The effects of restraint stress on ceramide metabolism disorders in the rat liver: the role of CerS6 in hepatocyte injury.

BACKGROUND: Stress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms. METHODS: The rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC-MS/MS. RESULTS: In comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT. CONCLUSIONS: CerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6.

LASS2 enhances chemosensitivity to cisplatin by inhibiting PP2A-mediated ß-catenin dephosphorylation in a subset of stem-like bladder cancer cells.

BACKGROUND: The benefits of first-line, cisplatin-based chemotherapy for muscle-invasive bladder cancer are limited due to intrinsic or acquired resistance to cisplatin. Increasing evidence has revealed the implication of cancer stem cells in the development of chemoresistance. However, the underlying molecular mechanisms remain to be elucidated. This study investigates the role of LASS2, a ceramide synthase, in regulating Wnt/ß-catenin signaling in a subset of stem-like bladder cancer cells and explores strategies to sensitize bladder cancer to cisplatin treatment. METHODS: Data from cohorts of our center and published datasets were used to evaluate the clinical characteristics of LASS2. Flow cytometry was used to sort and analyze bladder cancer stem cells (BCSCs). Tumor sphere formation, soft agar colony formation assay, EdU assay, apoptosis analysis, cell viability, and cisplatin sensitivity assay were used to investigate the functional roles of LASS2. Immunofluorescence, immunoblotting, coimmunoprecipitation, LC-MS, PCR array, luciferase reporter assays, pathway reporter array, chromatin immunoprecipitation, gain-of-function, and loss-of-function approaches were used to investigate the underlying mechanisms. Cell- and patient-derived xenograft models were used to investigate the effect of LASS2 overexpression and a combination of XAV939 on cisplatin sensitization and tumor growth. RESULTS: Patients with low expression of LASS2 have a poorer response to cisplatin-based chemotherapy. Loss of LASS2 confers a stem-like phenotype and contributes to cisplatin resistance. Overexpression of LASS2 results in inhibition of self-renewal ability of BCSCs and increased their sensitivity to cisplatin. Mechanistically, LASS2 inhibits PP2A activity and dissociates PP2A from ß-catenin, preventing the dephosphorylation of ß-catenin and leading to the accumulation of cytosolic phospho-ß-catenin, which decreases the transcription of the downstream genes ABCC2 and CD44 in BCSCs. Overexpression of LASS2 combined with a tankyrase inhibitor (XAV939) synergistically inhibits tumor growth and restores cisplatin sensitivity. CONCLUSIONS: Targeting the LASS2 and ß-catenin pathways may be an effective strategy to overcome cisplatin resistance and inhibit tumor growth in bladder cancer patients.

The Role of Longevity Assurance Homolog 2/Ceramide Synthase 2 in Bladder Cancer.

The human CERS2 gene encodes a ceramide synthase enzyme, known as CERS2 (ceramide synthase 2). This protein is also known as LASS2 (LAG1 longevity assurance homolog 2) and TMSG1 (tumor metastasis-suppressor gene 1). Although previously described as a tumor suppressor for different types of cancer, such as prostate or liver cancer, it has also been observed to promote tumor growth in adenocarcinoma. In this review, we focus on the influence of CERS2 in bladder cancer (BC), approaching the existing literature about its structure and activity, as well as the miRNAs regulating its expression. From a mechanistic point of view, different explanations for the role of CERS2 as an antitumor protein have been proposed, including the production of long-chain ceramides, interaction with vacuolar ATPase, and its function as inhibitor of mitochondrial fission. In addition, we reviewed the literature specifically studying the expression of this gene in both BC and biopsy-derived tumor cell lines, complementing this with an analysis of public gene expression data and its association with disease progression. We also discuss the importance of CERS2 as a biomarker and the presence of CERS2 mRNA in extracellular vesicles isolated from urine.

Cholesterol-dependent homeostatic regulation of very long chain sphingolipid synthesis.

Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking.

Ceramide synthase 4 overexpression exerts oncogenic properties in breast cancer.

