Invasion-related Genes Research Articles

Silencing of LINC00461 enhances radiosensitivity of lung adenocarcinoma cells by down-regulating HOXA10 via microRNA-195.

Lung adenocarcinoma is recognized as one of the most recurrent tumours in adults. Long non‐coding RNAs (lncRNAs) are non–protein‐coding transcripts and have been demonstrated to regulate biological functions during tumorigenesis. Our study aims to investigate the underlying molecular mechanisms of LINC00461/microRNA‐195 (miR‐195)/HOXA10 responsible for its involvement in lung adenocarcinoma. We firstly selected differentially expressed lncRNAs and genes by the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO). The functional role of LINC00461 in lung adenocarcinoma was then determined using ectopic expression, knockdown and reporter assay experiments. Besides, we detected the expression profiles of LINC00461, miR‐195, HOXA10 and apoptosis‐ and invasion‐related genes. Cell proliferation, migration and invasion were evaluated. In vivo tumour formation ability was analysed. Overexpressed LINC00461 and HOXA10 but down‐regulated miR‐195 were observed in primary and metastatic lung adenocarcinoma. LINC00461 negatively regulated miR‐195, while miR‐195 negatively regulated HOXA10. Forced LINC00461 expression decreased expression of miR‐195 and Bax, increased expression of HOXA10, MMP‐2, MMP‐9 and Bcl‐2, promoted cell proliferation, migration and invasion as well as tumour formation, and enhanced radiosensitivity of lung adenocarcinoma cells. However, these effects were reversed by lentivirus‐mediated miR‐195–forced expression, thereby suggesting that miR‐195 could antagonize the harmful effect of LINC00461 on lung adenocarcinoma cells. Collectively, the present study provides evidence supporting the inhibitory effect of LINC00461 silencing on lung adenocarcinoma, which suppresses lung adenocarcinoma cell migration, invasion and radiosensitivity via HOXA10 by binding to miR‐195, which provides a promising basis for the targeted intervention treatment for human lung adenocarcinoma.

Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level

The central role that erythrocyte invasion plays in Plasmodium falciparum survival and reproduction makes this process an attractive target for therapeutic or vaccine development. However, multiple invasion-related genes with complementary and overlapping functions afford the parasite the plasticity to vary ligands used for invasion, leading to phenotypic variation and immune evasion. Overcoming the challenge posed by redundant ligands requires a deeper understanding of conditions that select for variant phenotypes and the molecular mediators. While host factors including receptor heterogeneity and acquired immune responses may drive parasite phenotypic variation, we have previously shown that host-independent changes in invasion phenotype can be achieved by continuous culturing of the W2mef and Dd2 P. falciparum strains in moving suspension as opposed to static conditions. Here, we have used a highly biologically replicated whole transcriptome sequencing approach to identify the molecular signatures of variation associated with the phenotype switch. The data show increased expression of particular invasion-related genes in switched parasites, as well as a large number of genes encoding proteins that are either exported or form part of the export machinery. The genes with most markedly increased expression included members of the erythrocyte binding antigens (EBA), reticulocyte binding homologues (RH), surface associated interspersed proteins (SURFIN), exported protein family 1 (EPF1) and Plasmodium Helical Interspersed Sub-Telomeric (PHIST) gene families. The data indicate changes in expression of a repertoire of genes not previously associated with erythrocyte invasion phenotypes, suggesting the possibility that moving suspension culture may also select for other traits.

Cinobufacini Inhibits Colon Cancer Invasion and Metastasis via Suppressing Wnt/β-Catenin Signaling Pathway and EMT.

