MicroRNA-361-5p Alleviates Leydig Cell Apoptosis and Promotes Cell Growth by Targeting PIAS1 in Late-Onset Hypogonadism.

Late-onset hypogonadism (LOH) is an age-related syndrome characterized by deficiency of serum testosterone produced by Leydig cells. Previous evidence suggested that microRNA (miR)-361-3p can serve as a promising biomarker for LOH. Nonetheless, its detailed function and molecular mechanism in LOH remain unclarified. The 24-month-old male mice were selected as an animal LOH model, and mouse Leydig cell line TM3 was stimulated with H2O2. ELISA was employed for testosterone level evaluation. Hematoxylin-eosin staining was implemented for histologic analysis of mouse testicular tissues. Western blotting and RT-qPCR were utilized for evaluating molecular protein and RNA expression, respectively. Functional experiments were conducted to test miR-361-5p roles. Luciferase reporter assay was for verifying the interaction between miR-361-5p and protein inhibitor of activated STAT 1 (PIAS1). miR-361-5p displayed a decreased level in the testes of LOH mice. Overexpressing miR-361-5p attenuated Leydig cell loss in the testis and elevated serum and intratesticular testosterone levels in LOH mice. H2O2 stimulation impaired TM3 cell viability, proliferation and intracellular testosterone production and enhanced cell apoptosis. miR-361-5p targeted PIAS1 in TM3 cell. PIAS1 upregulation counteracted miR-361-5p overexpression-mediated alleviation of cell apoptosis and elevation of testosterone synthesis in H2O2-stimualetd TM3 cells. miR-361-5p ameliorates LOH progression by increasing testosterone production and alleviate Leydig cell apoptosis via downregulation of PIAS1.

Epigenetic activation of METTL14 promotes docetaxel resistance in prostate cancer by promoting pri-microRNA-129 maturation.

The development of resistance to Docetaxel (DTX) compromises its therapeutic efficacy and worsens the prognosis of prostate cancer (PCa), while the underlying regulatory mechanism remains poorly understood. In this study, METTL14 was found to be upregulated in DTX-resistant PCa cells and PCa tissues exhibiting progressive disease during DTX therapy. Furthermore, overexpression of METTL14 promoted the development of resistance to DTX in both in vitro and in vivo. Interestingly, it was observed that the hypermethylation of the E2F1 targeting site within DTX-resistant PCa cells hindered the binding ability of E2F1 to the promoter region of METTL14, thereby augmenting its transcriptional activity. Consequently, this elevated expression level of METTL14 facilitated m6A-dependent processing of pri-miR-129 and subsequently led to an increase in miR-129-5p expression. Our study highlights the crucial role of the E2F1-METTL14-miR-129-5p axis in modulating DTX resistance in PCa, underscoring METTL14 as a promising therapeutic target for DTX-resistant PCa patients.

Identification of the circRNA-miRNA-mRNA Regulatory Network in Bladder Cancer by Bioinformatics Analysis.

In recent years, increasing evidence shows that circular RNA (circRNA) disorder is closely related to tumorigenesis and cancer progression. However, the regulatory functions of most circRNAs in bladder cancer (BCa) remain unclear. This study was aimed at exploring the molecular regulatory mechanism of circRNAs in BCa. We obtained four datasets of circRNA, microRNA (miRNA), and messenger (mRNA) expression profiles from the Gene Expression Omnibus and The Cancer Genome Atlas microarray databases and identified 434, 367, and 4799/4841 differentially expressed circRNAs, miRNAs, and mRNAs, respectively. With these differentially expressed RNAs, we established a circRNA-miRNA-mRNA targeted interaction network. A total of 18, 24, and 51 central circRNAs, miRNAs, and mRNAs were identified, respectively. Among them, the top 10 mRNAs that had high connectivity with other circRNAs and miRNAs were regarded as hub genes. We detected the expression levels of these 10 mRNAs in 16 pairs of BCa tissues and adjacent normal tissues through quantitative real-time polymerase chain reaction. The differentially expressed mRNAs and central mRNAs were enriched in the processes and pathways that are associated with the growth, differentiation, proliferation, and apoptosis of tumor cells. The outstanding genes (CDCA4, GATA6, LATS2, RHOB, ZBTB4, and ZFPM2) also interacted with numerous drugs, indicating their potency as biomarkers and drug targets. The findings of this study provide a deep understanding of the circRNA-related competitive endogenous RNA regulatory mechanism in BCa pathogenesis.