BACKGROUND: Ceramide, a bioactive signaling sphingolipid, has long been implicated in cancer. Members of the ceramide synthase (CerS) family determine the acyl chain lengths of ceramides, with ceramide synthase 4 (CerS4) primarily generating C18-C20-ceramide. Although CerS4 is known to be overexpressed in breast cancer, its role in breast cancer pathogenesis is not well established. METHODS: To investigate the role of CerS4 in breast cancer, public datasets, including The Cancer Genome Atlas (TCGA) and two Gene Expression Omnibus (GEO) datasets (GSE115577 and GSE96058) were analyzed. Furthermore, MCF-7 cells stably overexpressing CerS4 (MCF-7/CerS4) as a model for luminal subtype A (LumA) breast cancer were produced, and doxorubicin (also known as Adriamycin [AD])-resistant MCF-7/ADR cells were generated after prolonged treatment of MCF-7 cells with doxorubicin. Kaplan-Meier survival analysis assessed the clinical significance of CERS4 expression, while Student's t-tests or Analysis of Variance (ANOVA) compared gene expression and cell viability in different MCF-7 cell lines. RESULTS: Analysis of the public datasets revealed elevated CERS4 expression in breast cancer, especially in the most common breast cancer subtype, LumA. Persistent CerS4 overexpression in MCF-7 cells activated multiple cancer-associated pathways, including pathways involving sterol regulatory element-binding protein, nuclear factor kappa B (NF-κB), Akt/mammalian target of rapamycin (mTOR), and ß-catenin. Furthermore, MCF-7/CerS4 cells acquired doxorubicin, paclitaxel, and tamoxifen resistance, with concomitant upregulation of ATP-binding cassette (ABC) transporter genes, such as ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2. MCF-7/CerS4 cells were characterized by increased cell migration and epithelial-mesenchymal transition (EMT). Finally, CERS4 knockdown in doxorubicin-resistant MCF-7/ADR cells resulted in reduced activation of cancer-associated pathways (NF-κB, Akt/mTOR, ß-catenin, and EMT) and diminished chemoresistance, accompanied by ABCB1 and ABCC1 downregulation. CONCLUSIONS: Chronic CerS4 overexpression may exert oncogenic effects in breast cancer via alterations in signaling, EMT, and chemoresistance. Therefore, CerS4 may represent an attractive target for anticancer therapy, especially in LumA breast cancer.

CerS6 triggered by high glucose activating the TLR4/IKKß pathway regulates ferroptosis of LO2 cells through mitochondrial oxidative stress.

Lipid metabolism disorders and mitochondrial dysfunction contribute to the progression of diabetes and chronic liver disease (CLD). Ferroptosis, as a form of cell death centered on reactive oxygen species (ROS) accumulation and lipid peroxidation, is closely related to mitochondrial dysfunction. However, whether there exists mechanistic links between these processes remains unknown. Here, to explore the molecular mechanism of diabetes complicated with CLD, we showed that high glucose could restrain the activity of antioxidant enzymes, promote mitochondrial ROS (mtROS) production, and induce a state of oxidative stress in the mitochondria of human normal liver (LO2) cells. We demonstrated that high glucose induced ferroptosis and promoted the development of CLD, which was reversed by the ferroptosis inhibitor Ferrostatin-1 (Fer-1). In addition, the mitochondria-targeting antioxidant Mito-TEMPO was used to intervene LO2 cells in high-glucose culture, and ferroptosis was found to be inhibited, whereas markers of liver injury and fibrosis improved. Furthermore, high glucose could promote ceramide synthetase 6 (CerS6) synthesis through the TLR4/IKKß pathway. The knockout of CerS6 in LO2 cells showed that mitochondrial oxidative stress was attenuated, ferroptosis was inhibited, and markers of liver injury and fibrosis were ameliorated. In contrast, the overexpression of CerS6 in LO2 cells showed the opposite changes and these changes were inhibited by Mito-TEMPO. In short, we positioned the study of lipid metabolism to a specific enzyme CerS6, with a high degree of specificity. Our findings revealed the mechanism by which the mitochondria act as a bridge linking CerS6 and ferroptosis, confirming that under high glucose conditions, CerS6 promotes ferroptosis through mitochondrial oxidative stress, eventually leading to CLD.

LncRNA CERS6-AS1 Is a Tumor Promoter in Cervical Cancer by Sponging miR-195-5p.