Cinobufacini is a well-known Chinese medicine extracted from Venenum Bufonis, also called Chan Su. It has been used clinically for various cancers, including colon cancer. However, the function of Cinobufacini on colon cancer invasion and metastasis, and its underlying molecular mechanism, is still not clear. In this study, we investigated the function and mechanism of Cinobufacini on colon cancer invasion and metastasis both in vitro and in vivo studies. Human colon cancer cells were cultured. CCK assay was used to detect the effect of Cinobufacini on colon cancer cells proliferation. The invasion and migration abilities were observed by transwell assays, and the expression of invasion and migration related genes MMP2, MMP9, and epithelial-to-mesenchymal transition (EMT) relate genes were observed by Western blot assays. An orthotopic xenograft model in nude mice was established using colon cancer HCT116 cells, and the function of Cinobufacini on colon cancer invasion and metastasis were observed in vivo. We found Cinobufacini significantly inhibited colon cancer cell proliferation in a dose/time-dependent manner; the invasion and migration abilities of colon cancer were decreased after treated with Cinobufacini. The metastasis and EMT related genes MMP9, MMP2, N-cadherin and Snail were obviously down-regulated, while the expression of E-cadherin was up-regulated after treatment with Cinobufacini. The Wnt/-catenin signaling pathway related genes were observed using WB,and results show that the expression of -catenin, wnt3a, c-myc, cyclin D1, and MMP7 were all down-regulated after being treated with cinobufacini, while the expression of APC was up-regulated. In vivo studies of the volume and weight of orthotopic xenograft tumors showed significantly shrinkage in the Cinobufacini group compared to the control group. The enterocoelia and liver metastasis tumors were significantly decreased, and the expression of MMP9, MMP2, and -catenin were also down-regulated, while E-cadherin was up-regulated in vivo after the treatment with Cinobufacini. Our data proves that Cinobufacini can inhibit colon cancer invasion and metastasis both in vitro and in vivo; the mechanism is related by suppressing the Wnt/-catenin signaling pathway and then inhibiting the EMT of CRC.

ANGI-02. GENOME-WIDE shRNA SCREEN IDENTIFIES CANDIDATE GENES DRIVING GLIOBLASTOMA INVASION

Abstract BACKGROUND A major hallmark of glioblastoma (GBM) is its invasive capacity, contributing to its aggressive behaviour. Invasive cells cannot be easily removed by surgery or irradiation and eventually result in lethal recurrence. A better understanding of the invasion process and the key molecular players underlying the invasive potential of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM cultures. The most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cells was compared with overexpression in non-invasive cells. RNAseq of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS A zinc-finger domain containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong impact on invasion in highly invasive GBM cells. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of one or both zinc-finger motifs decreased invasion indicating that both are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent novel promising targets to overcome invasive capacities in GBM.

ANGI-08. AN ANNEXIN A2-REGULATED PHENOTYPIC SHIFT IN GLIOMA

Abstract BACKGROUND Malignant glioma has a poor prognosis and is characterized by excessive proliferation, invasion, and angiogenesis. Previously, we established subclones of glioma cell lines with different invasive phenotypes to investigate the mechanisms of invasion in this malignancy. That study revealed annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we investigated the molecular mechanism of the phenotypic shift in glioma that is induced by ANXA2. METHODS We identified ANXA2 target genes using microarray analyses of our four established cell lines and RNA-seq data from the IVY Glioblastoma Atlas Project. Co-expression was then confirmed in human glioma cells. Additionally, qRT-PCR, immunoblotting, invasion assays, proliferation assays, and tube formation assays were performed to uncover the specific roles of these angiogenic invasion-related genes. Furthermore, we examined survival times and phenotypic shifts in vivo. RESULTS We identified oncostatin M receptor (OSMR) as an ANXA2-target gene using microarray and RNA-seq, and confirmed its expression correlated with ANXA2 in glioma cells. ANXA2-overexpressing glioma cells showed enhanced OSMR expression and STAT3 phosphorylation, while STAT3 knockdown reduced OSMR expression. When ANXA2 was overexpressed in glioma cells, invasion, angiogenesis, proliferation, and epithelial-mesenchymal transition were promoted; however, silencing OSMR attenuated the ANXA2-induced phenotypic shift. In vivo, ANXA2-overexpressing glioma cells shortened the survival time of tumor-bearing mice, whereas OSMR knockdown increased survival times. CONCLUSIONS We analyzed genes whose expression was regulated by ANXA2 in glioma using invasive glioma models. Through this analysis, we identified that ANXA2 and OSMR regulate a phenotypic shift, suggesting that OSMR could be a promising target to treat and prevent glioma invasion.

P11.26 Genome-wide shRNA screen identifies candidate genes driving glioblastoma invasion

Abstract BACKGROUND A major hallmark of glioblastoma (GBM) is its highly invasive capacity, contributing to its aggressive behaviour. Since invasive cells cannot be easily removed by surgery or irradiation, they are left behind and eventually result in lethal recurrence. Therefore, a better understanding of the invasion process and of the key molecular players underlying the invasive capacities of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM sphere cultures. The phenotype of the most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cell lines was compared with overexpression in non-invasive cells. RNA sequencing of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS Through a whole genome shRNA screen, a zinc-finger containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong decrease in invasion capacity in two highly invasive GBM cell lines. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of either one or both zinc-finger motifs led to decreased invasion indicating that the two zinc-finger motifs are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through a transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent a novel promising target to overcome invasive capacities in GBM.

