A novel de novo CLTC variant altering RNA splicing causes fetal developmental abnormalities.

BACKGROUND: About 31 individuals with CLTC variants have been reported worldwide, and all reported individuals have motor and mental retardation. CLTC is known to lead to intellectual developmental disorder, autosomal dominant 56. Few studies are focusing on the prenatal stage of the disease. METHOD: An ultrasound examination was performed to obtain the prenatal phenotype. Whole-exome sequencing was used to find the pathogenic variant. Multiple computational tools predicted the conservation and deleteriousness. Minigene assay and western blot were utilized to investigate the effect on splicing of mRNA and protein expression. RESULT: Here we found a novel de novo variant of CLTC in a fetus. The fetus manifested bilateral choroid plexus cysts of the brain, hyperechogenic kidneys, and ventricular septal defect. A heterozygous variant c.3249 + 1G > C was identified in the fetus. This position was conserved and the variant was predicted to be deleterious. Minigene assay revealed the presence of a truncating transcript with the retention of intron 20. Western blot result showed the c.3249 + 1G > C variant elicited degradation of the protein. CONCLUSION: To the best of our knowledge, our study identified a novel de novo variant of CLTC and provided the earliest clinical characteristic of the CLTC variant at the prenatal stage. The functional experiment suggested the variant caused the altering of the RNA splicing and the protein expression. We extended the mutational spectrum of CLTC and provided guidance on genetic counseling.

Expanding Our Knowledge of Molecular Pathogenesis in Histiocytoses: Solitary Soft Tissue Histiocytomas in Children With a Novel CLTC::SYK Fusion.

The histiocytoses comprise a histopathologically and clinically diverse group of disorders bearing recurrent genomic alterations, commonly involving the BRAF gene and mitogen-activated protein kinase pathway. In the current study, a novel CLTC :: SYK fusion in 3 cases of a histopathologically distinct histiocytic neoplasm arising as solitary soft tissue lesions in children identified by next-generation sequencing and fluorescence in situ hybridization is described. Morphologically, all 3 neoplasms were composed of sheets of cells with round-oval nuclei and vacuolated eosinophilic cytoplasm but, in contrast to classic juvenile xanthogranuloma (JXG), Touton giant cells were absent. A separate cohort of classic JXG cases subsequently profiled by fluorescence in situ hybridization were negative for the presence of a CLTC::SYK fusion suggesting that CLTC::SYK fusion-positive histiocytoma is genetically and histologically distinct from JXG. We postulate that the CLTC::SYK fusion leads to aberrant activation of the SYK kinase, which is involved in variable pathways, including mitogen-activated protein kinase. The identification of a novel CLTC::SYK fusion may pave the way for the development of targeted therapeutic options for aggressive disease.

Molecular investigation of ALK-rearranged epithelioid fibrous histiocytomas identifies CLTC as a novel fusion partner and evidence of fusion-independent transcription activation.

Epithelioid fibrous histiocytoma (EFH) is a rare cutaneous neoplasm, which is characterized by the presence of rearrangements involving the ALK gene. Although EFH was long considered a variant of fibrous histiocytoma, the identification of its unique genetic signature confirmed that it represents a distinct entity. The aim of the present study was to examine a cohort of ALK-immunoreactive EFH cases to further characterize gene fusion partners. Next generation sequencing detected ALK fusions in 11 EFH cases identified in the pathology archives of two different institutions. The most common fusion partner was DCTN1 (N = 4) followed by CLTC (N = 2) and VCL (N = 2), while the remaining cases harbored fusions involving SPECC1L, PPFIBP1, and PRKAR1A. In one case no fusion was detected by NGS and FISH despite suitable sample quality. Notably, IHC demonstrated positive ALK expression and the level of aligned ALK reads was comparable to the fusion-positive cases. To the best of our knowledge, this is the first report of CLTC as a fusion partner in EFH. The two CLTC rearranged cases in our cohort also represent the first two EFH cases in the literature that involve exon 19 of ALK, instead of exon 20. These findings underscore the remarkable plasticity of ALK as an oncogenic driver and further expand the list of its potential fusion partners in EFH. Lastly this is also the first report of ALK-immunoreactive EFH with no underlying fusion suggesting a fusion independent transcription mechanism as seen in other tumors.

