Laminin Subunit Alpha Research Articles

Identification of Novel Biomarkers for Pre-diabetic Diagnosis Using a Combinational Approach.

Reliable protein markers for pre-diabetes in humans are not clinically available. In order to identify novel and reliable protein markers for pre-diabetes in humans, healthy volunteers and patients diagnosed with pre-diabetes and stroke were recruited for blood collection. Blood samples were collected from healthy and pre-diabetic subjects 12 h after fasting. BMI was calculated from body weight and height. Fasting blood glucose (FBG), glycated hemoglobin (HbA1C), triglyceride (TG), total cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), insulin and albumin were assayed by automated clinical laboratory methods. We used a quantitative proteomics approach to identify 1074 proteins from the sera of pre-diabetic and healthy subjects. Among them, 500 proteins were then selected using Mascot analysis scores. Further, 70 out of 500 proteins were selected via volcano plot analysis according to their statistical significance and average relative protein ratio. Eventually, 7 serum proteins were singled out as candidate markers for pre-diabetes due to their diabetic relevance and statistical significance. Immunoblotting data demonstrated that laminin subunit alpha 2 (LAMA2), mixed-lineage leukemia 4 (MLL4), and plexin domain containing 2 (PLXDC2) were expressed in pre-diabetic patients but not healthy volunteers. Receiver operating characteristic curve analysis indicated that the combination of the three proteins has greater diagnostic efficacy than any individual protein. Thus, LAMA2, MLL4 and PLXDC2 are novel and reliable serum protein markers for pre-diabetic diagnosis in humans.

Inhibition of macrophage migration inhibitory factor (MIF) as a therapeutic target in bleomycin-induced pulmonary fibrosis rats.

Macrophage migration inhibitory factor (MIF) inhibition can attenuate pulmonary fibrosis, but the antifibrotic mechanism is unclear. Here we investigated the antifibrotic effect of MIF knockdown in rats with bleomycin (BLM)-induced pulmonary fibrosis. The results showed that MIF inhibition attenuated lung injury and extracellular matrix deposition; significantly reduced the levels of cytokines including transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hydroxyproline (hyp), fibroblast growth factor 23 (FGF23), and secreted phosphoprotein 1 (Spp1); and inhibited the expression of CD68, F4/80, and α-smooth muscle actin (α-SMA) protein. MIF inhibition is associated with reduction of proinflammatory mediators and macrophage infiltration in lungs. In addition, MIF knockdown in the day 14 group was significantly better than MIF knockdown in day 1 group in terms of the above mentioned cytokines TGF-β1, IL-17, TNF-α. MIF knockdown in day 14 group showed a better trend than MIF knockdown in day 1 group in inhibition of hyp and α-SMA formation. Furthermore, MIF inhibition downregulated the FGF23, Spp1, anti-integrin alpha 10 (Itga10), laminin subunit alpha 1 (Lama1), thrombospondin 2 (THBS2), and Serpin family B member 5 (SERPINB5) mRNA levels and the p-Smad2/3 protein level. MIF knockdown may inhibit fibrosis through the TGF-β1/Smads signaling pathway. In addition, MIF inhibition protects against vascular remodeling via Thbs2 and Serpinb5 signaling. In summary, our study showed that knockdown of MIF can significantly inhibit lung inflammation and fibrosis in rats with BLM-induced pulmonary fibrosis. The future development of inhibitors targeting MIF may contribute to the treatment of pulmonary fibrosis.

Prognostic value of midkine, syndecan-1, hyaluronan synthase-2, sestrin-1, laminin subunit alpha-4 and fibulin-3 for malignant pleural mesothelioma.