Identification of important invasion and proliferation related genes in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an end-stage hormonal syndrome. Although profound attempts have been made to illuminate the pathogenesis, the molecular mechanisms of ACC remain to be clarified. To identify the important genes in the progression of ACC, microarray datasets GSE19775 was downloaded from the gene expression omnibus database. The differentially-expressed genes (DEGs) were identified, and pathway and GO enrichment analyses were performed. The protein-protein interaction (PPI) network was constructed and the module analysis was performed using the protein interaction network analysis and Cytoscape. Also constructed target genes-miRNA regulatory network and target genes-TF regulatory network. Correlation of the hub genes were analyzed in The Cancer Genome Atlas. The prognostic values of hub genes were further validated by online tool UALCAN. Mutation analysis was done by online tool CBio Cancer Genomics Portal. A total of 884 DEGs were identified, with 441 in up regulation and 443 in down regulation. Pathways in catecholamine biosynthesis, aldosterone synthesis and secretion, pyrimidine deoxyribonucleosides salvage and systemic lupus erythematosus were the most significantly enriched for DEGs (up and down regulated). Blood vessel morphogenesis and cell cycle phase transition were the most significantly enriched term in biological processes, while extracellular matrix and chromosome, centromeric region were in cellular component and heparin binding and protein dimerization activity were in molecular function. Among the PPI networks and its module, target genes-miRNA regulatory network and target genes-TF regulatory network, hub genes were YWHAZ, FN1, GRK5, VCAM1, GATA6, TXNIP, HSPA1A, and F11R. Hub genes such as YWHAZ, STAT1, ICAM1, SH3BP5, CD83, FN1, TK1, HIST1H1C, CABLES1, and MCM3 were associated with poor overall survival, while hub genes such as STAT1, ICAM1, CD83, FN1, TK1, HIST1H1C, and MCM3 were highly expressed in stage 4. In conclusion, DEGs and hub genes diagnosed in this study may deepen our understanding of molecular mechanisms underlying the progression of ACC, and provide important targets for diagnosis and treatment of ACC.

MicroRNA-3662 targets ZEB1 and attenuates the invasion of the highly aggressive melanoma cell line A375.

BackgroundCutaneous melanoma is the most aggressive form of skin cancer. It accounts for approximately 5% of all cutaneous malignancies and is currently responsible for the majority of skin cancer-related deaths. However, the exact mechanisms responsible for the occurrence of melanoma, in particular the invasive growth in normal skin or muscle tissue, remain unknown.Materials and methodsmiR-3662, a microRNA is a potential tumor suppressor targeting zinc finger E-box binding homeobox 1 (ZEB1), which functions as a key regulator of the epithelial-mesenchymal transition (EMT) process. This microRNA was identified using an online database (miRDB) and expression was confirmed by Western blot analysis. Quantitative polymerase chain reaction (qPCR) was used to examine whether miR-3662 inhibits the EMT process in the aggressive melanoma cell line, A375, through the modification of the expression of invasion-related genes in A375 cells. The effects of miR-3662 on the in vivo growth of A375 cells were examined in a nude mouse model.ResultsUsing virtual screening of the miRDB database, miR-3662 was shown to target the 3ʹ untranslated region (UTR) of the ZEB1 gene. Expression of miR-3662 via a lentivirus vector significantly decreased protein levels of ZEB1 and inhibited the growth of A375 cells in vitro and in vivo. The reduction in ZEB1 expression induced by miR-3662 resulted in EMT inhibition in A375 cells and decreased the relative expression of metastasis genes.ConclusionDown-regulation of ZEB1’s expression via miR-3662 lentivirus vectors significantly decreased the in vitro and in vivo growth of the highly aggressive melanoma cell line A375.

Identification of Important Invasion-Related Genes in Non-functional Pituitary Adenomas.

