The hepatitis B virus promotes the progression of non-alcoholic fatty liver disease through incomplete autophagy.

Hepatitis B virus (HBV) infection is still a serious public health problem. In recent years, with the increasing incidence of chronic hepatitis B (CHB) combined with nonalcoholic fatty liver disease (NAFLD), a more in-depth exploration of the pathogenesis of CHB combined with NAFLD is required. HBV can induce autophagy and use to increase replication. The removal of fat by autophagy, also known as lipophagy, is also currently considered an alternative pathway for lipid metabolism in liver cells. This degradation of autophagy prevents hepatotoxicity and steatosis. However, it is not known whether there is a correlation between HBV-related autophagy and the progression of NAFLD. We explored how HBV affects disease progression in NAFLD should be " and determined whether it is associated with HBV-associated autophagy. In this study, we constructed HBV-TG mouse high-fat diet (HFD) models and controls, and the results showed that the presence of HBV promoted the occurrence of NAFLD. We also demonstrated that HBV promotes lipid droplet accumulation in hepatocytes using HBV-stable expression cell lines HepG2.2.15 and AML12-HBV. In addition, this study also found that exogenous OA supplementation reduced HBV replication. We further studied the mechanism and found that HBV-related autophagy can promote the absorption of liver cells to lipid droplets. It can reduce the decomposition of lipid droplets by inhibiting the function of autophagolysosome, and eventually lead to the accumulation of lipid droplets in hepatocytes. In a word, HBV promotes the progression of NAFLD by increasing lipid accumulation in hepatocytes through incomplete autophagy.

The potential role of exosomal miRNAs and membrane proteins in acute HIV-infected people.

Exosomes play an important role during human immunodeficiency virus (HIV) acute infection. Yet, information regarding its cargo and its association with HIV rapid progressors (RPs) and typical progressors (TPs) remain largely unknown. In this study, exosomal miRNAs sequencing and mass cytometry were used to identify differential exosomal miRNAs and membrane proteins that participate in the pathogenesis of TPs and RPs. We discovered that miR-144-5p, miR-1180-3p, miR-451a, miR-362-5p, and miR-625-5p are associated with the TPs and miR-362-5p with the RPs. Decreased autophagy, amino acid metabolism, immune response, and IL-6 are closely related to RPs. In addition, SP1 was selected as the most significant transcription factor (TF) associated with disease progression. CD49D, CD5, CCR5, CD40, CD14, and CD86 were selected as the differential exosomal membrane proteins between TPs and RPs. This study provides valuable information for clarifying the mechanism in people with acute HIV infection.

Inactivation of tumor suppressor TAp63 by hepatitis B virus X protein in hepatocellular carcinoma.

BACKGROUND: The hepatitis B virus X (HBx) protein plays a critical role in the initiation and progression of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). In the early stage of the disease, HBx facilitates tumor onset by inactivating the tumor suppressor p53. The p53-encoding gene, however, is frequently mutated or deleted as the cancer progresses to the late stage and, under such circumstance, the p53 homolog TAp63 can harness HCC growth by transactivating several important p53-target genes. METHODS: To determine whether HBx regulates TAp63, we performed co-immunoprecipitation assay, real-time quantitative polymerase chain reaction, immunoblotting, and flow cytometry analysis in p53-null cancer cell lines, Hep3B and H1299. RESULTS: HBx interacts with the transactivation domain of TAp63, as HBx was co-immunoprecipitated with TAp63 but not with ΔNp63. The interaction between HBx and TAp63 abolished transcriptional activity of TAp63, as evidenced by the reduction of the levels of its target genes p21 and PUMA , consequently leading to restricted apoptosis and augmented proliferation of HCC cells. CONCLUSION: HBV induces progression of HCC that harbors defective p53 by inhibiting the tumor suppressor TAp63.

Nrf2-Mediated Ferroptosis Inhibition Exerts a Protective Effect on Acute-on-Chronic Liver Failure.

