance and on the diurnal patterns of serum triglycerides and gut microbiota in high-fat diet- (HFD-) fed mice. The results showed that oral administration of miRNA-10a-5p significantly prevented body weight gain and improved glucose tolerance and insulin sensitivity in HFD-fed mice. Administration of miRNA-10a-5p also maintained the diurnal rhythm of Clock, Per2, and Cry1 expression, as well as serum glucose and triglyceride levels. Surprisingly, the diurnal oscillations of three genera of microbes, Oscillospira, Ruminococcus, and Lachnospiraceae, disrupted by HFD feeding, maintained by administration of miRNA-10a-5p. Moreover, a strong positive correlation was found between hepatic Clock expression and relative abundance of Lachnospiraceae, both in control mice (r = 0.877) and in mice administered miRNA-10a-5p (r = 0.853). Furthermore, we found that along with changes in Lachnospiraceae abundance, butyrate content in the feces maintained a diurnal rhythm after miRNA-10a-5p administration in HFD-fed mice. In conclusion, we suggest that miRNA-10a-5p may improve HFD-induced glucose intolerance and insulin resistance through the modulation of the diurnal rhythm of Lachnospiraceae and its metabolite butyrate. Therefore, miRNA-10a-5p may have preventative properties in subjects with metabolic disorders.

[Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway].

The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.

All-in-One Bifunctional Oxygen Electrode Films for Flexible Zn-Air Batteries.

As a promising energy-storage device, rechargeable Zn-air batteries have attracted considerable interests. Herein, a bifunctional oxygen electrode film prepared by adhering NiCo2 O4 nanosheets to a nitrogen and oxygen dual-doped carbon nanotubes film in a large scale is reported. The resulting self-supporting film electrode is multifunctional, which integrates a porous conducting structure for air diffusion and charge transfer, high-performance catalysts for oxygen reduction and evolution, and novel structural flexibility. The composite film demonstrates excellent oxygen reduction/evolution reaction catalytic activities with low Tafel slopes (50 mV dec-1 for oxygen reduction reaction; 92 mV dec-1 for oxygen evolution reaction). Without any additional current collector, gas diffusion layer, or binder, the obtained bifunctional film performs as an "all-in-one" air electrode in a Zn-air battery. A 50-cm-long cable-shaped Zn-air battery based on such a film air electrode exhibits high operating potentials (≈1.2 V at 0.25 mA cm-2 ), low charging-discharging overpotentials (≈0.7 V), and stable cycling performance. Moreover, the flexible cable Zn-air batteries show excellent stability under different deformation conditions. The proposed concept of constructing scalable, all-in-one, freestanding, and flexible air electrodes would pave the way to develop next-generation wearable and portable energy-storage devices.

Large-Stroke Electrochemical Carbon Nanotube/Graphene Hybrid Yarn Muscles.

Artificial muscles are reported in which reduced graphene oxide (rGO) is trapped in the helical corridors of a carbon nanotube (CNT) yarn. When electrochemically driven in aqueous electrolytes, these coiled CNT/rGO yarn muscles can contract by 8.1%, which is over six times that of the previous results for CNT yarn muscles driven in an inorganic electrolyte (1.3%). They can contract to provide a final stress of over 14 MPa, which is about 40 times that of natural muscles. The hybrid yarn muscle shows a unique catch state, in which 95% of the contraction is retained for 1000 s following charging and subsequent disconnection from the power supply. Hence, they are unlike thermal muscles and natural muscles, which need to consume energy to maintain contraction. Additionally, these muscles can be reversibly cycled while lifting heavy loads.

Crosslinked Carbon Nanotube Aerogel Films Decorated with Cobalt Oxides for Flexible Rechargeable Zn-Air Batteries.

Air electrodes with high catalytic activity are of great importance for rechargeable zinc-air batteries. Herein, a flexible, binder-free composite air electrode for zinc-air batteries is reported, which utilizes a lightweight, conductive, and crosslinked aerogel film of carbon nanotubes (CNTs) functioned as a 3D catalyst-supporting scaffold for bifunctional cobalt (II/III) oxides and as a current collector. The composite electrode shows high catalytic activities for both oxygen reduction reaction and oxygen evolution reaction, resulting from the synergistic effect of nitrogen-doped CNTs and spinel Co3 O4 nanoparticles. Solid-state Zn-air batteries assembled using such free-standing air electrodes (without the need of additional current collectors) are bendable and show low resistances, low charge/discharge overpotentials, and a high cyclic stability.