The prognosis of malignant pleural mesothelioma (MPM) is poor, with a limited survival time. In this study, we aimed to examine expression levels of genes selected from relevant literature and to utilize in silico methods to determine genes whose expression could reflect the prognosis of patients with MPM by ex-vivo validation experiments. The study group consisted of 54 MPM patients treated with chemotherapy. Expression of 6 genes - midkine (MDK), syndecan-1 (SDC1), hyaluronan synthase-2 (HAS2), sestrin-1 (SESN1), laminin subunit alpha-4 (LAMA4), and fibulin-3 (FBLN3) - was examined by qPCR in tumor tissues. Sestrin-1 and LAMA4 were identified using an in house R-based script: Unsupervised Survival Analysis Tool. Midkine, SDC1, HAS2, and FBLN3 were selected from current literature. We used two housekeeping genes, i.e. glucose-6-phosphate dehydrogenase and TATA-box binding protein, as controls. Of the patients, 43 (79.6%) had epithelioid mesothelioma. The median survival for all patients was 10 (±1.2 SE) months (95% CI: 7.7-12.3). In multivariate analyses, MDK (p = 0.007), HAS2 (p = 0.008) and SESN1 (p = 0.014) expression levels were related to survival time in the whole group. In epithelioid type MPM patients, MDK (p = 0.014), FBLN3 (p = 0.029), HAS2 (p = 0.014) and SESN1 (p = 0.045) expression was related to survival time in multivariate analyses. High HAS2 and SESN1 expressions and low MDK are potential biomarkers of good prognosis in MPM. High HAS2 and SESN1 expression and low MDK and FBLN3 can also be utilized as biomarkers of good prognosis for epithelioid MPM. Those results should be further investigated in sera, plasma, and pleural effusions.

Evolving Up-regulation of Biliary Fibrosis-Related Extracellular Matrix Molecules After Successful Portoenterostomy.

Successful portoenterostomy (SPE) improves the short‐term outcome of patients with biliary atresia (BA) by relieving cholestasis and extending survival with native liver. Despite SPE, hepatic fibrosis progresses in most patients, leading to cirrhosis and a deterioration of liver function. The goal of this study was to characterize the effects of SPE on the BA liver transcriptome. We used messenger RNA sequencing to analyze global gene‐expression patterns in liver biopsies obtained at the time of portoenterostomy (n = 13) and 1 year after SPE (n = 8). Biopsies from pediatric (n = 2) and adult (n = 2) organ donors and other neonatal cholestatic conditions (n = 5) served as controls. SPE was accompanied by attenuation of inflammation and concomitant up‐regulation of key extracellular matrix (ECM) genes. Highly overexpressed genes promoting biliary fibrosis and bile duct integrity, such as integrin subunit beta 6 and previously unreported laminin subunit alpha 3, emerged as candidates to control liver fibrosis after SPE. At a cellular level, the relative abundance of activated hepatic stellate cells and liver macrophages decreased following SPE, whereas portal fibroblasts (PFs) and cholangiocytes persisted. Conclusion: The attenuation of inflammation following SPE coincides with emergence of an ECM molecular fingerprint, a set of profibrotic molecules mechanistically connected to biliary fibrosis. The persistence of activated PFs and cholangiocytes after SPE suggests a central role for these cell types in the progression of biliary fibrosis.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell‐like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post‐inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i‐state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP‐2 gamma), SOX17 (SRY‐box transcription factor 17), and NANOS3 (nanos C2HC‐type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C‐X‐C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i‐state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Transcriptome sequencing identifies genes associated with invasion of ovarian cancer.

