Retinal VEGF-A Overexpression Is Not Sufficient to Induce Lymphangiogenesis Regardless of VEGF-C Upregulation and Lyve1+ Macrophage Infiltration.

Purpose: No lymphatic vessels have been identified in the retina. This study investigated whether pathological VEGF-A-overexpressing diabetic retina causes lymphangiogenesis. Methods: Three genetic mouse models of diabetic retinopathy (DR) (Akita [Ins2+/-], Kimba [vegfa+/+], and Akimba [Akita × Kimba] mice) were used. Retinas were examined by fundus photography, fluorescence angiography (FA), and immunostaining to detect lymphangiogenesis or angiogenesis. Lyve1-GFP (Lyve1EGFP/Cre) mice were used to examine Lyve1-expressing cells by immunostaining. Lymphatic-related factors were investigated in mouse retina and vitreous fluid from proliferative diabetic retinopathy (PDR) patients by RT-PCR and ELISA, respectively. Aged Kimba and Akimba mice were used to examine the retinal phenotype at the late phase of VEGF overexpression. Results: FA and immunostaining showed retinal neovascularization in Kimba and Akimba mice but not wild-type and Akita mice. Immunohistochemistry showed that lymphangiogenesis was not present in the retinas of Akita, Kimba, or Akimba mice despite the significant upregulation of lymphatic-related factors (Lyve1, podoplanin, VEGF-A, VEGF-C, VEGF-D, VEGFR2, and VEGFR3) in the retinas of Kimba and Akimba mice by RT-PCR (P < 0.005). Furthermore, lymphangiogenesis was not present in aged Kimba or Akimba mice. Significantly increased numbers of Lyve1-positive cells present in the retinas of Kimba and Akimba mice, especially in the peripheral areas, were CD11b positive, indicating a macrophage population (P < 0.005). VEGF-C in PDR vitreous with vitreous hemorrhage (VH) was higher than in PDR without VH or a macular hole. Conclusions: Retinal VEGF-A overexpression did not cause typical lymphangiogenesis despite upregulated lymphatic-related factors and significant Lyve1-positive macrophage infiltration.

Morphine-induced kinase activation and localization in the periaqueductal gray of male and female mice.

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.

Quantitative acetylome and phosphorylome analysis reveals Girdin affects pancreatic cancer progression through regulating Cortactin.

The actin-binding protein Girdin is involved in a variety of cellular processes, including pancreatic cancer. The objective of this study is to explore the role and the mechanism of Girdin in pancreatic cancer by quantitative acetylome and phosphorylome analysis. We firstly found that Girdin was overexpressed in pancreatic cancer tissue and increased expression of Girdin was associated with tumor size and stage of patients with pancreatic cancer. We established the shRNA knockdown of Girdin in PANC-1 and Aspc-1 cells, and we found that shGirdin inhibited proliferation, migration and invasion, and promoted apoptosis. Subsequently, we identified and quantified 5,338 phosphorylated sites in 2,263 proteins that changed in response to Girdin knockdown, and identified a similar set of Girdin-responsive acetylome data as well. Additional data revealed that down-regulation of Girdin affected Cortactin phosphorylation and acetylation, suggesting Cortactin as an important regulatory target of Girdin. Moreover, we found that overexpression of Cortactin could rescue the effect of shGirdin on proliferation, apoptosism, migration and invasion of pancreatic cancer cells. In general, our results provided new insights into the mechanisms of Girdin function including cell proliferation, migration and invasion, and offer biomarker candidates for clinical evaluation of Girdin.

CAPS1 Suppresses Tumorigenesis in Cholangiocarcinoma.