Although massive hepatocyte cell death and oxidative stress constitute major events of acute-on-chronic liver failure (ACLF), the relationship of ferroptosis with ACLF has yet to be explored. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of ferroptosis. However, if Nrf2 modulates ACLF through ferroptosis remains unknown. Here, the liver tissues of ACLF patients were collected and murine models of ACLF using carbon tetrachloride, D-galactosamine, and lipopolysaccharide as well as an H2O2-induced hepatocyte injury model were established. Upon ACLF, livers exhibited key features of ferroptosis, including lipid peroxidation (increase in malondialdehyde whereas a decrease in glutathione and nicotinamide adenine dinucleotide phosphate), and increased mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2). Ferroptosis inducer RSL-3 treatment aggravated liver damage, while ferroptosis inhibitor Ferrostatin-1 administration alleviated ACLF severity, manifesting with improved liver histopathological lesions and reduced serum ALT and AST. Compared with normal liver tissue, Nrf2 was upregulated in ACLF patients and murine models. Pharmacological activation of Nrf2 (Bardoxolone Methyl) attenuated liver damage, prevented lipid peroxidation, upregulated PTGS2 mRNA expression, and improved ferroptosis-specific mitochondrial morphology in vivo. In contrast, Nrf2 inhibitor ML385 exacerbated lipid peroxidation and liver injury. Collectively, Nrf2 plays a protective role in ACLF progression through repressing ferroptosis, which provides promising therapeutic cues for ACLF.

PINK1 ameliorates acute-on-chronic liver failure by inhibiting apoptosis through mTORC2/AKT signaling.

Acute-on-chronic liver failure (ACLF) is a lethal syndrome with a remarkable short-term death rate. Even worse, effective internal medicine therapies are currently lacking. Increasing evidence indicates apoptosis plays a critical role in the progression of liver failure. PINK1 has an essential function in maintaining cell survival. However, the role and underlying mechanism of PINK1 in apoptosis in ACLF are incompletely understood. Herein, our team discovered that PINK1 remarkably improved ACLF, featured by a reduction in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and an amelioration in the gross and microscopy histopathology appearance of hepatic tissues. Meanwhile, PINK1 affected cleaved caspase-3 expression via mTORC2/AKT, and this effect was eliminated after further intervention with Rictor or AKT. Overall, these findings indicate that PINK1 participates in the regulation of multiple biological functions, including hepatic cell growth and apoptosis in ACLF via the mTORC2/AKT signaling pathway. The present research offers a solid theory-wise foundation for the clinic applications of PINK1 as a valid target for ACLF treatment to reverse or postpone the development of ACLF.

LncRNA RNA Component of Mitochondrial RNA-Processing Endoribonuclease Promotes AKT-Dependent Breast Cancer Growth and Migration by Trapping MicroRNA-206.

The RNA component of mitochondrial RNA-processing endoribonuclease (RMRP) was recently shown to play a role in cancer development. However, the function and mechanism of RMRP during cancer progression remain incompletely understood. Here, we report that RMRP is amplified and highly expressed in various malignant cancers, and the high level of RMRP is significantly associated with their poor prognosis, including breast cancer. Consistent with this, ectopic RMRP promotes proliferation and migration of TP53-mutated breast cancer cells, whereas depletion of RMRP leads to inhibition of their proliferation and migration. RNA-seq analysis reveals AKT as a downstream target of RMRP. Interestingly, RMRP indirectly elevates AKT expression by preventing AKT mRNA from miR-206-mediated targeting via a competitive sequestering mechanism. Remarkably, RMRP endorses breast cancer progression in an AKT-dependent fashion, as knockdown of AKT completely abolishes RMRP-induced cancer cell growth and migration. Altogether, our results unveil a novel role of the RMRP-miR-206-AKT axis in breast cancer development, providing a potential new target for developing an anti-breast cancer therapy.

Single-cell transcriptomes reveal heterogeneity of high-grade serous ovarian carcinoma.

BACKGROUND: High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive histotype of epithelial ovarian cancer. The heterogeneity and molecular basis of this disease remain incompletely understood. METHODS: To address this question, we have performed a single-cell transcriptomics analysis of matched primary and metastatic HGSOC samples. RESULTS: A total of 13 571 cells are categorized into six distinct cell types, including epithelial cells, fibroblast cells, T cells, B cells, macrophages, and endothelial cells. A subset of aggressive epithelial cells with hyperproliferative and drug-resistant potentials is identified. Several new markers that are highly expressed in epithelial cells are characterized, and their roles in ovarian cancer cell growth and migration are further confirmed. Dysregulation of multiple signaling pathways, including the translational machinery, is associated with ovarian cancer metastasis through the trajectory analysis. Moreover, single-cell regulatory network inference and clustering (SCENIC) analysis reveals the gene regulatory networks and suggests the JUN signaling pathway as a potential therapeutic target for treatment of ovarian cancer, which is validated using the JUN/AP-1 inhibitor T-5224. Finally, our study depicts the epithelial-fibroblast cell communication atlas and identifies several important receptor-ligand complexes in ovarian cancer development. CONCLUSIONS: This study uncovers new molecular features and the potential therapeutic target of HGSOC, which would advance the understanding and treatment of the disease.