ObjectiveTo identify key genes in ovarian cancer using transcriptome sequencing in two cell lines: MCV152 (benign ovarian epithelial tumour) and SKOV-3 (ovarian serous carcinoma).MethodsDifferentially expressed genes (DEGs) between SKOV-3 and MCV152 were identified. Candidate genes were assessed for enrichment in gene ontology function and Kyoto Encyclopaedia of Genes and Genomes pathway. Candidate gene expression in SKOV-3 and MCV152 cells was validated using Western blots.ResultsA total of 2020 upregulated and 1673 downregulated DEGs between SKOV3 and MCV152 cells were identified that were significantly enriched in the cell adhesion function. Upregulated DEGs, such as angiopoietin 2 (ANGPT2), CD19 molecule (CD19), collagen type IV alpha 3 chain (COL4A3), fibroblast growth factor 18 (FGF18), integrin subunit beta 4 (ITGB4), integrin subunit beta 8 (ITGB8), laminin subunit alpha 3 (LAMA3), laminin subunit gamma 2 (LAMC2), protein phosphatase 2 regulatory subunit Bgamma (PPP2R2C) and spleen associated tyrosine kinase (SYK) were significantly involved in the extracellular matrix-receptor interaction pathway. Downregulated DEGs, such as AKT serine/threonine kinase 3 (AKT3), collagen type VI alpha 1 chain (COL6A1), colony stimulating factor 3 (CSF3), fibroblast growth factor 1 (FGF1), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 11 (ITGA11), MYB proto-oncogene, transcription factor (MYB), phosphoenolpyruvate carboxykinase 2, mitochondrial (PCK2), placental growth factor (PGF), phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), serum/glucocorticoid regulated kinase 1 (SGK1), toll like receptor 4 (TLR4) and tumour protein p53 (TP53) were involved in PI3K-Akt signalling. Expression of these DEGs was confirmed by Western blot analyses.ConclusionCandidate genes enriched in cell adhesion, extracellular matrix–receptor interaction and PI3K-Akt signalling pathways were identified that may be closely associated with ovarian cancer invasion and potential targets for ovarian cancer treatment.

Mex3a interacts with LAMA2 to promote lung adenocarcinoma metastasis via PI3K/AKT pathway

Lung adenocarcinoma (LUAD) is the main subtype of lung cancer. In this study, we found that RBP Mex3a was significantly upregulated in LUAD tissues and elevated Mex3a expression was associated with poor LUAD prognosis and metastasis. Furthermore, we demonstrated that Mex3a knockdown significantly inhibited LUAD cell migration and invasion in vitro and metastasis in nude mice. Transcriptome sequencing indicated that Mex3a affected gene expression linked to ECM-receptor interactions, including laminin subunit alpha 2(LAMA2). RNA immunoprecipitation (RIP) assay revealed Mex3a directly bound to LAMA2 mRNA and Mex3a increased the instability of LAMA2 mRNA in LUAD cells. Furthermore, we discovered that LAMA2 was surprisingly downregulated in LUAD and inhibited LUAD metastasis. LAMA2 knockdown partially reverse the decrease of cell migration and invasion caused by Mex3a knockdown. In addition, we found that both Mex3a and LAMA2 could influence PI3K-AKT pathway, which are downstream effectors of the ECM-receptor pathway. Moreover, the reduced activation of PI3K-AKT pathway in caused by Mex3a depletion was rescued by LAMA2 knockdown. In conclusion, we demonstrated that Mex3a downregulates LAMA2 expression to exert a prometastatic role in LUAD. Our study revealed the prognostic and prometastatic effects of Mex3a in LUAD, suggesting that Mex3a can serve as a prognostic biomarker and a target for metastatic therapy.

Laminin Subunit Alpha-4 and Osteopontin Are Glioblastoma-Selective Secreted Proteins That Are Increased in the Cerebrospinal Fluid of Glioblastoma Patients.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. The purpose of the present study was to identify GBM cell-selective secreted proteins by analyzing conditioned media (CM) from GBM, breast, and colon cancer cell lines using sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) and targeted proteomics. We identified 2371 proteins in the CM from GBM and the other cancer cell lines. Among the proteins identified, 15 showed significantly higher expression in the CM from GBM cell lines than in those from other cancer cell lines. These GBM-selective secreted proteins were further quantified in the cerebrospinal fluid (CSF) from patients with GBM. Laminin subunit alpha-4 (LAMA4) and osteopontin (OPN) had increased expression levels in the CSF from GBM patients compared to those from non-brain tumor patients. In addition, the areas under the curves in a receiver operating characteristic analysis of LAMA4 and OPN were greater than 0.9, allowing for discrimination of GBM patients from non-brain tumor patients. The CSF levels of LAMA4 and OPN were also significantly correlated with the GBM tumor volume. These results suggest that LAMA4 and OPN are secreted from GBM cells into the CSF and appear to be candidates as diagnostic markers and therapeutic targets for GBM.