BACKGROUND: CAPS1 (calcium-dependent activator protein for secretion) is a multi-domain protein involved in regulating exocytosis of synaptic vesicles and dense-core vesicles. However, the expression and function of CAPS1 in cholangiocarcinoma (CCA) remains unclear. In the present study, we explored the role of CAPS1 in CCA carcinogenesis. METHODS: CAPS1 expression was explored using western blotting and immunohistochemistry in four CCA cell lines and clinical samples from 90 cases of CCA. The clinical significance of CAPS1 was analyzed. The biological function of CAPS1 in CCA cells was detected in vitro and in vivo. The underlying mechanism of CAPS1 function was explored by detecting the expression of critical molecules in its associated signaling pathways. The mechanism of CAPS1 downregulation in tumor tissues was explored using in silico prediction and luciferase reporter assays. RESULTS: CAPS1 expression was reduced in CCA cell lines and human tumor tissues. Loss of CAPS1 in tumor tissues was closely associated with poor prognosis of patients with CCA. Moreover, CAPS1 expression correlated significantly with tumor-node-metastasis stage, lymph node metastasis, and vascular invasion. Lentivirus-mediated CAPS1 overexpression substantially prevented clone formation, cell proliferation, and cell cycle progression. CAPS1 overexpression also suppressed carcinogenesis in nude mice. Mechanistically, CAPS1 overexpression greatly accelerated the ERK and p38 MAPK signal pathways. In addition, microRNA miR-30e-5p negatively regulated CAPS1 expression. CONCLUSION: These data showed that CAPS1 functions as a tumor suppressor in CCA. Reduced CAPS1 expression could indicate poor prognosis of patients with CCA.

MAP­1B, PACS­2 and AHCYL1 are regulated by miR­34A/B/C and miR­449 in neuroplasticity following traumatic spinal cord injury in rats: Preliminary explorative results from microarray data.

Spinal cord injury (SCI) is a specific type of damage to the central nervous system causing temporary or permanent changes in its function. The present aimed to identify the genetic changes in neuroplasticity following SCI in rats. The GSE52763 microarray dataset, which included 15 samples [3 sham (1 week), 4 injury only (1 week), 4 injury only (3 weeks), 4 injury + treadmill (3 weeks)] was downloaded from the Gene Expression Omnibus database. An empirical Bayes linear regression model in limma package was used to identify the differentially expressed genes (DEGs) in injury vs. sham and treadmill vs. non­treadmill comparison groups. Subsequently, time series and enrichment analyses were performed using pheatmap and clusterProfile packages, respectively. Additionally, protein­protein interaction (PPI) and transcription factor (TF)­microRNA (miRNA)­target regulatory networks were constructed using Cytoscape software. In total, 159 and 105 DEGs were identified in injury vs. sham groups and treadmill vs. non­treadmill groups, respectively. There were 40 genes in cluster 1 that presented increased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and decreased expression levels in the injury + treadmill group compared with the injury only groups; conversely, 52 genes in cluster 2 exhibited decreased expression levels in the injury (1 week/3 weeks) groups compared with the sham group, and increased expression levels in the injury + treadmill group compared with the injury only groups. Enrichment analysis indicated that clusters 1 and 2 were associated with immune response and signal transduction, respectively. Furthermore, microtubule associated protein 1B, phosphofurin acidic cluster sorting protein 2 and adenosylhomocysteinase­like 1 exhibited the highest degrees in the regulatory network, and were regulated by miRNAs including miR­34A, miR­34B, miR­34C and miR­449. These miRNAs and their target genes may serve important roles in neuroplasticity following traumatic SCI in rats. Nevertheless, additional in­depth studies are required to confirm these data.

p120 inhibits LPS/TNFα-induced endothelial Ang2 synthesis and release in an NF-κB independent fashion.

Adherens junction protein p120 is thought to be crucial for maintaining vascular integrity, which is important in many pathologies and diseases including atherosclerosis, vascular malformations, hemorrhagic stroke, sepsis and others. However, the mechanisms responsible for this is not completely understood. In this study, using an unbiased proteomics approach, followed by other experimental techniques, we identified that in HUVECs p120 overexpression inhibits LPS/TNFα-induced angiopoietin-2 (Ang2) expression, a key switch of endothelial destabilization. Interestingly, p120 overexpression did not inhibit LPS/TNFα-induced expression of adhesion molecules/cytokines including VCAM-1, ICAM-1, E-selectin, MCP-1, IL-8 and IL-6 in our experimental system. Furthermore, this p120-mediated repression of Ang2 is in an NF-κB independent manner, possibly via transcription factor Ets1. Our results demonstrate that p120 influences vascular integrity by secreted signals, providing new insights into the mechanisms of p120-mediated vascular stability.

The expression of Eps15 homology domain 1 is negatively correlated with disease-free survival and overall survival of osteosarcoma patients.