HBV antigen and DNA loss from mouse serum is associated with novel vaccine-induced HBV surface antigen-specific cell-mediated immunity and cytokine production.

Therapeutic vaccination is a promising strategy for controlling chronic hepatitis B virus (HBV). Here, we tested whether several novel vaccination strategies could be used to induce HBV-specific adaptive immune responses and control/eradicate HBV in a mouse model. Robust HBV antigen-specific antibody responses were elicited by several vaccination strategies using a novel particle vaccine (HBSS1), which expresses a fusion of the S (amino acids [aa] 1-223) and preS1 (aa 21-47) antigens, and/or a recombinant adenovirus rAdSS1 vaccine. However, antigen-specific cell-mediated immunity and high levels of production of multiple cytokines were elicited only by heterologous prime-boost immunization; i.e., priming with the HBSS1 vaccine followed by a rAdSS1 boost. Furthermore, the most rapid loss of serum HBsAg, HBeAg and DNA was achieved by the novel vaccination regimen (priming with HBSS1 formulated with adjuvants [alum plus PolyI:C]), which was strongly associated with more potent and functional HBsAg-specific CD4+ and CD8+ T-cell responses and increased production of interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor-α, IL-12, and IFN-γ-induced protein (IP)-10. Thus, our novel heterogeneous prime-boost vaccine regimen shows promise as a therapeutic strategy against HBV.

Kaempferol protects mice from d-GalN/LPS-induced acute liver failure by regulating the ER stress-Grp78-CHOP signaling pathway.

Kaempferol is a flavonoid compound that has many functions, such as anti-inflammation and antioxidation. Acute liver failure (ALF) is a life-threatening illness accompanied by serious inflammation and extensive hepatocyte apoptosis. The aim of this study was to examine the therapeutic potential of kaempferol and its mechanism in ALF. In a murine ALF model induced by d-galactosamine (d-GalN, 700 mg/kg) / lipopolysaccharide (LPS, 10 µg/kg), mice were pretreated with kaempferol at 2 h before d-GalN/LPS administration and then sacrificed 6 h after d-GalN/LPS injection. Lethality, liver damage, endoplasmic reticulum(ER) stress, hepatocyte viability and apoptosis were evaluated. Whether pretreatment of kaempferol protected hepatocytes from ER stress-induced apoptosis was detected in vitro. Pretreatment of kaempferol decreased lethality, prolonged the survival time and significantly protected against liver injury, which was indicated by decreased transaminase levels and the well-preserved liver structure. The protective effect of kaempferol on the ALF mouse model was achieved by inhibiting hepatocyte apoptosis. Moreover, pretreatment of kaempferol increased the expression of glucose-regulated/binding immunoglobulin protein 78 (Grp78), decreased the expression of C/EBP-homologous protein (CHOP), and protected hepatocytes from ER stress-induced apoptosis in vitro. Our results showed that pretreatment of Grp78 siRNA partially negated the hepatic protection from kaempferol and reversed the inhibition of CHOP protein expression in d-GalN/LPS-induced ALF mice. In conclusion, kaempferol inhibits hepatocyte apoptosis to protect mice from liver failure by regulating the ER stress-Grp78-CHOP signaling pathway. Therefore, kaempferol may be used to treat ALF.

The immune response of rhesus macaques to novel vaccines comprising hepatitis B virus S, PreS1, and Core antigens.

Therapeutic vaccines represent a unique approach to hepatitis B virus (HBV) treatment and have the potential to induce long-term control of infection. This study explored the immune responses of rhesus macaques to novel vaccines comprising the S, PreS1, and Core antigens of the HBV that showed promise as prophylactic and therapeutic approaches in a mouse model. The tested vaccines included two DNA vaccines (pVRC-SS1, pVRC-CS1), an HBV particle subunit (HBSS1) vaccine and the recombinant vaccinia virus- (RVJ-) based vaccines (RVJSS1 and RVJCS1) in which SS1 containing S (1-223 aa) and PreS1 (21-47 aa), CS1 containing Core (1-144 aa) and PreS1 (1-42 aa). The humoral immunity and cell-mediated immunity (CMI) induced by vaccines comprising the S, PreS1, and Core antigens of HBV were investigated in a longitudinal study that continued up to 98 weeks after the firstvaccination. In rhesus macaques, anti-PreS1 antibody was induced more rapidly than anti-S or anti-Core antibody after DNA vaccination. The antibody and cell-mediated immune responses against S, PreS1, and C were significantly enhanced in macaques boosted with RVJSS1 and RVJCS1, whereas the cell-mediated response to C was most robust and durable. The immune response to S, PreS1, and C was restored by HBSS1 boosting and detected in macaques until weeks 74 and 98 after the first vaccination. Additionally, robust neutralizing activity was detected at week 52. In conclusion, novel HBV vaccine candidates, especially those used for therapeutic applications should incorporate the PreS1 and Core antigens.