The anti-cancer properties of heparin and its derivatives: a review and prospect.

Heparin, including unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) and heparin derivatives, are commonly used in venous thromboembolism treatment and reportedly have beneficial effects on cancer survival. Heparin can affect the proliferation, adhesion, angiogenesis, migration and invasion of cancer cells via multiple mechanisms. The main mechanisms involve inhibition of heparanase, P-/L-selectin, angiogenesis, and interference with the CXCL12-CXCR4 axis. Here we summarize the current experimental evidence regarding the anti-cancer role of heparin and its derivatives, and conclude that there is evidence to support heparin's role in inhibiting cancer progression, making it a promising anti-cancer agent.

MiR-129-5p promotes docetaxel resistance in prostate cancer by down-regulating CAMK2N1 expression.

This study focuses on the effect of miR-129-5p on docetaxel-resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT-PCR in PCa patient tissues and cell lines including PC-3 and PC-3-DR. Cells transfected with miR-129-5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR-129-5p and CAMK2N1 levels were identified by qRT-PCR and dual-luciferase reporter assay. CAMK2N1 was found to be down-expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up-regulation of miR-129-5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR-129-5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR-129-5p contributed to the resistance of PC-3-DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR-129-5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.

The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain.

Mammalian patatin-like phospholipase domain-containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure-function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.

Molecular identification of transmembrane protein 68 as an endoplasmic reticulum-anchored and brain-specific protein.

Acyltransferases catalyze essential reactions in the buildup and remodeling of glycerophospholipids and contribute to the maintenance and diversity of cellular membranes. Transmembrane protein 68 (TMEM68) is an evolutionarily conserved protein of unknown function, that forms a distinct subgroup within the glycerophospholipid acyltransferase family. In the current study we expressed murine TMEM68 for the first time in mammalian cells to characterize its subcellular localization, topology, and possible biological function(s). We show that TMEM68 is an integral membrane protein and orients both, the N- and C-terminus towards the cytosol. Live cell imaging demonstrated that TMEM68 is localized mainly at the endoplasmic reticulum (ER), but not at cellular lipid droplets (LDs). The positioning of TMEM68 at the ER was dependent on its first transmembrane domain (TMD), which by itself was sufficient to target cytosolic green fluorescence protein (GFP) to the ER. In contrast, a second TMD was dispensable for ER localization of TMEM68. Finally, we found that among multiple murine tissues the expression level of TMEM68 transcripts was highest in brain. We conclude that TMEM68 is an integral ER membrane protein and a putative acyltransferase involved in brain glycerolipid metabolism.

The destruction box is involved in the degradation of the NTE family proteins by the proteasome.

Neuropathy target esterase (NTE) and NTE-related esterase (NRE) are endoplasmic reticulum (ER) membrane-anchored proteins belonging to the NTE protein family. NTE and NRE are degraded by macroautophagy and by the ubiquitin-proteasome pathway. However, the regulation of NTE and NRE by proteasome has not been well understood. Western blotting showed that the deletion of the regulatory region of NTE and NRE led to protein accumulation compared with that of the corresponding wild-type proteins. Further, deletion and site-directed mutagenesis experiments demonstrated that the destruction (D) box was required for the proteasomal degradation of NTE and NRE. However, unlike the deletion of the regulatory region, the deletion of the D box did not affect the subcellular localisation of NTE or NRE or disrupt the ER. Moreover, the deletion of the D box or the regulatory region of NTE has similar inhibitory effects on cell growth, which are greater than those produced by the full-length NTE. Here, for the first time, we show that the D box is involved in the regulation of NTE family proteins by the proteasome but not in their subcellular localisation. In addition, these results suggest that the NTE overexpression-mediated inhibition of cell growth is related to active protein levels but not to its ER disruption effect.