BACKGROUND: Osteosarcoma was locally aggressive and frequently metastasizes to the lung. However, the etiology of osteosarcoma was unknown. Thus, exploring the mechanisms behind the occurrence of osteosarcoma was important for its prediction and prevention. To investigate the usefulness of mammalian Eps15 homology domain 1 (EHD1) as a prognostic marker for osteosarcoma, the expression of EHD1 in 57 osteosarcoma patients was measured using immunohistochemistry techniques and correlated with the clinicopathological features of patients. METHODS: Correlations of EHD1 expression levels with clinicopathological features of patients were assessed using the Pearson χ2 test for categorical variables and the Student t test for continuous variables. Cumulative disease-free survival (DFS) curves and overall survival (OS) curves were plotted using the Kaplan-Meier method, and the relationship between each of the variables and survival was assessed by log-rank tests using univariate analysis. Subsequently, the parameters were tested using the multivariate Cox proportional hazards model, which was used to identify independent variables for predicting survival. EHD1 expression [P = 0.020; HR, 5.582; 95% confidence intervals (CI), 1.314-23.72] was an independent prognostic indicator of DFS in osteosarcoma patients; tumor size and EHD1 expression of osteosarcomas were independent prognostic indicators of OS in osteosarcoma patients. RESULTS: EHD1 protein expression was a positive expression in examined tumor tissues. The median OS time of patients with high expression of EHD1 was 46.8 months (95% CI, 29.8-63.8 months), and the median OS time of patients with low expression of EHD1 was 58.8 months (95% CI, 31.6-86.0 months). The prognosis for patients with low expression of EHD1 in osteosarcomas was significantly better than that for patients with high expression of EHD1 (log-rank test, P = 0.019). CONCLUSION: The expression of EHD1 was negatively correlated with DFS and OS of osteosarcoma patients; therefore, the expression of EHD1 is a prognostic marker for prediction and prevention of osteosarcomas.

Circular RNA circVAPA is up-regulated and exerts oncogenic properties by sponging miR-101 in colorectal cancer.

Circular RNAs (circRNAs) are a novel class of non-coding RNAs with distinct properties and diverse physiological and pathological functions. However, the functions of circRNAs in colorectal cancer (CRC) remain elusive. This study aimed to investigate the functional roles of circVAPA in CRC. High-throughput RNA sequencing was performed in 4 paired CRC tissues, and circVAPA (hsa_circ_0006990), was identified as a potential functional circRNA. Using quantitative real-time polymerase chain reaction (qRT-PCR), circVAPA was found to be up-regulated in CRC patients' tissues and plasma. Furthermore, circVAPA level was associated with unfavorable clinicopathologic features in CRC. The area under curve (AUC) of ROC was 0.724, suggesting that plasma level of circVAPA could serve as a promising biomarker for CRC detection. Sanger sequencing confirmed the back-splice junction sequences of circVAPA. Actinomycin D and RNase R treatments suggested that circVAPA was highly stable compared with its linear counterpart, and qRT-PCR for the circVAPA level in nuclear and cytoplasmic fractions indicated that circVAPA was predominantly localized in the cytoplasm. Gain-of-function and loss-of-function studies in CRC cell lines indicated that circVAPA could promote CRC cell proliferation, migration, invasion, and inhibit apoptosis. miRanda software (v3.3a) was used to predict target miRNAs of circVAPA. Moreover, target miRNAs associated with the KEGG pathway of COLORECTAL CANCER (Entry: map05210; https://www.kegg.jp/) were screened using DIANA-miRPath v.3 platform (Reverse Search module; TarBase v7.0 method). The analyses by miRanda and miRPath suggested that circVAPA could potentially bind to hsa-miR-101-3p (miR-101) associated with the COLORECTAL CANCER pathway. Luciferase reporter assay confirmed a direct interaction between circVAPA and miR-101. Furthermore, circVAPA had no effect on the expression level of miR-101, and miR-101 over-expression had the similar tumor-suppressing effects as circVAPA silencing. The tumor-promoting effect of circVAPA over-expression could be reversed by the up-regulation of miR-101. These data demonstrated that circVAPA promoted CRC progression by sponging miR-101. In conclusion, we have verified that circVAPA is up-regulated in CRC patients' tissues and plasma, and exerts oncogenic properties by sponging miR-101 in CRC. CircVAPA could serve as a promising biomarker and a therapeutic target for CRC.

Muscle LIM Protein Is Expressed in the Injured Adult CNS and Promotes Axon Regeneration.