Non-functioning pituitary adenomas (NFPAs) are locally invasive with high morbidity. The objective of this study was to diagnose important genes and pathways related to the invasiveness of NFPAs and gain more insights into the underlying molecular mechanisms of NFPAs. The gene expression profiles of GSE51618 were downloaded from the Gene Expression Omnibus database with 4 non-invasive NFPA samples, 3 invasive NFPA samples, and 3 normal pituitary gland samples. Differentially expressed genes (DEGs) are screened between invasive NFPA samples and normal pituitary gland samples, followed by pathway and ontology (GO) enrichment analyses. Subsequently, a protein-protein interaction (PPI) network was constructed and analyzed for these DEGs, and module analysis was performed. In addition, a target gene-miRNA network and target gene-TF (transcription factor) network were analyzed for these DEGs. A total of 879 DEGs were obtained. Among them, 439 genes were upregulated and 440 genes were downregulated. Pathway enrichment analysis indicated that the upregulated genes were significantly enriched in cysteine biosynthesis/homocysteine degradation (trans-sulfuration) and PI3K-Akt signaling pathway, while the downregulated genes were mainly associated with docosahexaenoate biosynthesis III (mammals) and chemokine signaling pathway. GO enrichment analysis indicated that the upregulated genes were significantly enriched in animal organ morphogenesis, extracellular matrix, and hormone activity, while the downregulated genes were mainly associated with leukocyte chemotaxis, dendrites, and RAGE receptor binding. Subsequently, ESR1, SOX2, TTN, GFAP, WIF1, TTR, XIST, SPAG5, PPBP, AR, IL1R2, and HIST1H1C were diagnosed as the top hub genes in the upregulated and downregulated PPI networks and modules. In addition, HS3ST1, GPC4, CCND2, and SCD were diagnosed as the top hub genes in the upregulated and downregulated target gene-miRNA networks, while CISH, ISLR, UBE2E3, and CCNG2 were diagnosed as the top hub genes in the upregulated and downregulated target gene-TF networks. The new important DEGs and pathways diagnosed in this study may serve key roles in the invasiveness of NFPAs and indicate more molecular targets for the treatment of NFPAs.

CHD7 promotes glioblastoma cell motility and invasiveness through transcriptional modulation of an invasion signature

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.

Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida

BackgroundSpironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive.ResultsTo investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis.ConclusionWhile oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

Bisphenol A exposure alters placentation and causes preeclampsia-like features in pregnant mice involved in reprogramming of DNA methylation of WNT2.

Preeclampsia leads to adverse outcomes for pregnant women. Bisphenol A (BPA) is an environmental endocrine disruptor and has been shown to be positively associated with increased risk of preeclampsia in human studies. We investigated whether BPA exposure causes preeclampsia-like features in pregnant mice and the associated underlying mechanisms. Experiments were performed in animal models and cell cultures. In pregnant mice, BPA-exposed mice exhibited preeclampsia-like features including hypertension, disruption of the circulation, and the placental angiogenesis biomarkers fms-related tyrosine kinase 1 and placenta growth factor, and glomerular atrophy; urinary protein was not affected. These preeclampsia-like features correlated with increased retention of smooth muscle cells and reduced vessel areas at the junctional zone of the placenta. In addition, there were disrupted expression of invasion-related genes including increased tissue inhibitors of metalloproteinases, decreased metalloproteinases, and Wnt family member WNT2/β-catenin, which correlated with increased DNA methylation in its promoter region and upregulation of DNA methyltransferase (Dnmt)1. BPA exposure impeded the interaction between the human cytotrophoblast cell line, HTR-8/SVneo, and endothelium cells. BPA exposure down-regulated WNT2 expression, and elevated the DNA methylation of WNT2; these results were consistent with in vivo observations. Inhibition of DNMT in HTR-8/SVneo cells resulted in reduced DNA methylation and increased expression of WNT2. Taken together, these data demonstrate that BPA exposure alters trophoblast cell invasion and causes abnormal placental vessel remodeling, both of which lead to the development of preeclampsia-like features in pregnant mice. Our results suggest that this phenomenon involves the epigenetic reprogramming and down-regulation of WNT2 mediated by DNMT1.-Ye, Y., Tang, Y., Xiong, Y., Feng, L., Li, X. Bisphenol A exposure alters placentation and causes preeclampsia-like features in pregnant mice involved in reprogramming of DNA methylation of WNT2.

Fabrication and Characterization of Chitosan-Hyaluronic Acid Scaffolds with Varying Stiffness for Glioblastoma Cell Culture.