LAMA4 upregulation is associated with high liver metastasis potential and poor survival outcome of Pancreatic Cancer.

Rationale: Pancreatic cancer is one of the most difficult cancers to manage and its poor prognosis stems from the lack of a reliable early disease biomarker coupled with its highly metastatic potential. Liver metastasis accounts for the high mortality rate in pancreatic cancer. Therefore, a better understanding of the mechanism(s) underlying the acquisition of the metastatic potential in pancreatic cancer is highly desirable.Methods: Microarray analysis in wild-type and highly liver metastatic human pancreatic cancer cell lines was performed to identify gene expression signatures that underlie the metastatic process. We validated our findings in patient samples, nude mice, cell lines and database analysis.Results: We identified a metastasis-related gene, laminin subunit alpha 4 (LAMA4), that was upregulated in highly liver metastatic human pancreatic cancer cell lines. Downregulation of LAMA4 reduced the liver metastatic ability of pancreatic cancer cells in vivo. Furthermore, LAMA4 expression was positively correlated with tumor severity and in silico analyses revealed that LAMA4 was associated with altered tumor microenvironment. In particular, our in vitro and in vivo results showed that LAMA4 expression was highly correlated with cancer-associated fibroblasts (CAFs) level which may contribute to pancreatic cancer metastasis. We further found that LAMA4 had a positive effect on the recruitment and activity of CAFs.Conclusions: These data provide evidence for LAMA4 as a possible biomarker of disease progression and poor prognosis in pancreatic cancer. Our findings indicate that LAMA4 may contribute to pancreatic cancer metastasis via recruitment or activation of CAFs.

Long Non-coding RNA LINC00628 Interacts Epigenetically with the LAMA3 Promoter and Contributes to Lung Adenocarcinoma

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular progress in lung adenocarcinoma. In this study, to identify cancer-related lncRNAs and genes, we screened for those lncRNAs that were differentially expressed in lung adenocarcinoma, which revealed LINC00628 overexpression and low expression of laminin subunit alpha 3 (LAMA3). This was further validated in the cancerous tissues from patients diagnosed with lung adenocarcinoma. Thereafter, we explored the functional relevance of LINC00628 and LAMA3 in lung adenocarcinoma by analyzing the recruitment of DNA methyltransferase (DNMT) and the cellular processes of lung adenocarcinoma cells following treatments that induced LINC00628 overexpression or LINC00628 silencing or with 5-azacytidine (5-Aza, a DNMT inhibitor). The results showed that LINC00628 silencing decreased cell proliferation, migration, and invasion as well as the drug resistance of lung adenocarcinoma cells to vincristine (VCR). The results were opposite in the cells with LAMA3 demethylation induced by 5-Aza treatment. Further research indicated that LINC00628 recruited DNMT1, DNMT3A, and DNMT3B to promote the methylation of LAMA3 promoter, thereby decreasing its expression. Moreover, an in vivo experiment was performed in nude mice to assess the tumor growth ability and drug resistance of human lung adenocarcinoma cells. It was observed that LINC00628 silencing or 5-Aza treatment inhibited the in vivo tumor growth ability of the human lung adenocarcinoma cells and reduced their resistance to VCR. Altogether, our results provide evidence of a mechanism by which LINC00628 silencing exerts an inhibitory role in lung adenocarcinoma by modulating the DNA methylation of LAMA3, indicative of a novel molecular target for treatment of lung adenocarcinoma patients showing resistance to VCR.