Muscle LIM protein (MLP) has long been regarded as a muscle-specific protein. Here, we report that MLP expression is induced in adult rat retinal ganglion cells (RGCs) upon axotomy, and its expression is correlated with their ability to regenerate injured axons. Specific knockdown of MLP in RGCs compromises axon regeneration, while overexpression in vivo facilitates optic nerve regeneration and regrowth of sensory neurons without affecting neuronal survival. MLP accumulates in the cell body, the nucleus, and in axonal growth cones, which are significantly enlarged by its overexpression. Only the MLP fraction in growth cones is relevant for promoting axon extension. Additional data suggest that MLP acts as an actin cross-linker, thereby facilitating filopodia formation and increasing growth cone motility. Thus, MLP-mediated effects on actin could become a therapeutic strategy for promoting nerve repair.

MiR-144-3p inhibits the proliferation and metastasis of pediatric Wilms' tumor cells by regulating Girdin.

OBJECTIVE: The aim of this study was to investigate the role of miR-144-3p in the proliferation and metastasis capacity of pediatric Wilms' tumor (WT) cells and to explore the underlying mechanism. PATIENTS AND METHODS: The quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was performed to measure the expression level of miR-144-3p in pediatric WT tissues and cell lines (G401). A bioinformatics software was utilized to predict the interaction between miR-144-3p and Girdin. Subsequently, the interaction was further verified by dual luciferase reporter (DLR) gene assay and Western blot. The proliferation and colony formation ability of G401 cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and colony formation assay, respectively. Finally, the effect of miR-144-3p on cell invasion and migration was analyzed by transwell assay. RESULTS: In the current study, we found that the expression level of miR-144-3p was significantly reduced in pediatric WT tissues and cells, whereas Girdin expression was upregulated. On-line target gene prediction software was applied to screen Girdin, which was considered as a downstream target gene of miR-144-3p. The interaction between miR-144-3p and Girdin was further verified by dual Luciferase reporter gene assay and Western blot. Subsequent experiments demonstrated that the proliferation and metastasis ability of cells was remarkably suppressed after up-regulating the expression of miR-144-3p. However, an addition of Girdin could reverse the effect of miR-144-3p. CONCLUSIONS: MiR-144-3p, which was up-regulated in pediatric WT, might inhibit the proliferation and metastasis of the cells by directly targeting Girdin. This further indicated that miR-144-3p could be a potential therapeutic target for the treatment of pediatric WT.

Lymphangiogenesis and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Tumor lymphangiogenesis plays a key role in lymph node (LN) metastasis in oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and their relationship to nodal metastasis and other clinicopathological variables. PATIENTS AND METHODS: Podoplanin and LYVE-1 expression of the primary tumor and normal tissue were investigated by means of a quantitative real-time PCR assay and immunohistochemistry in samples from 33 cases of OSCC. RESULTS: The mRNA high expression levels of both genes had a statistically significantly higher rate of LN metastasis (p<0.01) and histological grade (p<0.01 for podoplanin, p<0.05 for LYVE-1). High expression of each gene, as shown by immunohistochemistry, had a statistically significant higher rate of LN metastasis (p<0.01 for podoplanin, p<0.05 for LYVE-1). CONCLUSION: Podoplanin and LYVE-1 were strongly associated with LN metastasis.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

Combination of cytoplasmic and nuclear girdin expression is an independent prognosis factor of breast cancer.

Girdin is an actin-binding protein playing key roles in the development of various carcinomas. Although online tools have predicted nuclear localization of girdin with a high probability, convincing proof has rarely been provided until now. The purpose of this study was to discover girdin's precise subcellular distribution and the potential prognostic value corresponding to its localization. The subcellular distribution of girdin was detected in a human breast cancer cell line and in >800 samples of human breast tissue by clinical pathologic analysis. In this study, we discovered for the first time that girdin could attach to chromatin and interact with topoisomerase-IIα in nucleus. Cytoplasmic and nuclear girdin exhibited different roles in prognosis of breast cancer: cytoplasmic girdin expression was an independent prognostic factor for progression-free survival (PFS), whereas nuclear girdin expression was an independent prognostic factor for overall survival (OS). More important, combination cytoplasmic and nuclear girdin was an independent prognosis factor of both OS and PFS. In conclusion, our research results strongly recommend combination analysis of cytoplasmic and nuclear girdin for a precise prognostic prediction in breast cancer.-Zhang, H., Yu, F., Qin, F., Shao, Y., Chong, W., Guo, Z., Liu, X., Fu, L., Gu, F., Ma, Y. Combination of cytoplasmic and nuclear girdin expression is an independent prognosis factor of breast cancer.

Cervical cancer cell-derived angiopoietins promote tumor progression.