Novel CLTC variants cause new brain and kidney phenotypes.

Heterozygous variants in CLTC, which encode the clathrin heavy chain protein, cause neurodevelopmental delay of varying severity, and often accompanied by dysmorphic features, seizures, hypotonia, and ataxia. To date, 28 affected individuals with CLTC variants have been reported, although their phenotypes have not been fully elucidated. Here, we report three novel de novo CLTC (NM_001288653.1) variants in three individuals with previously unreported clinical symptoms: c.3662_3664del:p.(Leu1221del) in individual 1, c.2878T>C:p.(Trp960Arg) in individual 2, and c.2430+1G>T:p.(Glu769_Lys810del) in individual 3. Consistent with previous reports, individuals with missense or small in-frame variants were more severely affected. Unreported symptoms included a brain defect (cystic lesions along the lateral ventricles of the brain in individuals 1 and 3), kidney findings (high-echogenic kidneys in individual 1 and agenesis of the left kidney and right vesicoureteral reflux in individual 3), respiratory abnormality (recurrent pneumonia in individual 1), and abnormal hematological findings (anemia in individual 1 and pancytopenia in individual 3). Of note, individual 1 even exhibited prenatal abnormality (fetal growth restriction, cystic brain lesions, high-echogenic kidneys, and a heart defect), suggesting that CLTC variants should be considered when abnormal prenatal findings in multiple organs are detected.

Deregulation of CLTC interacts with TFG, facilitating osteosarcoma via the TGF-beta and AKT/mTOR signaling pathways.

Although the treatment of osteosarcoma has improved, the overall survival rate of this common type of osseous malignancies has not changed for four decades. Thus, new targets for better therapeutic regimens are urgently needed. In this study, we found that high expression of clathrin heavy chain (CLTC) was an independent prognostic factor for tumor-free survival (HzR, 3.049; 95% CI, 1.476-6.301) and overall survival (HzR, 2.469; 95% CI, 1.005-6.067) of patients with osteosarcoma. Down-regulation of CLTC resulted in tumor-suppressive effects in vitro and in vivo. Moreover, we found that CLTC was transcriptionally regulated by a transcription factor-specificity protein 1 (SP1), which binds to the CLTC promoter at the -320 to -314-nt and +167 to +173-nt loci. Mechanistic investigations further revealed that CLTC elicited its pro-tumor effects by directly binding to and stabilizing trafficking from the endoplasmic reticulum to the Golgi regulator (TFG). Importantly, overexpression of TFG rescued both the tumor-suppressive effect and inhibition of the TGF-ß and AKT/mTOR pathways caused by CLTC down-regulation, which indicated that the activity of CLTC was TFG-dependent. Immunohistochemistry analysis confirmed that CLTC expression was positively correlated with TFG expression. These findings collectively highlight CLTC as a new prognostic biomarker for patients with osteosarcoma, and the interruption of the SP1/CLTC/TFG axis may serve as a novel therapeutic strategy for osteosarcoma.

Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network.

G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.

Circ_0074027 contributes to the progression of non-small cell lung cancer via microRNA-362-3p/clathrin heavy chain axis.

Circular RNAs are involved in the occurrence and development of different types of cancers. We aimed to illustrate the expression profile and mechanism of circ_0074027 in non-small cell lung cancer (NSCLC). Quantitative real-time PCR was employed to detect the expression of circ_0074027, paired like homeodomain 1 (PITX1) mRNA (mPITX1) and microRNA-362-3p (miR-362-3p). Western blot assay was utilized to measure the levels of clathrin heavy chain (CLTC), cyclin D1, BCL2-associated X, apoptosis regulator Bax (Bax), vimentin and matrix metallopeptidase 9. The clonogenicity, apoptosis and metastasis of NSCLC cells were examined by colony formation assay, flow cytometry and transwell migration and invasion assays. The target relationship between miR-362-3p and circ_0074027 or CLTC was predicted by starBase website and was validated by dual-luciferase reporter assay. Murine xenograft assay was applied to explore the function of circ_0074027 in vivo. We found that The enrichment of circ_0074027 and CLTC protein was elevated, and a significant reduction in the expression of miR-362-3p was observed in NSCLC tissues and cells relative to adjacent normal tissues and human bronchial epithelial cells 16HBE. Circ_0074027 possessed a stable circular structure. Circ_0074027 and CLTC could accelerate the colony formation and metastasis and suppress the apoptosis of NSCLC cells. Circ_0074027/miR-362-3p/CLTC axis was first found to regulate the malignance of NSCLC cells. The biological influence caused by circ_0074027 depletion on NSCLC cells was alleviated by the accumulation of CLTC. Circ_0074027 acted as an oncogene to promote the growth of NSCLC tumors in vivo. In conclusion, Circ_0074027 contributed to the progression of NSCLC through promoting the proliferation and motility while hampering the apoptosis of NSCLC cells via miR-362-3p/CLTC axis.

