ABSTRACT
ATP synthase catalyzes the synthesis of ATP by consuming the proton electrochemical gradient, which is essential for maintaining the life activity of organisms. The peripheral stalk belongs to ATP synthase and plays an important supporting role in the structure of ATP synthase, but their regulation in filamentous fungi are not yet known. Here, we characterized the subunits of the peripheral stalk, FfATPh, FfATP5, and FfATPb, and explored their functions on development and pathogenicity of Fusarium Fujikuroi. The FfATPh, FfATP5, and FfATPb deletion mutations (∆FfATPh, ∆FfATP5, and ∆FfATPb) presented deficiencies in vegetative growth, sporulation, and pathogenicity. The sensitivity of ∆FfATPh, ∆FfATP5, and ∆FfATPb to fludioxonil, phenamacril, pyraclostrobine, and fluazinam decreased. In addition, ∆FfATPh exhibited decreased sensitivity to ionic stress and osmotic stress, and ∆FfATPb and ∆FfATP5 were more sensitive to oxidative stress. FfATPh, FfATP5, and FfATPb were located on the mitochondria, and ∆FfATPh, ∆FfATPb, and ∆FfATP5 disrupted mitochondrial location. Furthermore, we demonstrated the interaction among FfATPh, FfATP5, and FfATPb by Bimolecular Fluorescent Complimentary (BiFC) analysis. In conclusion, FfATPh, FfATP5, and FfATPb participated in regulating development, pathogenicity, and sensitivity to fungicides and stress factors in F. fujikuroi.
Subject(s)
Fungicides, Industrial , Fusarium , Fungicides, Industrial/pharmacology , Virulence , Fusarium/genetics , Nitric Oxide Synthase , Adenosine TriphosphateABSTRACT
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells (LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase (ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. METHODS: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/ß-catenin signaling by ACLY. Rescue experiments were performed to investigate whether ß-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. RESULTS: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of ß-catenin, and Lys49 acetylation of ß-catenin might mediate ACLY-regulated ß-catenin level in HCC cells. CONCLUSIONS: ACLY is a potent regulator of Wnt/ß-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , ATP Citrate (pro-S)-Lyase/genetics , Adenosine Triphosphate , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Multienzyme Complexes , Oxo-Acid-Lyases , Wnt Signaling Pathway , beta Catenin/geneticsABSTRACT
Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Hepatitis B virus/physiology , Liver Neoplasms/genetics , Liver Neoplasms/virology , Cell Transformation, Neoplastic , CpG Islands , DNA, Viral/genetics , Female , Genome, Human , Genome, Viral , Hepatitis B, Chronic/genetics , Humans , Kaplan-Meier Estimate , Liver Cirrhosis/genetics , Liver Cirrhosis/virology , Male , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Analysis, RNA , Virus IntegrationABSTRACT
BACKGROUND & AIMS: Considerable evidence suggests that adrenergic signaling played an essential role in tumor progression. However, its role in hepatocellular carcinoma (HCC) and the underlying mechanisms remain unknown. METHODS: The effect of adrenaline in hepatocarcinogenesis was observed in a classical diethylnitrosamine-induced HCC mouse model. Effects of ADRB2 signaling inhibition in HCC cell lines were analyzed in proliferation, apoptosis, colony formation assays. Autophagy regulation by ADRB2 was assessed in immunoblotting, immunofluorescence and immunoprecipitation assays. In vivo tumorigenic properties and anticancer effects of sorafenib were examined in nude mice. Expression levels of ADRB2 and hypoxia-inducible factor-1α (HIF1α) in 150 human HCC samples were evaluated by immunohistochemistry. RESULTS: We uncovered that adrenaline promoted DEN-induced hepatocarcinogenesis, which was reversed by the ADRB2 antagonist ICI118,551. ADRB2 signaling also played an essential role in sustaining HCC cell proliferation and survival. Notably, ADRB2 signaling negatively regulated autophagy by disrupting Beclin1/VPS34/Atg14 complex in an Akt-dependent manner, leading to HIF1α stabilization, reprogramming of HCC cells glucose metabolism, and the acquisition of resistance to sorafenib. Conversely, inhibition of ADRB2 signaling by ICI118,551, or knockdown ADRB2 expression, led to enhanced autophagy, HIF1α destabilization, tumor growth suppression, and improved anti-tumor activity of sorafenib. Consistently, ADRB2 expression correlated positively with HIF1α in HCC specimens and was associated with HCC outcomes. CONCLUSIONS: Our results uncover an important role of ADRB2 signaling in regulating HCC progression. Given the efficacy of ADRB2 modulation on HCC inhibition and sorafenib resistance, adrenoceptor antagonist appears to be a putative novel treatment for HCC and chemoresistance. LAY SUMMARY: ADRB2 signaling played an essential role in sustaining hepatocellular carcinoma cell proliferation and survival. ADRB2 signaling negatively regulated autophagy, leading to hypoxia-inducible factor-1α stabilization, reprogramming of hepatocellular carcinoma cells glucose metabolism, and the acquisition of resistance to sorafenib. Adrenoceptor antagonist appears to be a putative novel treatment for hepatocellular carcinoma and chemoresistance.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Autophagy , Disease Progression , Drug Resistance, Neoplasm , Humans , Mice , Mice, Nude , Niacinamide/analogs & derivatives , Phenylurea Compounds , Receptors, Adrenergic, beta-2 , Signal Transduction , SorafenibABSTRACT
Objective To observe the effects of miR-429 on inflammatory infiltration, differentiation and proliferation of hepatocelluar carcinoma (HCC). Methods HCCLM3 cells were sorted into positive and -negative cells using magnetic activated cell sorting (MACS) according to EpCAM marker. The EpCAM-positive and negative cells were transfected with AntagomiR- 429 and miR-429mimic to construct over-expressed and down-regulated cells of miR-429, respectively. To validate the effect of transfection, control cells and constructed cells were tested by flow cytometry (FC) and real-time polymerase chain reaction (PCR). The proliferative curves of transfected cells were plotted using cell counting kit-8 (CCK8) assay. Furthermore, tumor- bearing mice model was established by subcutaneous inoculation with transfected cells and related control cells into non-obese diabetic, severe combined immunodeficient mice (NOD-SCID). Mice were sacrificed after 6 weeks, and protein expressions of Ki67, AFP and F4/80 in tumors were determined via immunohistochemistry (IHC) to examine the status of proliferation, differentiation and inflammatory infiltration, respectively. Results The proportion of EpCAM-positive cells was decreased by down-regulating miR-429, and EpCAM-negative cells could partially transfer to EpCAM-positive cells by miR-429 ove- expression. In vivo study showed that over-expressing miR-429 in EpC AM-negative cells cound enhance the tumor forming ability; whereas EpCAM-positive cells had an inhibited tumor-formation when down-regulating miR-429. Interestingly, F4/80, a marker of macrophage, was strongly stained in tumor tissue with miR-429 over-expression. It was also found that in tumor tissue, Ki67 and AFP staining was suppressed with miR-429 down-regulation. Meanwhile, compared to the control, miR-429 significantly accelerated the proliferation of HCCLM3 in vitro compared with the control (P$0. 05). Conclusion miR-429 can regulate the proportion of EpCAM-positive cells of HCC in vitro and in vivo, and greatly increase the inflammatory infiltration and proliferative ability, accompanied by decreased differentiation.