Metastatic or recurrent cervical cancer has limited treatment options and a high rate of mortality. Although anti-vascular endothelial growth factor drugs have shown great promise as a therapeutic target for treatment of advanced cervical cancer, drug resistance and class-specific side effects negate long-term benefits. The identification of alternative anti-angiogenic factors will be critical for future drug development for advanced or recurrent cervical cancer. In this study, we found that angiopoietins and Tie receptors were highly expressed in cervical cancer cells. Tie-2 expression in tumor cells predicted poorer prognosis. Wound closure assay and Transwell assay showed that upregulated or downregulated Ang-1 and Ang-2 expression promoted or reduced cervical cancer cell lines migration and invasion, respectively. In subcutaneous xenograft models of cervical cancer, downregulation of Ang-1 and Ang-2 attenuated tumor growth. The expression of vimentin and endomucin and microvessel density were all significantly decreased in the siAng-1 group and siAng-2 group relative to the infection control group. Our data support that dual inhibition of Ang-1 and Ang-2 may be an alternative target for anti-angiogenic adjuvant therapy in advanced or recurrent cervical squamous cell cancer.

All-trans retinoic acid suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the second common cancer in Henan province and is well-known for aggressiveness and dismal prognosis. Adjuvant therapies, chemotherapy, radiotherapy and endoscopic treatment have not improved survival rates in patients with late stage esophageal carcinoma. All-trans retinoic acid (ATRA) is the active ingredient of Vitamin A and affects a wide spectrum of biological processes including development, growth, neural function, immune function, reproduction, and vision. It is one of the most potent therapeutic agents used for treating cancers, especially lung adenocarcinomas. ATRA inhibits metastatic potential and angiogenesis in several tumor models. We investigated the effects of ATRA on the expression of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2) and receptor Tie-2 in EC1 cells in vitro. We also assessed the growth and migration of EC1 cells in vitro. ATRA treatment caused 29.5% and 40.3% reduction of the growth of EC1 cells after 24 hours and 48 hours, relative to the control. ATRA plus fluorouracil treatment reduced the viability more strongly than either drug alone, indicating an additive effect. Moreover, ATRA decreased EC1 migration by 87%. Furthermore, ATRA treatment led to a marked decrease of the transcript levels of Ang-1, Ang-2, Tie-2, VEGF, and VEGF receptors, as assessed by real-time RT-PCR. Importantly, the protein levels of Ang-1, Ang-2 and Tie-2 were reduced by ATRA treatment. In vivo, we found ATRA treatment suppressed the tumor growth and improved the cachexia of mice. Importantly, ATRA treatment decreased the expression of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors of EC1 cells. Collectively, our findings demonstrate that ATRA exhibits a dose- and temporal-dependent effect on the metastatic behavior, suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and the progression of xenograft tumors of EC1 cells.

Local Adaptation of Sun-Exposure-Dependent Gene Expression Regulation in Human Skin.

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual's gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

MicroRNA-21 promotes proliferation, migration, and invasion of colorectal cancer, and tumor growth associated with down-regulation of sec23a expression.

BACKGROUND: MicroRNA-21 (miR-21) is up-regulated in many cancers, including colorectal cancer (CRC). Nevertheless, the function of miR-21 in CRC and the mechanism underlying that function is still unclear. METHODS: After analyzing the expression of miR-21 and Sec23A in CRC cell lines, we transfected the highest miR-21 expressing cell line, SW-480, with a plasmid containing an miR-21 inhibitor and the lowest miR-21 expressing cell line, DLD-1, with a plasmid containing an miR-21 mimic and measured the effects on the expression of Sec23A and on cell proliferation, migration, and invasion. We also evaluated the effect of knocking down Sec23A on miR-21 expression and its effects on cell proliferation, migration, and invasion. Finally, we assessed the effect of miR-21 in a xenograft tumor model in mice. Tumor tissues from these mice were subjected to immunohistochemical staining to detect the expression of Sec23A. RESULTS: Genetic deletion of miR-21 suppressed the proliferation, migration, and invasion of SW-480 cells, while over-expression of miR-21 promoted proliferation, migration, and invasion of DLD-1 cells. Inhibition of miR-21 increased the expression of Sec23A protein in SW-480 cells while over-expression of miR-21 significantly suppressed the expression of Sec23A protein and Sec23A mRNA in DLD-1 cells. Knockdown of Sec23A increased the expression of miR-21 in SW480 and DLD-1 cells and their proliferation (DLD-1 only), migration, and invasion. Over-expression of miR-21 promoted tumor growth in BALB/c nude mice and suppressed tumor expression of Sec23A. CONCLUSION: These findings provide novel insight into the molecular functions of miR-21 in CRC, which may serve as a potential interesting target.