Kaempferol induces apoptosis in HepG2 cells via activation of the endoplasmic reticulum stress pathway.

Kaempferol is a flavonoid compound that has gained importance due to its antitumor properties; however, the underlying mechanisms remain to be fully understood. The present study aimed to investigate the molecular mechanisms of the antitumor function of kaempferol in HepG2 hepatocellular carcinoma cells. Kaempferol was determined to reduce cell viability, increase lactate dehydrogenase activity and induce apoptosis in a concentration­ and time­dependent manner in HepG2 cells. Additionally, kaempferol­induced apoptosis possibly acts via the endoplasmic reticulum (ER) stress pathway, due to the significant increase in the protein expression levels of glucose­regulated protein 78, glucose­regulated protein 94, protein kinase R­like ER kinase, inositol­requiring enzyme 1α, partial activating transcription factor 6 cleavage, caspase­4, C/EBP homologous protein (CHOP) and cleaved caspase­3. The pro­apoptotic activity of kaempferol was determined to be due to induction of the ER stress­CHOP pathway, as: i) ER stress was blocked by 4­phenyl butyric acid (4­PBA) pretreatment and knockdown of CHOP with small interfering RNA, which resulted in alleviation of kaempferol­induced HepG2 cell apoptosis; and ii) transfection with plasmid overexpressing CHOP reversed the protective effect of 4­PBA in kaempferol­induced HepG2 cells and increased the apoptotic rate. Thus, kaempferol promoted HepG2 cell apoptosis via induction of the ER stress­CHOP signaling pathway. These observations indicate that kaempferol may be used as a potential chemopreventive treatment strategy for patients with hepatocellular carcinoma.

Endoplasmic reticulum stress-activated glycogen synthase kinase 3ß aggravates liver inflammation and hepatotoxicity in mice with acute liver failure.

Endoplasmic reticulum stress (ER stress) has been increasingly recognized as an important mechanism in various liver diseases. However, its intrinsic physiological role in acute liver failure (ALF) remains largely undetermined. This study aimed to examine how ER stress orchestrates glycogen synthase kinase 3ß (GSK3ß) and inflammation to affect ALF. In a murine ALF model induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS), 4-phenylbutyric acid (4-PBA) is to be administered to relieve ER stress. The lethality rate, liver damage, cytokine expression, and the activity of GSK3ß were evaluated. How to regulate LPS-induced inflammation and TNF-α-induced hepatocyte apoptosis by ER stress was investigated in vitro. In vivo, ER stress was triggered in the liver with the progression of mice ALF model. ER stress was essential for the development of ALF because ER stress inhibition by 4-PBA ameliorated the liver damage through decreasing liver inflammation and hepatocyte apoptosis. 4-PBA also decreased GSK3ß activity in the livers of ALF mice. In vitro, ER stress induced by tunicamycin synergistically increased LPS-triggered pro-inflammatory cytokine induction and promoted the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway in bone marrow-derived macrophages; moreover, tunicamycin also cooperated with TNF-α to increase hepatocyte apoptosis. ER stress promoted LPS-triggered inflammation depending on GSK3ß activation because inhibition of GSK3ß by SB216763, the specific inhibitor of GSK3ß, resulted in downregulation of pro-inflammatory genes. ER stress contributes to liver inflammation and hepatotoxicity in ALF, particularly by regulating GSK3ß, and is therefore a potential therapeutic target for ALF.

EphA3, induced by PC-1/PrLZ, contributes to the malignant progression of prostate cancer.