OBJECTIVE: CERS6 antisense RNA 1 (CERS6-AS1), a long non-coding RNA (lncRNA), plays a role in the malignant progression of a variety of cancers. However, it is unclear whether it affects the malignant behavior of cervical cancer (CC) cells. METHODS: CERS6-AS1 and miR-195-5p expression was estimated in CC via qRT-PCR. CCK-8, caspase-3 activity, scratch, and Transwell assays were performed to detect CC cell viability, caspase-3 activity, migration, and invasion in vitro. A tumor xenograft experiment was designed to study the growth of CC tumors in vivo. RIP and luciferase reporter experiments verified the relationship between CERS6-AS1 and miR-195-5p. RESULTS: CERS6-AS1 overexpression and poor miR-195-5p levels were observed in CC. Inhibition of CERS6-AS1 impaired the viability, invasion, and migration of CC cells, promoted apoptosis, and suppressed tumor growth. In terms of the underlying mechanism, CERS6-AS1, as a competitive endogenous RNA (ceRNA), participated in the regulation of miR-195-5p levels in CC cells. Functionally, miR-195-5p interference attenuated the inhibitory effect of CERS6-AS1 on the malignant behaviors of CC cells. CONCLUSION: CERS6-AS1 acts as an oncogene in CC, in vivo and in vitro, by negatively regulating miR-195-5p.

CERS6 antisense RNA 1 promotes colon cancer via upregulating mitochondrial calcium uniporter.

BACKGROUND: Colon cancer (CC) belongs to a common cancer of digestive system. Long non-coding RNAs (lncRNAs) are dysregulated in numerous cancers and affect their development. The function of lncRNA CERS6 antisense RNA 1 (CERS6-AS1) in CC remains unclear. MATERIALS AND METHODS: CERS6-AS1 expression in colon adenocarcinoma tissues and CC cell lines was assessed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction analysis. The function of CERS6-AS1 in CC was analysed by 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling, wound healing, Transwell and immunofluorescence assays. Mechanistic analyses including RNA pull down, RNA-binding protein immunoprecipitation and luciferase reporter assay revealed the interaction between RNAs. RESULTS: CERS6-AS1 expression was aberrantly upregulated in colon adenocarcinoma tissues and CC cell lines. CERS6-AS1 knockdown inhibited CC cell malignant phenotypes and in vivo tumour growth. CERS6-AS1 served as the competing endogenous RNA of microRNA-16-5p in CC, and microRNA-16-5p inhibition partly rescued the effects of CERS6-AS1 depletion on CC development. Mitochondrial calcium uniporter was targeted by microRNA-16-5p. Mitochondrial calcium uniporter upregulation completely remedied the influence of CERS6-AS1 silencing in CC progression. Moreover, CERS6-AS1 enhanced the stability of mitochondrial calcium uniporter messenger RNA via recruiting RNA-binding protein embryonic lethal abnormal vision like 1. CONCLUSION: CERS6-AS1 promotes the development of CC via upregulating mitochondrial calcium uniporter expression.

LncRNA CERS6-AS1, sponging miR-6838-5p, promotes proliferation and invasion in cervical carcinoma cells by upregulating FOXP2.

Cervical cancer (CC) is a common disease in women characterized by high recurrence rate. LncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) has been found to play a crucial role in the progression of breast cancer and pancreatic cancer. Nevertheless, the regulatory role of CERS6-AS1 in CC remains largely unclear. Here, we found that the expression of CERS6-AS1 was upregulated in CC tissues and cell lines compared with adjacent tissues and normal human cervical epithelial cells. CERS6-AS1 overexpression promoted proliferation and invasion, and inhibited apoptosis in CC cells, while silencing of CERS6-AS1 led to the opposite results. CERS6-AS1 was verified as a sponge of miR-6838-5p by RNA pull-down and luciferase reporter gene assays. Functional investigations revealed that CERS6-AS1 knockdown inhibited proliferation and invasion, and promoted apoptosis in CC cells, which was reversed by miR-6838-5p inhibitor. Furthermore, forkhead box P2 (FOXP2) was identified as a target for miR-6838-5p, and overexpression of miR-6838-5p decreased the expression level of FOXP2. Besides, CERS6-AS1 was able to sponge miR-6838-5p to accelerate CC cell proliferation and invasion and inhibited cell apoptosis through upregulating FOXP2 expression. In general, CERS6-AS1 was able to regulate CC cell proliferation, invasion and apoptosis by the miR-6838-5p/FOXP2 axis, which suggested that CERS6-AS1 may be a potential target for the treatment of CC.

The role of lncRNA CERS6-AS1 in cancer and its molecular mechanisms: A systematic review and meta-analysis.