Identification mouse patatin-like phospholipase domain containing protein 1 as a skin-specific and membrane-associated protein.

Patatin-like phospholipase domain containing protein 1 (PNPLA1) mutations have been identified to be associated with autosomal recessive congenital ichthyosis (ARCI) in recent years. However, its molecular characters have not been achieved until now. In the current study, the full length coding cDNA sequence of mouse PNPLA1 (mPNPLA1) was identified firstly. There were several putative transmembrane domains (TMDs) in mPNPLA1 by bioinformation analysis. mPNPLA1 was further found to be expressed exclusively in the membrane fraction in mammalian cells. However, it did not colocalized with the endoplasmic reticulum (ER) or lipid droplets (LDs). Moreover, the mRNA levels of mPNPLA1 was detected to be highly expressed in the skin, while very weak or even less in other mouse tissues by quantitative PCR. In addition, based on experiments with inhibitors and inducer of protein degradation pathways, mPNPLA1 was demonstrated to be degraded by macroautophagy, but not by the proteasome. These results indicated PNPLA1 was a skin-specific and membrane-associated protein for the first time, suggesting that it may mainly play a role in the skin.

Identification of human patatin-like phospholipase domain-containing protein 1 and a mutant in human cervical cancer HeLa cells.

Recently members of mammalian patatin-like phospholipase domain containing (PNPLA) protein family have attracted attention for their critical roles in diverse aspects of lipid metabolism and signal pathway. Until now little has been known about the characteristics of PNPLA1. Here, the full length coding cDNA sequence of human PNPLA1 (hPNPLA1) was cloned for the first time, which encoded a polypeptide with 532 amino acids containing the whole patatin domain. Tissue expression profiles analysis showed that low mRNA levels of hPNPLA1 existed in various tissues, except high expression in the digestive system, bone marrow and spleen. Subcellular distribution of hPNPLA1 tagged with green fluorescence protein mainly localized to lipid droplets. Furthermore, a nonsense mutation of PNPLA1 in human cervical cancer HeLa cells was identified. The hPNPLA1 mutant encoded a protein of 412 amino acids without the C-terminal domain and did not colocalize to lipid droplets, which suggested that the C-terminal region of hPNPLA1 affected lipid droplet binding. These results identified hPNPLA1 and a mutant in HeLa cells, and provided insights into the structure and function of PNPLA1.

Degradation of mouse NTE-related esterase by macroautophagy and the proteasome.

NTE-related esterase (NRE) is a novel endoplasmic reticulum-anchored lysophospholipase with high homology to neuropathy target esterase (NTE). However, little is known about the regulation of NRE protein. In the current study, we investigated the degradation pathways of mouse NRE (mNRE) in mammalian cells. Based on experiments with inhibitors and inducer of protein degradation pathways, we provide here the first evidence that mNRE is degraded by macroautophagy as well as by the proteasome. Moreover, the contribution of protein domains to the degradation of mNRE was investigated, which showed that the transmembrane and regulatory domain played a role in the degradation of mNRE by macroautophagy and the proteasome respectively. In contrast the C-terminal catalytic domain was not involved in both degradation pathways of mNRE. These findings showed for the first time that the degradation pathways in controlling mNRE quantity and may provide further insight into structure and regulation of mNRE.

Identification of two novel splicing variants of murine NTE-related esterase.

NTE-related esterase (NRE) is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE), which plays a role in energy metabolism. Here, we reported two alternative splicing variants of the murine NRE (mNRE) gene, termed mNREV1 and mNREV2. Genomic organization analysis indicated that 5' splice site of mNRE intron 33 was changed in both mNREV1 and mNREV2, and mNRE exon 21 was deleted in mNREV2. mNREV1 had the same protein domains with mNRE, while mNREV2 lacked the patatin domain in the C-terminal catalytic region. Green fluorescent protein-mNREV1 or mNREV2 fusion proteins localized to the endoplasmic reticulum. mNREV1 and mNRE exhibited equal hydrolytic activity to the substrate phenyl valerate, whereas mNREV2 did not have any catalytic activity. The expression profiles of mNRE and its splicing isoforms in white adipose tissue, cardiac muscle, skeletal muscle, and testis tissues were further analyzed by real time quantitative-PCR in fed and fasted states, which indicated that the major isoform of mNRE mRNA generated switched from mNREV2 to mNREV1 during fasting. Thus there was a nutritional regulation of mNRE expression at the mRNA levels via alternative splicing.