A Tale of 2 Morphologies: Diagnostic Pitfalls in TFEB-Associated Renal Cell Carcinomas, Including a Novel NEAT1-TFEB Fusion.

AIMS: Translocation-associated renal cell carcinomas (RCCs) have been extensively subcharacterized in recent years, such that each is largely recognized by the 2016 World Health Organization as categorical neoplastic entities in the genitourinary tract. Those belonging to the t(6;11) family of tumors classically have a fusion between TFEB and MALAT1/α, and display a particular histomorphology. Specifically, they show a biphasic population of both small and large epithelioid cells, the smaller component of which surrounds basement membrane-type material. Despite this apt description, the tumors have variable morphology and mimic other RCCs including those with TFE3 translocations. Therefore, a high degree of suspicion is required to make the correct diagnosis. METHODS: The 2 cases described in this article were of strikingly different appearance, and initially considered consistent with other non-translocation-associated renal tumors. These included clear cell RCC (CCRCC), perivascular epithelioid cell tumor (PEComa), and other eosinophilic RCCs (mainly papillary RCC type 2). RESULTS: Using RNA sequencing techniques, they were found to harbor distinct pathogenic rearrangements involving the TFEB gene, namely, fusions with CLTC and NEAT1 (the latter partnering heretofore never reported). CONCLUSIONS: These alterations manifested in 2 notably dissimilar lesions, underscoring the importance of including this family of carcinomas in the differential of any renal neoplasm that does not display immunophenotypic characteristics consistent with its morphology.

A novel CLTC-FOSB gene fusion in pseudomyogenic hemangioendothelioma of bone.

Pseudomyogenic hemangioendothelioma, an uncommon mesenchymal neoplasm composed of plump spindled and/or epithelioid endothelial cells, may present multicentrically and tends to locally recur but rarely metastasizes. Morphologic resemblance to epithelioid sarcoma and other spindle cell neoplasms may result in diagnostic confusion. Molecular characterization of pseudomyogenic hemangioendothelioma has revealed these neoplasms often harbor a rearrangement of the FOSB gene with SERPINE1 or ACTB as recurrent fusion gene partners. Herein, a case of a fibular pseudomyogenic hemangioendothelioma with minimal extension into the adjacent soft tissue arising in a 17 year-old male is presented. The neoplasm exhibited sheets of epithelioid cells with abundant eosinophilic cytoplasm and variably eccentric nuclei. RNA sequencing revealed a novel CLTC-FOSB fusion transcript that was subsequently confirmed by direct sequencing of reverse transcription-polymerase chain reaction products demonstrating an in-frame fusion between exon 17 of the clathrin heavy chain (CLTC) gene and exon 2 of the FOSB (FosB proto-oncogene, AP-1 transcription factor subunit) gene. CLTC-FOSB fusion has not been described in a neoplasm before.

Tatton-Brown-Rahman syndrome: Six individuals with novel features.

Tatton-Brown Rahman syndrome (TBRS) is an overgrowth-intellectual disability syndrome caused by heterozygous variants in DNMT3A. Seventy-eight individuals have been reported with a consistent phenotype of somatic overgrowth, mild to moderate intellectual disability, and similar dysmorphisms. We present six individuals with TBRS, including the youngest individual thus far reported, first individual to be diagnosed with tumor testing and two individuals with variants at the Arg882 domain, bringing the total number of reported cases to 82. Patients reported herein have additional clinical features not previously reported in TBRS. One patient had congenital diaphragmatic hernia. One patient carrying the recurrent p.Arg882His DNMT3A variant, who was previously reported as having a phenotype due to a truncating variant in the CLTC gene, developed a ganglioneuroblastoma at 18 months and T-cell lymphoblastic lymphoma at 6 years of age. Four patients manifested symptoms suggestive of autonomic dysfunction, including central sleep apnea, postural orthostatic hypotension, and episodic vasomotor instability in the extremities. We discuss the molecular and clinical findings in our patients with TBRS in context of existing literature.