The invasive and recurrent nature of glioblastoma multiforme (GBM) is linked to a small subpopulation of cancer cells, which are self-renewing, resistant to standard treatment regimens, and induce formation of new tumors. Matrix stiffness is implicated in the regulation of cell proliferation, drug resistance, and reversion to a more invasive phenotype. Therefore, understanding the relationship between matrix stiffness and tumor cell behavior is vital to develop appropriate in vitro tumor models. Here, chitosan-hyaluronic acid (CHA) polyelectrolyte complex scaffolds are fabricated with statistically significant stiffness variances to characterize the effect of scaffold stiffness on morphology, proliferation, drug resistance, and gene expression in human glioblastoma cells (U-87 MG). All scaffolds support GBM proliferation over a 12-day culture period, yet larger spheroids are observed in scaffolds with higher stiffness. Additionally, GBM cells cultured in stiffer CHA scaffolds prove significantly more resistant to the common chemotherapeutic temozolomide. Moreover, the stiffer 8% CHA scaffolds exhibit an increase in expression of drug resistance and invasion related genes compared to 2D culture. CHA scaffolds present a tunable microenvironment for enhanced tumor cell malignancy and may provide a valuable in vitro microenvironment for studying tumor progression and screening anticancer therapies.

Moesin Up-regulation Is Associated with Enhanced Tumor Progression Imaged Non-invasively in an Orthotopic Mouse Model of Human Glioblastoma.

Glioblastoma is a recalcitrant and poorly understood disease. The aim of the present study was to investigate the effect of moesin up-regulation on tumor progression in an orthotopic nude-mouse model of human glioblastoma. U87-GFP glioblastoma cells, transfected with either U87-H4645 (moesin up-regulated) or U87-H149 (vector control) were orthotopically implanted into the brains of nude mice. Moesin expression in the tumors was analyzed with RT-PCR and western blotting. Real-time fluorescence imaging was used to longitudinally and non-invasively quantitate tumor growth. The expression of cancer-related genes β-catenin, CD44, MMP-2, ICAM-1, and PCNA in the tumor was analyzed by RT-PCR, western blotting and immunohistochemistry in both sublines. The expression levels of moesin mRNA and protein were significantly increased in the glioblastoma derived from transfected U87-H4645 cells compared to the vector control and untransfected cells. Tumor growth rate and final tumor weight were significantly increased in the animals with the glioblastoma derived from transfected U87-H4645 cells, compared to untransfected and vector control (p<0.01). mRNA expression of β-catenin, CD44, ICAM-1, and MMP-2 in the glioblastoma derived from the transfected U87-H4645 tumors was significantly increased compared with tumors derived from untransfected and vector-control U87 cells (p<0.01). Furthermore, a similar increase in the expression of these proteins was observed by western blotting or immunohistochemistry. Up-regulation of moesin expression in glioblastoma cells resulted in more aggressive orthotopic glioblastoma growth in nude mice. This effect may be mediated by the regulation of several proliferation-, adhesion-, and invasion-related cancer genes, which may serve as future therapeutic targets for this recalcitrant disease.

ShRNA-mediated knock-down of CXCL8 inhibits tumor growth in colorectal liver metastasis

CXCL8 belongs to proinflammatory chemokines that are predominantly involved in neutrophil chemotaxis and degranulation. Several studies have suggested that secretion of CXCL8 from cancer cells have a profound effect on tumor microenvironment. In this study, in continuation to our previous work of understanding the global picture of invasion related genes in colorectal liver metastases, we clearly show an up-regulation of CXCL8 expression in the tumor cells at the invasion front as compared to the tumor cells in the inner parts of the tumor. Furthermore, ShRNA mediated down-regulation of CXCL8 resulted in inhibition of cell proliferation, viability and invasion in vitro and a near complete growth reduction of tumor in vivo. We showed that CXCL8 secreted by tumor cells at the invasion front were able to promote migration through angiogenesis by upregulating VEGFA and invasion via the AKT/GSK3β/β-catenin/MMP7 pathway by upregulating BCL-2 confirming the key role of CXCL8 during tumor progression.

MicroRNA‐944 inhibits migration and invasion in melanoma cells by targeting Notch2

Melanoma is a malignant tumor of melanocytes. The incidence of melanoma is increasing in worldwide. miR‐944 is a primates‐specific microRNA, which plays a role as putative tumor suppressor in cancer cells. However, the function of miR‐944 in melanoma is not reported yet. In this study, we observed that the migration and invasion in melanoma cells transfected with miR‐944 was decreased compared to that in control cells. The expression of cellular invasion‐related genes was decreased in miR‐944 transfected cells. By using bioinformatics program, Notch2 was selected as a target of miR‐944. The overexpression of miR‐944 inhibited the expression of Notch2 in melanoma cells. By reporter assay, we found miR‐944 binding to the 3′UTR of Notch2. Knockdown of Notch2 in melanoma cells also suppressed cellular migration and decreased the expression of cellular invasion‐related genes. Taken together, our results show that miR‐944 suppressed migration of melanoma cells by targeting Notch2. These finding may have important implications concerning the molecular mechanisms of invasion and migration in melanoma.Support or Funding InformationThis research was supported by a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) (No. 2017R1A2B4009615).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RING-Finger Protein 6 Amplification Activates JAK/STAT3 Pathway by Modifying SHP-1 Ubiquitylation and Associates with Poor Outcome in Colorectal Cancer.