BACKGROUND: LncRNAs have the potential to play a regulatory role in different processes of cancer development and progression. We conducted a systematic review and meta-analysis of evidence on the clinical significance and prognostic value of lncRNA CERS6-AS1 in cancer. METHODS: This systematic review was conducted following PRISMA guidelines. Medline and Embase databases were searched using the relevant key terms covering lncRNA CERS6-AS1 and cancer. We pooled the estimated effect sizes and their 95 % confidence interval (CI) using random-effects models in STATA 16.0 (StataCorp, College Station, TX, USA). RESULTS: Eleven articles on pancreatic, colorectal, gastric, papillary thyroid, breast, and hepatocellular cancers fulfilled our eligibility criteria. Studies consistently found that lncRNA CERS6-AS1 expression is upregulated in all assessed cancers. Based on our meta-analysis, its aberrant expression was directly associated with unfavorable clinical outcomes, including higher stage (pooled Odds ratios (95 % CI): 3.15 (2.01-4.93; I2 = 0.0 %), tumor size (1.97 (1.27-3.05; I2 = 37.8 %), lymph node metastasis (6.48 (4.01-10.45; I2 = 0.40 %), and poor survival (Pooled log-rank test P-value < 0.001) in patients. Regarding potential mechanisms, functional studies revealed that LncRNA CERS6-AS1 is involved in cancer growth mainly by sponging miRNAs and regulating their downstream targets. CONCLUSION: Available evidence suggests that LncRNA CERS6-AS1 is upregulated in different cancers and has an oncogenic role. LncRNA CERS6-AS1 expression level might predict cancer prognosis, highlighting its potential application as a prognostic biomarker for cancer.

LASS2 overexpression enhances early apoptosis of lung cancer cells through the caspase­dependent pathway.

In a previous study by the authors, the longevity assurance homolog 2 (LASS2) gene was determined to inhibit activity of vacuolar H+­ATPase (V­ATPase) by combining with the C subunit (ATP6L) of V­ATPase. However, the influence of LASS2 overexpression and silencing on apoptosis of human lung cancer cells 95D or 95C remains unclear. Thus, the effect of LASS2 on apoptosis and its potential mechanisms were investigated in 95D and 95C cells. Using the lentiviral transfection method, lentiviral vectors of LASS2 overexpression and silencing were transfected into 95D and 95C cells, respectively. The apoptotic ability of tumor cells was observed by flow cytometry. The expression levels of LASS2, Bcl­2, Bax, cytochrome c, caspase­9, and caspase­3 were detected by western blotting. CCK­8 assay was used to detect the growth ability of tumor cells in vitro. Flow cytometric analysis revealed that LASS2 overexpression could promote the early apoptosis of lung cancer cells 95D. CCK­8 assay demonstrated that LASS2 overexpression inhibited the proliferation of 95D cells. Additionally, LASS2 overexpression decreased the expression of Bcl­2, induced the release of cytochrome c from mitochondria, and promoted the activation of caspase­9 and caspase­3. There was a significant difference in the expression of Bcl­2, cytochrome c, caspase­9 and caspase­3 in the LASS2­overexpression group compared with the normal and negative control groups. Alternatively, the aforementioned experiments in lung cancer cells 95C following LASS2 silencing produced the opposite effects. LASS2 may induce early apoptosis of lung cancer cells by influencing the caspase­dependent mitochondrial pathway.

Ceramide synthase 6 antisense RNA 1 contributes to the progression of breast cancer by sponging miR-16-5p to upregulate ubiquitin-conjugating enzyme E2C.

Breast cancer (BC) is the most dangerous female mortality all over the world, described by unavoidable spread and metastaticity of BC cells. Increasing evidences verified that lncRNA play a major role in the tumorgenesis and development of BC cell. The purpose of this study is to investigate the roles of lncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) and ubiquitin-conjugating enzyme E2C (UBE2C) in BC and explore the regulatory association among miR-16-5p, CERS6-AS1, and UBE2C in BC. The CERS6-AS1 and UBE2C expression levels were determined by real time quantitative PCR in cell lines and tissues of BC. The function of CERS6-AS1 and UBE2C in the apoptosis, proliferation, and migration was confirmed by cell counting kit-8, Transwell, and flowcytometry tests. We performed tumor xenograft assay to validate the roles of CERS6-AS1 in vivo. The expression of UBE2C proteins was evaluated by Western Blot analysis. Moreover, the relationship among UBE2C, CERS6-AS1, and miR-16-5p was verified by luciferase report assay. It was found that CERS6-AS1 and UBE2C were meaningfully upregulated in BC, and knockdown of both CERS6-AS1 and UBE2C inhibited the BC cell proliferation and migration, whereas induced apoptosis. Mechanistically, CERS6-AS1 could facilitate BC progression by sponging miR-16-5p for upregulation of the UBE2C expression. The CERS6-AS1/miR-16-5p/UBE2C axis might be a prospective therapeutic target in the BC treatment by sponging miR-16-5p to upregulate UBE2C, which might contribute to the development of BC.