The role of cell cycle-dependent neuropathy target esterase in cell proliferation.

Neuropathy target esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G1 and lowest at G2/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase.

A mammalian family of lipid hydrolases, designated "patatin-like phospholipase domain containing (PNPLA)" recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

Neuropathy target esterase: an essential enzyme for neural development and axonal maintenance.

Neuropathy target esterase (NTE) is an endoplasmic reticulum-anchored protein conserved across species. The N-terminal regulatory region of NTE contains three cyclic nucleotide binding domains while the C-terminal catalytic domain has a patatin domain. The NTE gene is expressed in mouse early at embryonic day 7 and its expression is maintained throughout embryonic development. NTE protein is mainly distributed in the nervous system with a pattern that is more restricted to large neurons in older animals. NTE regulates phospholipid metabolism and is known to be a phospholipase B. Knockout of NTE is embryonic lethal in mice, indicating that NTE is essential for embryonic survival. Neuronal specific NTE knockouts survive to adulthood, but show vacuolation and neuronal loss characteristic of neurodegenerative diseases. Recently, mutations in human NTE have been shown to cause a hereditary spastic paraplegia called NTE-related motor neuron disorder, suggesting a critical role for NTE in the nervous system.

Trichlorfon induces apoptosis in SH-SY5Y neuroblastoma cells via the endoplasmic reticulum?

This study investigated the role of the endoplasmic reticulum pathway in apoptosis induced by trichlorfon in SH-SY5Y human neuroblastoma cells. Flow cytometric analysis demonstrated that trichlorfon and its degradation product dichlorvos-induced apoptosis in a dose-dependent manner and Hoechst 33342 staining experiments revealed trichlorfon/dichlorvos-induced nucleus condensation. Western blot analysis indicated decreased expression of caspase-12 and increased activated caspase-12 in trichlorfon-treated cells compared to a control, suggesting that trichlorfon may induce apoptosis in SH-SY5Y partly via the endoplasmic reticulum. Intracellular Ca(2+) level ([Ca(2+)](i)) in SH-SY5Y cells increased after treatment with trichlorfon but was significantly reduced by pre-treatment with a combination of a calcium channel blocker, an inositol trisphosphate receptor inhibitor, and a ryanodine receptor inhibitor. Percent apoptosis and activated caspase-3 and caspase-12 decreased in pre-treated cells compared to those treated with trichlorfon alone. Trichlorfon-induced apoptosis was also inhibited by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). These results suggest that endoplasmic reticulum stress, which is related to calcium, may be involved in the cytotoxicity of trichlorfon.

Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance.

Chemotherapy resistance in solid tumors is broad and encompasses diverse unrelated drugs. Three-dimensional multicellular spheroids (MCSs) are a good model for studying in vitro drug resistance. In the current study, we investigated the role of focal adhesion kinase (FAK) in 5-fluorouracil (5-FU) chemoresistance in colon carcinoma MCS culture cells. The expression of FAK was inhibited significantly by specific small hairpin RNA targeting FAK. The suppression of FAK expression did not affect the growth of spheroid cells. However, silencing of FAK combined with 5-FU treatment significantly decreased the 50% inhibitory concentration (IC(50)) of 5-FU and markedly increased the population of apoptosis cells, which was associated with the reduction of the levels of Akt and nuclear factor-kappa B (NF-kappaB). Moreover, knockdown of FAK could inhibit tumor growth and increase the sensitivity of the tumor to 5-FU in the nude mouse xenograft. These results indicate that while not affecting cellular proliferation in the absence of 5-FU, RNA interference targeting FAK potentiated 5-FU-induced cytotoxicity in vitro and in vivo, and partially reversed multicellular resistance, which may contribute to its chemosensitizing effect through efficiently suppressing Akt/NF-kappaB activity.