Clathrin switches transforming growth factor-ß role to pro-tumorigenic in liver cancer.

BACKGROUND & AIMS: Upon ligand binding, tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), are recruited into clathrin-coated pits for internalization by endocytosis, which is relevant for signalling and/or receptor degradation. In liver cells, transforming growth factor-ß (TGF-ß) induces both pro- and anti-apoptotic signals; the latter are mediated by the EGFR pathway. Since EGFR mainly traffics via clathrin-coated vesicles, we aimed to analyse the potential role of clathrin in TGF-ß-induced signalling in liver cells and its relevance in liver cancer. METHODS: Real-Time PCR and immunohistochemistry were used to analyse clathrin heavy-chain expression in human (CLTC) and mice (Cltc) liver tumours. Transient knockdown (siRNA) or overexpression of CLTC were used to analyse its role on TGF-ß and EGFR signalling in vitro. Bioinformatic analysis was used to determine the effect of CLTC and TGFB1 expression on prognosis and overall survival in patients with hepatocellular carcinoma (HCC). RESULTS: Clathrin expression increased during liver tumorigenesis in humans and mice. CLTC knockdown cells responded to TGF-ß phosphorylating SMADs (canonical signalling) but showed impairment in the anti-apoptotic signals (EGFR transactivation). Experiments of loss or gain of function in HCC cells reveal an essential role for clathrin in inhibiting TGF-ß-induced apoptosis and upregulation of its pro-apoptotic target NOX4. Autocrine TGF-ß signalling in invasive HCC cells upregulates CLTC expression, switching its role to pro-tumorigenic. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CONCLUSIONS: This work describes a novel role for clathrin in liver tumorigenesis, favouring non-canonical pro-tumorigenic TGF-ß pathways. CLTC expression in human HCC samples could help select patients that would benefit from TGF-ß-targeted therapy. LAY SUMMARY: Clathrin heavy-chain expression increases during liver tumorigenesis in humans (CLTC) and mice (Cltc), altering the cellular response to TGF-ß in favour of anti-apoptotic/pro-tumorigenic signals. A positive correlation between TGFB1 and CLTC was found in HCC cells and patients. Patients expressing high levels of TGFB1 and CLTC had a worse prognosis and lower overall survival. CLTC expression in HCC human samples could help select patients that would benefit from therapies targeting TGF-ß.

Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4).

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Clathrin heavy chain phosphorylated at T606 plays a role in proper cell division.

Clathrin regulates mitotic progression, in addition to membrane trafficking. However, the detailed regulatory mechanisms of clathrin during mitosis remain elusive. Here, we demonstrate novel regulation of clathrin during mitotic phase of the cell cycle. Clathrin heavy chain (CHC) was phosphorylated at T606 by its association partner cyclin G-associated kinase (GAK). This phosphorylation was required for proper cell proliferation and tumor growth of cells implanted into nude mice. Immunofluorescence analysis showed that the localization of CHC-pT606 signals changed during mitosis. CHC-pT606 signals localized in the nucleus and at the centrosome during interphase, whereas CHC signals were mostly cytoplasmic. Co-immunoprecipitation suggested that CHC formed a complex with GAK and polo-like kinase 1 (PLK1). Depletion of GAK using siRNA induced metaphase arrest and aberrant localization of CHC-pT606, which abolished Kiz-pT379 (as a phosphorylation target of PLK1) signals on chromatin at metaphase. Taken together, we propose that the GAK_CHC-pT606_PLK1_Kiz-pT379 axis plays a role in proliferation of cancer cells.

Neurotransmitter trafficking defect in a patient with clathrin (CLTC) variation presenting with intellectual disability and early-onset parkinsonism.