Our previous study revealed the potential linkage of PC-1/PrLZ, a novel isolated prostate-specific gene, to the progression of prostate cancer (PCa) in vitro and in vivo. To gain more insight into the mechanism of PC-1-induced promotion of PCa, expression analysis of differential genes induced by PC-1 was scanned by microarray. Among all the differentially expressed genes, EphA3 was altered to the greatest extent. EphA3 has been identified to be associated with multiple tumor progression. However, little is known concerning the function of EphA3 in PCa. In the present study, we aimed to ascertain whether EphA3 is induced by PC-1 and the functional significance of EphA3 expression in PCa. We found that overexpression of PC-1 increased the amount of EphA3 and that knockdown of PC-1 led to a decrease in EphA3 in PCa cells. The functional significance and mechanisms by which EphA3 contributes to PCa was investigated in vitro using cell lines, and in vivo using a mouse model and clinical specimens. The results showed that EphA3 enhanced the proliferation and survival of LNCaP cells and suppression of EphA3 inhibited the survival of C4-2B cells. EphA3 enhanced the tumor development of LNCaP cells in null mice. A positive correlation between the levels of EphA3 and the Gleason grade of PCa was identified in clinical PCa specimens. In addition, cellular localization changed with Gleason grade. We further detected that EphA3 increased phosphorylation of Akt (Ser473 and Thr308), indicating that EphA3 activated the Akt pathway. Taken together, EphA3 was induced by PC-1 and contributed to the malignant progression of prostate cancer. Our results provide the first demonstration that EphA3 is a novel promoter of human prostate cancer development and progression.

Analysis of differentially expressed genes in LNCaP prostate cancer progression model.

The LNCaP/C4-2 human prostate cancer progression model was established to mimic phenotypic and genotypic changes during prostate cancer development from androgen dependence to androgen independence, from nonmetastasis to metastasis. In this study, cDNA microarrays were performed using a microarray chip from Affymetrix to characterize and compare gene expression profiles in LNCaP and C4-2, which may provide novel insight into the molecular mechanism mediating prostate cancer progression. Three hundred eighteen genes consistently exhibited differential expression in LNCaP and C4-2 in 2-time microarray data. Based on their function, the differentially expressed genes can be grouped into several subcategories, including growth factors and signal transducers, oncogenes and tumor suppressors, tumor-specific antigens, transcriptional factors, transporters, and factors involved in invasion, metastasis, and metabolism. Some genes are novel and unexplored in prostate cancer progression and are of potential interest for follow-up investigation. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to corroborate the microarray results, and 76 differentially expressed genes were validated out of 104 candidates. Expression pattern analyses were performed in these 76 differentially expressed genes, and a series of genes was found to be positively or negatively correlated to prostate cancer progression in the LNCaP prostate cancer progression model and to possess predominant prostate cell specificity. ELF5/ESE-2b and long-chain acyl coenzyme A dehydrogenase (ACADL) expressions were found to be positively associated with malignant progression in LNCaP, C4-2, and C4-2B, and predominantly expressed in prostate cancer cells. Functional evaluation revealed that ELF5/ESE-2b and ACADL expressions contributed to the malignant phenotypes of prostate cancer cells. Accordingly, our microarray data may provide clues for finding novel genes involved in prostate cancer progression to androgen independent and metastasis, and shed light on finding new targets for diagnosis and therapy of prostate cancer.

The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP.

Radiation therapy is a relatively effective therapeutic method for localized prostate cancer (PCa) patients. However, radioresistance occurs in nearly 30% of patients treated with potentially curative doses. Therapeutic synergy between radiotherapy and androgen ablation treatment provides a promising strategy for improving the clinical outcome. Accordingly, the androgen deprivation-induced signaling pathway may also mediate radiosensitivity in PCa cells. The C4-2 cell line was derived from the androgen-sensitive LNCaP parent line under androgen-depleted condition and had acquired androgen-refractory characteristics. In our study, the response to radiation was evaluated in both LNCaP and C4-2. Results showed that C4-2 cells were more likely to survive from irradiation and appeared more aggressive in their resistance to radiation treatment compared with LNCaP, as measured by clonogenic assays and cell viability and cell cycle analyses. Gene expression analyses revealed that a set of genes involved in cell cycle arrest and DNA repair were differentially regulated in LNCaP and C4-2 in response to radiation, which was also consistent with the radiation-resistant property observed in C4-2 cells. These results strongly suggested that the radiation-resistant property may develop with progression of PCa to androgen-independent status. Not only can the LNCaP and C4-2 PCa progression model be applied for investigating androgen-refractory progression, but it can also be used to explore the development of radiation resistance in PCa.

Ubiquitous mitochondrial creatine kinase is overexpressed in the conditioned medium and the extract of LNCaP lineaged androgen independent cell lines and facilitates prostate cancer progression.

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.