Induction of specific CD8(+) T cells against hepatocellular carcinoma-associated neoantigens

Objective: To predict the tumor neoantigen peptides in hepatocellular carcinoma (HCC), and examine their specific immune effects against the tumor cells without injury to normal cells. Methods: The data of whole-genome sequencing and exome sequencing of HCC tumor and matched non-tumor liver tissues were analyzed to confirm the HCC-associated somatic mutations. Based on the HLA phenotype of the patients, we used NetMHC software to predict the neoantigen epitopes with high binding affinity to their MHC-I molecules. The predicted peptides with mutation sites included were synthesized. GPL10687 platform was applied to examine the gene expression difference between tumor and normal tissues of the selected genes in GSE25097, one of the GEO databases. The quantitative real-time PCR (qRT-qPCR) and immunohistochemistry were used to confirm the expressions in tumors and normal tissues of the selected genes. By using the predicted peptides, we induced the generation of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) and examined their specific effects against tumor cells. Results: The mutation frequency of TP53 (tumor protein p53) was 40%, and LAMA3 (Laminin Subunit Alpha 3) was 8% in the analyzed HCC tissues. In GSE25097 database, TP53 and LAMA3 mRNA levels in tumors were 1.57±0.02 and 1.37±0.10, which were significantly increased than those in matched no-tumor tissue (0.54±0.01 and 0.36±0.01, P<0.05). The differences of expression levels of TP53 and LAMA3 in tumor and no-tumor tissues were validated by using qRT-qPCR and immunohistochemistry in 10 HCC tissues. The mRNA levels of TP53 and LAMA3 in tumors were 0.24±0.03 and 0.13±0.06, which were significantly elevated than those in matched no-tumor tissue (0.11±0.01 and 0.01±0.01, P<0.05). Among the Chinese population, HLA-A2 and HLA-A11 and HLA-A24 accounted for 70%, representing the major MHC-I molecules. The CTLs induced by predicted peptides showed cytotoxicity to the targets pulsed with mutated peptide, with no effect on the target pulsed with normal peptide and on normal cells. Conclusions: TP53 and LAMA3 existed relative higher mutation frequency in HCC, and expressed higher in tumor tissues. The induced CTLs by predicted peptides derived from mutation-associated protein could specific kill the target cells without injury to normal cells.

MicroRNA-200b is downregulated and suppresses metastasis by targeting LAMA4 in renal cell carcinoma

BackgroundMetastasis is the primary cause of tumor death in renal cell carcinoma (RCC). Improved diagnostic markers of metastasis are critically needed for RCC. MicoRNAs are demonstrated to be stable and significant biomarkers for several malignancies. In this study, we aimed to explore the metastasis related microRNAs and its mechanism in RCC. MethodsThe relationship between microRNAs expression and prognosis and metastasis of RCC patients were explored by data mining through expression profiles from The Cancer Genome Atlas (TCGA). A total of 80 RCC tissues and adjacent normal kidney tissues were obtained from Department of Urology, Peking University First Hospital. Expression of microRNA-200b (miR-200b) in RCC tissues and cell lines were determined by bioinformatic data mining and quantitative real-time PCR (qRT-PCR). The effects of miR-200b on cell proliferation, migration and invasion were determined by cell counting kit-8 and colony formation assay, wound healing assay and Boyden chamber assay. Mouse cell-derived xenograft and patient-derived xenograft model were also performed to evaluate the effects of miR-200b on tumor growth and metastasis in vivo. The molecular mechanism of miR-200b function was investigated using bioinformatic target predication and high-throughput cDNA sequencing (RNA-seq) and validated by luciferase reporter assay, qRT-PCR, Western blot and immunostaining in vitro and in vivo. FindingsOur findings indicates that miR-200b is frequently downregulated and have potential utility as a biomarker of metastasis and prognosis in RCC. Interestingly, ectopic expression of miR-200b in the Caki-1 and OSRC-2 cell lines suppresses cell migration and invasion in vitro as well as tumor metastases in vivo. However, miR-200b has no effect on cell proliferation in vitro and tumor growth in vivo. In addition, bioinformatics target predication and RNA-seq results reveals that Laminin subunit alpha 4 (LAMA4) is one target of miR-200b and significantly inhibited by miR-200b in vitro and in vivo. InterpretationThese results demonstrate a previously undescribed role of miR-200b as a suppressor of tumor metastasis in RCC by directly destabilizing LAMA4 mRNA.

Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering.

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1β) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.

Comparative proteomic analysis provides new insight into differential transmission of two begomoviruses by a whitefly

BackgroundViruses in the genus Begomovirus (Family Geminiviridae) include many important economic plant viruses transmitted by whiteflies of the Bemisia tabaci species complex. In general, different begomoviruses may be acquired and transmitted by the same whitefly species with different efficiencies. For example, the species Mediterranean (MED) in this whitefly species complex transmits tomato yellow leaf curl virus (TYLCV) at a higher efficiency than papaya leaf curl China virus (PaLCuCNV). However, the proteomic responses of whitefly to the infection of different begomoviruses remain largely unknown.MethodsWe used iTRAQ-based proteomics coupled with RT-qPCR to investigate and compare responses of the MED whitefly to the infection of TYLCV and PaLCuCNV.ResultsTotally, 259, 395 and 74 differently expressed proteins (DEPs) were identified in the comparisons of TYLCV-infected vs. un-infected, PaLCuCNV-infected vs. un-infected, and TYLCV-infected vs. PaLCuCNV-infected whiteflies, respectively. These proteins appear associated with catabolic process, metabolic process, transport, defense response, cell cycle, and receptor. The comparisons of TYLCV-infected vs. un-infected and PaLCuCNV-infected vs. un-infected shared some similar DEPs, indicating possible involvement of laminin subunit alpha, dystroglycan, integrin alpha-PS2 and cuticle proteins in viral transport as well as the role of putative defense proteins 3 and PITH in anti-viral response. However, 20S proteasome subunits associated with regulation of virus degradation and accumulation were up-regulated in PaLCuCNV-infected but not in TYLCV-infected whiteflies, which may be related to the constraints of PaLCuCNV accumulation in MED.ConclusionsThese findings provide valuable clues for unravelling the roles of some whitefly proteins in begomovirus transmission.

Expression and methylation status of LAMA2 are associated with the invasiveness of nonfunctioning PitNET.

The laminin subunit alpha 2 (LAMA2) gene encodes an alpha 2 chain, which constitutes one of the subunits of laminin 2 (merosin) and laminin 4 (s-merosin). In the current study, we investigated the relationship between LAMA2 promoter methylation status and the invasiveness of clinically nonfunctioning pituitary adenomas (PitNETs). Specimens from patients with nonfunctioning PitNET were classified into three groups according to preoperative computed tomography (CT)/magnetic resonance imaging findings: a normal group (n = 6), non-invasive group (n = 11) and invasive group (n = 6). LAMA2 expression was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting, and the methylation status of the LAMA2 promoter region was observed using sodium bisulfite sequencing. Furthermore, 5-aza-2-deoxycytidine was used to explore the relationship between decreased LAMA expression and methylation in PitNET cells. According to the RT-qPCR and western blotting results, LAMA2 expression was downregulated in invasive PitNET, while the methylation of the LAMA2 promoter was increased. Methylation of the LAMA2 promoter decreased the expression of LAMA2. Thus, changes in LAMA2 expression due to promoter methylation were inversely correlated with the invasiveness of PitNET and the protein functions as a tumor suppressor. In addition, overexpression and demethylation of LAMA2 suppressed the invasion of PitNET cells, partially by exerting effects on the PTEN-PI3K/AKT signaling pathway and matrix metalloproteinase-9 (MMP-9). Furthermore, a xenograft model was also generated, and LAMA2 overexpression significantly suppressed tumor growth in vivo. Thus, LAMA2 expression and methylation patterns might be used as biomarkers to predict the prognosis of patients with PitNET.