Objective: The E3 ubiquitin ligase RNF6 (RING-finger protein 6) plays a crucial role in carcinogenesis. However, the copy number and expression of RNF6 were rarely reported in colorectal cancer. We aimed to explore the mechanical, biological, and clinical role of RNF6 in colorectal cancer initiation and progression.Design: The copy number and expression of RNF6 were analyzed from Tumorscape and The Cancer Genome Atlas (TCGA) datasets. Gene expressions were examined by real-time PCR, Western blot, and immunohistochemical staining. Gene expression profiling studies were performed to identify pivotal genes regulated by RNF6. Biological function of RNF6 on tumor growth and metastasis was detected in vivo and in vitro Role of RNF6 in modulating SHP-1 expression was examined by coimmunoprecipitation and confocal microscopy, respectively.Results: The copy number of RNF6 was significantly amplified in colorectal cancer, and the amplification was associated with RNF6 expression level. Amplification and overexpression of RNF6 positively correlated with patients with colorectal cancer with poor prognosis. The gene set enrichment analysis (GSEA) revealed cell proliferation, and invasion-related genes were enriched in RNF6 high-expressed colorectal cancer cells as well as in patients from TCGA dataset. Downregulation of RNF6 impaired the colorectal cancer cell proliferation and invasion in vitro and in vivo RNF6 may activate the JAK/STAT3 pathway and increase pSTAT3 levels by inducing the ubiquitination and degradation of SHP-1.Conclusions: Genomic amplification drives RNF6 overexpression in colorectal cancer. RNF6 may be a novel biomarker in colorectal carcinogenesis, and RNF6 may increase pSTAT3 level via promoting SHP-1 ubiquitylation and degradation. Targeting the RNF6/SHP-1/STAT3 axis provides a potential therapeutic option for RNF6-amplified tumors. Clin Cancer Res; 24(6); 1473-85. ©2017 AACR.

Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.

Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum. We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Shufeng Jiedu Capsule and its active ingredients induce apoptosis, inhibit migration and invasion, and enhances doxorubicin therapeutic efficacy in hepatocellular carcinoma

BackgroundShufeng Jiedu Capsule (SFJDC), a traditional Chinese medicine, has been used widely as antiviral, antibacterial, antitumor, and anti-inflammatory drugs. Previous studies indicated that some active ingredients of Shufeng Jiedu Capsule, such as resveratrol and quercetin, could suppress hepatocellular carcinoma (HCC) cells through various signaling pathways. However, anti-HCC activity of SFJDC as a complementary medicine remains unexplored. Here, we use a combination of Shufeng Jiedu Capsule and doxorubicin to treat HCC cells and investigated the effects and mechanisms of SFJDC and its ingredientsin vitro. MethodsIn this study, two HCC cell lines, HepG2 and HepG2.2.15, were employed and all cells were separated into seven groups: doxorubicin group, SFJDC group, combination of doxorubicin and SFJDC group, resveratrol group, quercetin group, resveratrol and quercetin group, and control group. Through this research, the cellular functional experiments, such as MTT assay, Hoechst 33,258 staining, would healing assay, and transwell assay, were took to observe the effects of those agents on proliferation, apoptosis, migration and invasion of cells. Then, apoptosis and invasion related genes and proteins were detected by real-time PCR and western blot to illuminate the signaling pathways. ResultsThe combination group induced more significant apoptosis and inhibition of migration and invasion by affecting proteins and mRNA of apoptosis, migration, and invasion related elements, such as Bcl-2, Bax, mTOR, and NF-κB. Furthermore, the research suggested SFJDC, as a mixture of a number of ingredients, had stronger activities than particular component or simple mixture of a few components. ConclusionsSFJDC and its active ingredients could play a role as complementary medicine to increase antitumor effect of doxorubicin by targeting mitochondrial, Akt/mTOR, and NF-κB signaling pathways.