Generation of a ceramide synthase 6 mouse lacking the DDRSDIE C-terminal motif.

The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide in vitro. In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths.

Danhe granule ameliorates nonalcoholic steatohepatitis and fibrosis in rats by inhibiting ceramide de novo synthesis related to CerS6 and CerK.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhe granule (DHG) is used by Chinese doctors to treat blood stasis, phlegm and dampness. Its lipid-lowering ability has been investigated in our previous research. However, the anti-liver inflammatory and fibrotic effects and mechanism of action of DHG in non-alcoholic steatohepatitis (NASH) have not been explored. AIM OF THE STUDY: To evaluate the ameliorative effects of DHG on liver inflammation and fibrosis in a methionine/choline-deficient (MCD) diet-induced NASH rat model, and its underlying mechanism. MATERIALS AND METHODS: Sprague-Dawley rats were fed an MCD diet for two weeks and then treated with or without DHG by oral gavage for eight weeks. Their body weight and liver index were measured. The serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities as well as the liver triglyceride (TG) and free fatty acid (FFA) levels were tested using reagent kits. Inflammatory cytokines, including Tnf-α, Il-ß and Il-6, and fibrosis genes, including Acta2, Col1a1, Col1a2 and Tgf-ß were examined by real-time quantitative PCR (RT-qPCR). Hematoxylin-eosin (H&E), Oil Red O, Masson's and Sirius Red staining were used to observe liver changes. The plasma and liver ceramide levels were analyzed using HPLC-QQQ-MS/MS. The expression of serine palmitoyl-CoA transferase (Spt), ceramide synthase 6 (Cers6), dihydroceramide desaturase 1 (Des1), glucosylceramide synthase (Gcs), and ceramide kinase (Cerk) mRNA was assayed by RT-qPCR, while the protein expression of CerS6, DES1, GCS, CerK, and casein kinase 2α (CK2α) was tested by western blotting (WB). CerS6 degradation was evaluated using a cycloheximide (CHX) assay in vitro. RESULTS: The liver index decreased by 20% in DHG groups and the serum ALT and AST decreased by approximately 50% and 30%, respectively in the DHG-H group. The liver Oil Red O staining, TG, and FFA changes showed that DHG reduced hepatic lipid accumulation by approximately 30% in NASH rats. H&E, Masson's and Sirius Red staining and the mRNA levels of Tnf-α, Il-ß, Il-6, Acta2, Col1a1, Col1a2 and Tgf-ß revealed that DHG alleviated liver inflammation and fibrosis in NASH rats. The ceramide (Cer 16:0), and hexosylceramide (HexCer 16:0, HexCer 18:0, HexCer 22:0, HexCer 24:0 and HexCer 24:1) levels decreased by approximately 17-56% in the plasma of the DHG-M and H rats. The Cer 16:0 content in the liver decreased by 20%, 50%, and 70% with the DHG-L, M, and H treatments; additionally, the dhCer 16:0, Cer 18:0, HexCer 18:0, HexCer 20:0 Cer 22:0-1P, Cer 24:0-1p, Cer 24:1-1p, and Cer 26:1-1p levels decreased in the DHG groups. The mRNA and protein expression levels of DES1, GCS, Cerk, CerS6, and CHX assay indicated that DHG decreased the mRNA and protein expression levels of CerK and reduced CerS6 protein expression by promoting its degradation. Additionally, DHG attenuated the protein expression of CK2α which could increase CerS6 enzymatic activity by phosphorylating its C-terminal region. CONCLUSION: DHG ameliorated the levels of liver FFA and TG and inflammation and fibrosis in MCD-induced rats, which were associated with decreasing ceramide species in the plasma and liver by reducing the expression levels of CerS6 and CerK.

Ceramide synthase 3 affects invasion and metastasis of hepatocellular carcinoma via the SMAD6 gene. / ç¥žç»é °èƒºåˆæˆé ¶3通过SMAD6åŸºå› å½±å“è‚ç»†èƒžç™Œçš„ä¾µè¢­å’Œè½¬ç§».