Role of ATG10 expression quantitative trait loci in non-small cell lung cancer survival.

The aim of this article was to evaluate whether genetic variants in autophagy-related genes affect the overall survival (OS) of non-small cell lung cancer (NSCLC) patients. We analyzed 14 single nucleotide polymorphisms (SNPs) in core autophagy-related genes for OS in 1,001 NSCLC patients. Three promising SNPs in ATG10 were subsequently annotated by the expression quantitative trait loci (eQTL) and methylation quantitative trait loci (meQTL) analyses based on Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. We observed that the variants of rs10514231, rs1864182 and rs1864183 were associated with poor lung cancer survival (HR = 1.33, 95% CI = 1.07-1.65; HR = 1.43, 95% CI = 1.13-1.81; HR = 1.38, 95% CI = 1.14-1.68, respectively) and positively correlated with ATG10 expression (all p < 0.05) from GTEx and TCGA datasets. The elevated expression of ATG10 may predict shorter survival time in lung cancer patients in TCGA dataset (HR = 2.10, 95% CI = 1.33-3.29). Moreover, the variants of rs10514231 and rs1864182 were associated with the increased methylation levels of cg17942617 (meQTL), which in turn contributed to the elevated ATG10 expression and decreased survival time. Further functional assays revealed that ATG10 facilitated lung cancer cell proliferation and migration. Our findings suggest that eQTL/meQTL variations of ATG10 could influence lung cancer survival through regulating ATG10 expression.

Characterization of α-taxilin as a novel factor controlling the release of hepatitis C virus.

Although it is well established that the release of HCV (hepatitis C virus) occurs through the secretory pathway, many aspects concerning the control of this process are not yet fully understood. α-Taxilin was identified as a novel binding partner of syntaxin-4 and of other members of the syntaxin family, which are part of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and so are involved in intracellular vesicle traffic. Since α-taxilin prevents t-SNARE (target SNARE) formation by binding exclusively to free syntaxin-4, it exerts an inhibitory effect on the vesicular transport. HCV-replicating Huh7.5 cells and HCV-infected primary human hepatocytes and liver samples of patients suffering from chronic HCV contain significantly less α-taxilin compared with the controls. HCV impairs the expression of α-taxilin via NS5A-dependent interruption of the Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signal transduction cascade. Moreover, the half-life of α-taxilin is significantly reduced in HCV-replicating cells. Whereas modulation of α-taxilin expression does not significantly affect genome replication, the overexpression of α-taxilin prevents the release of HCV. In contrast with this, silencing of α-taxilin expression leads to increased release of infectious viral particles. This is due to the negative effect of α-taxilin on t-SNARE formation that leads to impaired vesicular trafficking. Accordingly, overexpression of the t-SNARE component syntaxin-4 increases release of HCV, whereas silencing leads to an impaired release. These data identify α-taxilin as a novel factor that controls the release of HCV and reveal the mechanism by which HCV controls the activity of α-taxilin.

Increased Expression of Serglycin in Specific Carcinomas and Aggressive Cancer Cell Lines.

In the present pilot study, we examined the presence of serglycin in lung, breast, prostate, and colon cancer and evaluated its expression in cell lines and tissues. We found that serglycin was expressed and constitutively secreted in culture medium in high levels in more aggressive cancer cells. It is worth noticing that aggressive cancer cells that harbor KRAS or EGFR mutations secreted serglycin constitutively in elevated levels. Furthermore, we detected the transcription of an alternative splice variant of serglycin lacking exon 2 in specific cell lines. In a limited number of tissue samples analyzed, serglycin was detected in normal epithelium but was also expressed in higher levels in advanced grade tumors as shown by immunohistochemistry. Serglycin staining was diffuse, granular, and mainly cytoplasmic. In some cancer cells serglycin also exhibited membrane and/or nuclear immunolocalization. Interestingly, the stromal cells of the reactive tumor stroma were positive for serglycin, suggesting an enhanced biosynthesis for this proteoglycan in activated tumor microenvironment. Our study investigated for first time the distribution of serglycin in normal epithelial and cancerous lesions in most common cancer types. The elevated levels of serglycin in aggressive cancer and stromal cells may suggest a key role for serglycin in disease progression.