INTRODUCTION: Clathrins play a key role in endocytosis, recycling, and trafficking as well as the generation of presynaptic vesicles. We report a new clinical condition associated with a de novo variant in the CLTC gene, which encodes the clathrin heavy polypeptide. CASE REPORT: This 30-year-old woman presented with a developmental disorder during childhood that progressed to mild cognitive decline in late childhood and relapsing-remitting hypokinetic-rigid syndrome with severe achalasia, weight loss, and mood disorder in adulthood. 123I-Ioflupane SPECT was normal. Blood phenylalanine was slightly increased and PAH sequencing revealed compound heterozygosity for two variants, p.[Asp151Glu]:[Thr380Met]. CSF examination unexpectedly detected a remarkable reduction of homovanillic, 5-hydroxyindolacetic, and 5-methylthetrahydrofolic acids, which could not be ascribed to any alteration of tetrahydrobiopterin and related biogenic amine pathways. METHODS: Trio-based exome sequencing was performed. RESULT: A de novo missense variant (c.2669C > T/p.Pro890Leu) was detected in CLTC. Treatment with biogenic amine precursors was ineffective, while the inhibitor of MAO-A selegiline resulted in persistent clinical improvement. CONCLUSIONS: We suggest CLTC defect as a new disorder of biogenic amine trafficking, resulting in neurodevelopmental derangement and movement disorder. Neurotransmitter depletion in CSF may be a biomarker of this disease, and selegiline a possible treatment option.

Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting.

BACKGROUND: Copy number variation (CNV) has been implicated in the genetics of multiple human diseases. Spinal muscular atrophy (SMA) and 22q11.2 deletion syndrome (22q11.2DS) are two of the most common diseases which are caused by DNA copy number variations. Genetic diagnostics for these conditions would be enhanced by more accurate and efficient methods to detect the relevant CNVs. METHODS: Competitive PCR with limited deoxynucleotide triphosphates (dNTPs) and high-resolution melting (HRM) analysis was used to detect 22q11.2DS, SMA and SMA carrier status. For SMA, we focused on the copy number of SMN1 gene. For 22q11.2DS, we analyzed CNV for 3 genes (CLTCL1, KLHL22, and PI4KA) which are located between different region-specific low copy repeats. CFTR was used as internal reference gene for all targets. Short PCR products with separated Tms were designed by uMelt software. RESULTS: One hundred three clinical patient samples were pretested for possible SMN1 CNV, including carrier status, using multiplex ligation-dependent probe amplification (MLPA) commercial kit as gold standard. Ninety-nine samples consisting of 56 wild-type and 43 22q11.2DS samples were analyzed for CLTCL1, KLHL22, and PI4KA CNV also using MLPA. These samples were blinded and re-analyzed for the same CNVs using the limited dNTPs PCR with HRM analysis and the results were completely consistent with MLPA. CONCLUSIONS: Limited dNTPs PCR with HRM analysis is an accurate method for detecting SMN1 and 22q11.2 CNVs. This method can be used quickly, reliably, and economically in large population screening for these diseases.

Spindle Assembly Disruption and Cancer Cell Apoptosis with a CLTC-Binding Compound.

AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 µmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation. Mol Cancer Res; 16(9); 1361-72. ©2018 AACR.

Differential proteome of haemocyte subpopulations responded to white spot syndrome virus infection in Chinese shrimp Fenneropenaeus chinensis.

In our previous study, the differentially expressed proteins have been identified by proteomic analysis in total haemocytes of shrimp (Fenneropenaeus chinensis) after white spot syndrome virus (WSSV) infection. To further investigate the differential response of haemocyte subpopulations to WSSV infection, granulocytes and hyalinocytes were separated from healthy and WSSV-infected shrimp by immunomagnetic bead (IMB) method, respectively. Then two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to analyze the differentially expressed proteins in haemocyte subpopulations between healthy and WSSV-infected shrimp. The results of flow cytometry (FCM) showed that about 98% of granulocytes and about 96% of hyalinocytes in purity were obtained. Quantitative intensity analysis revealed that 26 protein spots in granulocytes and 24 spots in hyalinocytes were significantly changed post WSSV infection. Among them, 24 proteins in granulocytes and 23 proteins in hyalinocytes were identified by MS analysis, which could be divided into eight categories according to Gene Ontology. The identification of prophenoloxidase (proPO), proPO 2 and peroxiredoxin in WSSV-infected granulocytes was consistent with the facts that the proPO-activating system and peroxiredoxin were mainly existed in granulocytes. The phagocytosis of hyalinocytes seemed to be enhanced during the infection, because several proteins that involved in phagocytosis, including clathrin heavy chain, ADP ribosylation factor 4 and Alpha2 macroglobulin were up-regulated in hyalinocytes upon WSSV infection. Our results also reflected the vital biological significance of calcium ion binding proteins in granulocytes and ATPase/GTPase in hyalinocytes during WSSV infection. The data in this study verified the roles of granulocytes and hyalinocytes involved in WSSV infection, and differentially expressed proteins identified in granulocytes and hyalinocytes had a close correlation with their function characteristics.