OBJECTIVES: Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC. METHODS: A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3. RESULTS: Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P<0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P<0.01), TNM stage (95% CI 2.3-5.2, P<0.05), poor histological grade (95% CI 1.4-6.8, P<0.05), and CerS3 (95% CI 1.5-3.9, P<0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression (P<0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P<0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P<0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 (SMAD6) gene as an oncogenic gene could promote the HCC metastasis. CONCLUSIONS: Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Epidermal 1-O-acylceramides appear with the establishment of the water permeability barrier in mice and are produced by maturating keratinocytes.

1-O-Acylceramides (1-OACs) have a fatty acid esterified to the 1-hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1-OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1-OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1-OAC levels indicating they are one source of epidermal 1-OACs. In search of potential enzymes responsible for 1-OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl-CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1-OACs did not decrease in any mouse model. In Cers3 and Dgat2-deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1-OAC synthesis with an increase in 1-OACs with N-linked non-hydroxylated fatty acids and a 60% decrease compared to control in levels of 1-OACs with N-linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1-OACs, we conclude that either there is functional redundancy in forming 1-OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1-OAC synthesis, the implications of which are discussed.

CERS6-AS1 promotes cell proliferation and represses cell apoptosis in pancreatic cancer via miR-195-5p/WIPI2 axis.

Pancreatic cancer (PC) is a lethal malignancy that threatens human health. Long noncoding RNAs (lncRNAs) act as important mediators in PC development. Our study aimed to investigate the function and mechanism of lncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) in PC. As shown by RT-qPCR, CERS6-AS1 was significantly upregulated in PC cells and tissues. Silencing CERS6-AS1 suppressed PC cell viability and proliferation while enhancing cell apoptosis according to colony formation assays, EdU assays, and flow cytometry analyses. Mechanistically, CERS6-AS1 interacted with miR-195-5p to elevate the expression level of the WD repeat domain phosphoinositide interacting 2 (WIPI2), which is a downstream target gene of miR-195-5p in PC. Moreover, miR-195-5p expression was negatively associated with CERS6-AS1 expression (or WIPI2 expression) in PC tissues. Rescue assays revealed that WIPI2 overexpression rescued the effects of CERS6-AS1 deficiency on cell viability, proliferation, and apoptosis. In summary, CERS6-AS1 facilitates PC cell proliferation while inhibiting PC cell apoptosis by upregulating WIPI2 via miR-195-5p. This study might provide promising insight into the role of CERS6-AS1 in PC development.

CERS6-AS1 contributes to the malignant phenotypes of colorectal cancer cells by interacting with miR-15b-5p to regulate SPTBN2.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) act as tumor promoters or suppressors in various types of cancer. Previous investigations suggest that ceramide synthase 6 (CERS6) antisense RNA 1 (CERS6-AS1) acts as an oncogene in breast cancer; however, its role in colorectal cancer is unknown. This study aimed to explore the molecular mechanism of CERS6-AS1 in colorectal cancer. Gene expression in colorectal cancer was examined using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The viability and proliferation of colorectal cancer cells were measured by Cell Counting Kit-8 assays and colony formation assays. The migratory and invasive capacities of the colorectal cancer cells were assessed by Transwell assay. Cell stemness was examined by sphere-formation assay. Mechanistically, RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays were performed to explore the relationship among CERS6-AS1, miR-15b-5p and spectrin beta, non-erythrocytic 2 (SPTBN2). Moreover, a xenograft tumor model was established to investigate the role of CERS6-AS1 in vivo. We found that CERS6-AS1 and SPTBN2 were highly expressed in colorectal cancer tissues and cells. CERS6-AS1 depletion inhibited cell viability, proliferation, migration, and invasion; the epithelial-mesenchymal transition process and stemness. It suppressed xenograft tumor growth in colorectal cancer. Moreover, SPTBN2 levels were positively regulated by CERS6-AS1 and negatively regulated by miR-15b-5p in colorectal cancer cells. Rescue assays revealed that SPTBN2 reversed the inhibitory effect of CERS6-AS1 deficiency on the malignant behaviors of colorectal cancer cells. Overall, the lncRNA CERS6-AS1 facilitates malignant phenotypes of colorectal cancer cells by targeting miR-15b-5p to upregulate SPTBN2.

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.