Motor neuron diseases and neurotoxic substances: a possible link?

The motor neuron diseases (MNDs) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. Exploring the molecular mechanisms underlying MND phenotypes has been hampered by their multifactorial nature and high incidence of sporadic cases, although genetic factors are considered to play a considerable role at present. However, environmental factors, especial exposure to neurotoxic substances, could induce neurotoxicity with the same phenotypes of specific MNDs. Organophosphate-induced delayed neuropathy (OPIDN) is a neurodegenerative disorder characterized by ataxia and progression to paralysis, with a concomitant distal axonal degeneration and secondary demyelination of central and peripheral axons. The inhibition and subsequent aging of neuropathy target esterase (NTE) by organophosphate has been proposed to be the initiating event in OPIDN. NTE is characterized to be a lysophospholipase/phospholipase B mostly in the nervous system to regulate phospholipid homeostasis. Brain-specific deletion of mouse NTE contributes to the behavioral defects characterized by neuronal loss. Recently, mutations in human NTE have also been shown to cause a hereditary spastic paraplegia called NTE-related motor neuron disorder with the same characteristics of OPIDN, which supported the role of NTE abnormalities in OPIDN, and raised the possibility that NTE pathway disturbances contribute to other MNDs. Together with the identified association of paraoxonase polymorphisms with amyotrophic lateral sclerosis, there is a possibility that neurotoxic substances contribute to MND in genetically vulnerable people by gene-environment interactions.

Identification and characterization of chicken neuropathy target esterase.

Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) and adult hens are the animal model of OPIDN. However, little has been known about the sequence and characteristics of chicken NTE. Here, we firstly identified the full length cDNA of chicken NTE (cNTE), which contained an open reading frame of 3966 nucleotides encoding 1321 amino acids. Chicken NTE had two distinct regions, one was the regulatory domain (cNTER) and the other was the catalytic domain (cNEST). Over-expression of cNTER in mammalian cells did not show any NTE activity, whereas cNEST had NTE activity. Cells expressing cNTER tagged with green fluorescent protein (GFP) showed accumulation of cNTER-GFP in an endoplasmic reticulum-like localization pattern. In addition, macroautophagy and the proteasome pathways were found to be involved in the degradation of cNTER, but not cNEST. These results first showed that cNTE was an ER-anchored protein and degraded by macroautophagy as well as the proteasome.

Degradation of neuropathy target esterase by the macroautophagic lysosomal pathway.

AIMS: Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in humans and some sensitive animals. NTE was recently identified as a novel phospholipase B that is anchored to the cytoplasmic side of the endoplasmic reticulum. However, little is known about the degradation of NTE. In this study, we have investigated the role of the macroautophagic-lysosomal pathway in NTE degradation in neuronal and non-neuronal cells. MAIN METHODS: Macroautophagy inhibitors and activators were used to interrupt the lysosomal pathway, and NTE protein level was followed using western blotting analysis. A fluorescent microscopy assay was used to determine the co-localization of NTE and lysosomes. KEY FINDINGS: Western blotting analysis showed that the macroautophagy inhibitors 3-methyladenine and ammonium chloride increased the levels of a heterologously expressed NTE-GFP fusion protein as well as endogenous NTE. Starvation had the opposite effect. The role of macroautophagy in NTE degradation was further supported by the co-localization of exogenous NTE with lysosomes in starved COS7 cells. Furthermore, the contribution of NTE activity and protein domains to the degradation of NTE by macroautophagy was investigated, showing that both the transmembrane and regulatory domains played a role in the degradation of NTE and that the catalytic domain, and thus NTE activity, was not involved. SIGNIFICANCE: Our findings clearly demonstrate, for the first time, that the macroautophagy/lysosome pathway plays a role in controlling NTE quantity, providing a further understanding of the function of NTE.