Effect of Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) Blockade in Rats with Cecal Ligation and Puncture (CLP)-Induced Sepsis.

BACKGROUND Blocking of TREM-1 signaling improves survival of mice with sepsis induced by Pseudomonas aeruginosa. However, whether TREM-1 blockade has beneficial effects in polymicrobial sepsis is poorly understood. Here, we aimed to investigate the effect of modulation of the TREM-1 pathway in rats with polymicrobial sepsis induced by cecal ligation and puncture (CLP). MATERIAL AND METHODS Normal Sprague-Dawley (SD) rats with sepsis induced by CLP were allocated randomly to received scramble peptide or LP17 via the jugular vein. Serum level of sTREM-1, IL6, TNF-α, and IL-1ß were detected by ELISA assay. The mRNA and protein levels of JAK2 and STAT3 were detected by real-time PCR and Western blot analysis. RESULTS STREM-1 concentration was greatly and progressively increased in rats with CLP-induced sepsis, and the increase was attenuated by TREM-1 inhibitory peptide LP17. More than 60% survival was observed in rats at the experiment endpoint after LP17 treatment. TREM-1 blockade also attenuated the increased level of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß, and thus attenuated systematic and distant inflammatory responses. Furthermore, TREM-1 blockade significantly attenuated the increased levels of pJAK2 and pSTAT3. CONCLUSIONS TREM-1 blockade by the use of an inhibitory peptide LP17 could prolong survival of rats with polymicrobial sepsis and attenuate systematic inflammatory responses through the JAK2/STAT3 signaling pathway. Our results suggest that modulation of TREM-1 by a synthetic peptide might be a potential therapeutic option for polymicrobial sepsis.

Oxygen Evolution Assisted Fabrication of Highly Loaded Carbon Nanotube/MnO2 Hybrid Films for High-Performance Flexible Pseudosupercapacitors.

To date, it has been a great challenge to design high-performance flexible energy storage devices for sufficient loading of redox species in the electrode assemblies, with well-maintained mechanical robustness and enhanced electron/ionic transport during charge/discharge cycles. An electrochemical activation strategy is demonstrated for the facile regeneration of carbon nanotube (CNT) film prepared via floating catalyst chemical vapor deposition strategy into a flexible, robust, and highly conductive hydrogel-like film, which is promising as electrode matrix for efficient loading of redox species and the fabrication of high-performance flexible pseudosupercapacitors. The strong and conductive CNT films can be effectively expanded and activated by electrochemical anodic oxygen evolution reaction, presenting greatly enhanced internal space and surface wettability with well-maintained strength, flexibility, and conductivity. The as-formed hydrogel-like film is quite favorable for electrochemical deposition of manganese dioxide (MnO2 ) with loading mass up to 93 wt% and electrode capacitance kept around 300 F g(-1) (areal capacitance of 1.2 F cm(-2) ). This hybrid film was further used to assemble a flexible symmetric pseudosupercapacitor without using any other current collectors and conductive additives. The assembled flexible supercapacitors exhibited good rate performance, with the areal capacitance of more than 300 mF cm(-2) , much superior to other reported MnO2 based flexible thin-film supercapacitors.

Immobilization of Lipase by Adsorption Onto Magnetic Nanoparticles in Organic Solvents.

In order to improve the performance of lipase in organic solvents, a simple immobilization method was developed by adsorption of lipase onto Fe3O4@ SiO2magnetic nanoparticles in organic solvent. Among the solvents tested, toluene was found to be the most effective solvent for the immobilization. A maximum immobilization yield of 97% and relative activity of 124% were achieved in toluene at 30 °C. The optimal temperature, enzyme loading and water activity were 30 °C, 1.25 mg/mg support and 0.48 aw, respectively. The residual activity of immobilized lipase was 67% after 10 cycles of use. The advantages of the immobilized lipase including easy recovery, high stability, and enhanced activity of immobilized lipase in organic solvents show potential industrial applications in anhydrous solvents.

Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. METHODS: Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. RESULTS: CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CONCLUSIONS: CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.

The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma.

BACKGROUND & AIMS: Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. We investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. METHODS: Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. RESULTS: The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells. CONCLUSIONS: SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features.

Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma.

UNLABELLED: Recent evidence suggests that hepatocellular carcinoma (HCC) is organized by a subset of cells with stem cell features (cancer stem cells; CSCs). CSCs are considered a pivotal target for the eradication of cancer, and liver CSCs have been identified by the use of various stem cell markers. However, little information is known about the expression patterns and characteristics of marker-positive CSCs, hampering the development of personalized CSC-targeted therapy. Here, we show that CSC markers EpCAM and CD90 are independently expressed in liver cancer. In primary HCC, EpCAM+ and CD90+ cells resided distinctively, and gene-expression analysis of sorted cells suggested that EpCAM+ cells had features of epithelial cells, whereas CD90+ cells had those of vascular endothelial cells. Clinicopathological analysis indicated that the presence of EpCAM+ cells was associated with poorly differentiated morphology and high serum alpha-fetoprotein (AFP), whereas the presence of CD90+ cells was associated with a high incidence of distant organ metastasis. Serial xenotransplantation of EpCAM+ /CD90+ cells from primary HCCs in immune-deficient mice revealed rapid growth of EpCAM+ cells in the subcutaneous lesion and a highly metastatic capacity of CD90+ cells in the lung. In cell lines, CD90+ cells showed abundant expression of c-Kit and in vitro chemosensitivity to imatinib mesylate. Furthermore, CD90+ cells enhanced the motility of EpCAM+ cells when cocultured in vitro through the activation of transforming growth factor beta (TGF-ß) signaling, whereas imatinib mesylate suppressed TGFB1 expression in CD90+ cells as well as CD90+ cell-induced motility of EpCAM+ cells. CONCLUSION: Our data suggest the discrete nature and potential interaction of EpCAM+ and CD90+ CSCs with specific gene-expression patterns and chemosensitivity to molecular targeted therapy. The presence of distinct CSCs may determine the clinical outcome of HCC.

An intronic enhancer of Cebpa regulates adipocyte differentiation and adipose tissue development via long-range loop formation.

Cebpa is a master transcription factor gene for adipogenesis. However, the mechanisms of enhancer-promoter chromatin interactions controlling Cebpa transcriptional regulation during adipogenic differentiation remain largely unknown. To reveal how the three-dimensional structure of Cebpa changes during adipogenesis, we generated high-resolution chromatin interactions of Cebpa in 3T3-L1 preadipocytes and 3T3-L1 adipocytes using circularized chromosome conformation capture sequencing (4C-seq). We revealed dramatic changes in chromatin interactions and chromatin status at interaction sites during adipogenic differentiation. Based on this, we identified five active enhancers of Cebpa in 3T3-L1 adipocytes through epigenomic data and luciferase reporter assays. Next, epigenetic repression of Cebpa-L1-AD-En2 or -En3 by the dCas9-KRAB system significantly down-regulated Cebpa expression and inhibited adipocyte differentiation. Furthermore, experimental depletion of cohesin decreased the interaction intensity between Cebpa-L1-AD-En2 and the Cebpa promoter and down-regulated Cebpa expression, indicating that long-range chromatin loop formation was mediated by cohesin. Two transcription factors, RXRA and PPARG, synergistically regulate the activity of Cebpa-L1-AD-En2. To test whether Cebpa-L1-AD-En2 plays a role in adipose tissue development, we injected dCas9-KRAB-En2 lentivirus into the inguinal white adipose tissue (iWAT) of mice to suppress the activity of Cebpa-L1-AD-En2. Repression of Cebpa-L1-AD-En2 significantly decreased Cebpa expression and adipocyte size, altered iWAT transcriptome, and affected iWAT development. We identified functional enhancers regulating Cebpa expression and clarified the crucial roles of Cebpa-L1-AD-En2 and Cebpa promoter interaction in adipocyte differentiation and adipose tissue development.