Clathrin heavy chain 22 contributes to the control of neuropeptide degradation and secretion during neuronal development.

The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.

MiR-199a-5p suppresses tumorigenesis by targeting clathrin heavy chain in hepatocellular carcinoma.

The deregulation of microRNA (miRNA) is frequently associated with a variety of cancers, including hepatocellular carcinoma (HCC). In this study, we investigated the expression and possible role of miR-199a-5p in HCC. The expression of miR-199a-5p was measured by quantitative RT-PCR in HCC. The effect of miR-199a-5p was evaluated by cell viability and colony formation assays in HCC cell lines and tumor cell growth assay in xenograft nude mice. Quantitative real time PCR results showed that miR-199a-5p was down-regulated in 77.9 % (67/86) of HCC tissues compared with adjacent nontumor tissues. MiR-199a-5p mimic reduced cell viability and colony formation by induction of cell arrest in HCC cell lines and inhibited tumor cell growth in xenograft nude mice, but miR-199a-5p inhibitor increased cell viability and colony formation in HCC cell lines and tumor cell growth in xenograft nude mice. Furthermore, CLTC was defined as a potential direct target of miR-199a-5p by MiRanda and TargetScan predictions. The dual-luciferase reporter gene assay results showed that CLTC was a direct target of miR-199a-5p. The use of miR-199a-5p mimic or inhibitor could decrease or increase CLTC protein levels in HCC cell lines. We conclude that the frequently down-regulated miR-199a-5p can regulate CLTC and might function as a tumor suppressor in HCC. Therefore, miR-199a-5p may serve as a useful therapeutic agent for miRNA-based HCC therapy.

Reduced expression of miR­205­5p promotes apoptosis and inhibits proliferation and invasion in lung cancer A549 cells by upregulation of ZEB2 and downregulation of erbB3.

Previous studies have demonstrated that microRNA (miR)-205-5p expression is significantly increased in non­small cell lung cancer tissues and is associated with tumor differentiation grade. The aim of the present study was to explore the effects of miR­205­5p on viability, apoptosis and invasion of lung cancer A549 cells. The hsa­miR­205­5p small interfering RNA (siRNA) inhibitor was transfected into A549 cells and expression of miR­205­5p was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Cell viability, apoptosis and invasion were assayed by Cell Counting kit­8, Annexin V/propidium iodide double staining and Transwell assay, respectively. Target genes of miR­205­5p were predicted using bioinformatics analysis. Expression of mRNA and protein levels of candidate target genes following miR­205­5p inhibition were detected using RT­qPCR and western blot analysis respectively. The results demonstrated that relative survival rates of A549 cells were significantly inhibited in miR­205­5p siRNA­transfected cells at 24 and 48 h compared with control cells. Apoptosis was markedly increased in the miR­205­5p siRNA cells compared with control cells. The number of invaded cells following miR­205­5p siRNA silencing was significantly decreased compared with control cells. Bioinformatics analysis revealed that erb­B2 receptor kinase 3 (erbB3), zinc finger E­box binding homeobox 2 (ZEB2), clathrin heavy chain (CLTC) and mediator complex subunit 1 (MED1) may be potential target genes of miR­205­5p. Reduced expression of miR­205­5p significantly increased the expression of ZEB2 mRNA and protein, inhibited the expression of erbB3 protein, but had no significant effect on the expression levels of CLTC and MED1. In summary, reduced expression of miR­205­5p promoted apoptosis and inhibited proliferation and invasion in lung cancer A549 cells through upregulation of ZEB2 and downregulation of erbB3. The present results suggested that the increased miR­205­5p expression observed in non­small cell lung cancer tissues may contribute to increased proliferation and invasion of lung cancer cells and thus to cancer progression.