IDH mutation status is associated with distinct vascular gene expression signatures in lower-grade gliomas.

BackgroundVascular gene expression patterns in lower-grade gliomas (LGGs; diffuse World Health Organization [WHO] grades II–III gliomas) have not been thoroughly investigated. The aim of this study was to molecularly characterize LGG vessels and determine if tumor isocitrate dehydrogenase (IDH) mutation status affects vascular phenotype.MethodsGene expression was analyzed using an in-house dataset derived from microdissected vessels and total tumor samples from human glioma in combination with expression data from 289 LGG samples available in the database of The Cancer Genome Atlas. Vascular protein expression was examined by immunohistochemistry in human brain tumor tissue microarrays (TMAs) representing WHO grades II–IV gliomas and nonmalignant brain samples. Regulation of gene expression was examined in primary endothelial cells in vitro.ResultsGene expression analysis of WHO grade II glioma indicated an intermediate stage of vascular abnormality, less severe than that of glioblastoma vessels but distinct from normal vessels. Enhanced expression of laminin subunit alpha 4 (LAMA4) and angiopoietin 2 (ANGPT2) in WHO grade II glioma was confirmed by staining of human TMAs. IDH wild-type LGGs displayed a specific angiogenic gene expression signature, including upregulation of ANGPT2 and serpin family H (SERPINH1), connected to enhanced endothelial cell migration and matrix remodeling. Transcription factor analysis indicated increased transforming growth factor beta (TGFβ) and hypoxia signaling in IDH wild-type LGGs. A subset of genes specifically induced in IDH wild-type LGG vessels was upregulated by stimulation of endothelial cells with TGFβ2, vascular endothelial growth factor, or cobalt chloride in vitro.ConclusionIDH wild-type LGG vessels are molecularly distinct from the vasculature of IDH-mutated LGGs. TGFβ and hypoxia-related signaling pathways may be potential targets for anti-angiogenic therapy of IDH wild-type LGG.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Identification of Two Novel LAMA2 Mutations in a Chinese Patient with Congenital Muscular Dystrophy.

Merosin-deficient CMD type 1A (MDC1A), caused by mutations of laminin subunit alpha 2 (LAMA2), is a predominant subtype of congenital muscular dystrophy (CMD). Herein, we described a Chinese patient with MDC1A who was admitted to hospital 17 days after birth because of marasmus and feeding difficulties. Mutations were identified by targeted capture and next generation sequencing (NGS) and further confirmed by Sanger sequencing. Paternity was confirmed by short tandem repeat analysis. Physical examination showed malnutrition, poor suck and appendicular hypotonia. Her serum CK levels were 2483 and 1962 U/L at 2 and 4 months of age, respectively. Brain magnetic resonance imaging performed at 1 month of age presented hyperintensity on T2-weighted images, T1-weighted images in parietal and occipital lobes, and diffusion-weighted image (DWI) as well as hypointensity on fluid attenuated inversion recovery (FLAIR) image; however, the cerebellum and corpus arenaceum were normal. At 7 months of age, delayed developmental milestones were observed, and she failed to turn her body over and raise her head up. A point mutation (c.1782+2T > G) and a frameshift duplication (c.8217dupT) in the LAMA2 gene were identified by targeted capture and NGS and further confirmed by Sanger sequencing. Moreover, genotyping with multiple short tandem repeat markers confirmed paternity to demonstrate that the point mutation is de novo. The frameshift duplication (c.8217dupT), inherited from her mother, was predicted to cause a substitution of Pro (P) to Ser (S) at the 2740th amino-acid residue and generate a prematurely truncated protein. The in silico analysis suggests that the mutation (c.1782+2T > G) may lead to aberrant splicing of LAMA2. Our case further confirms the heterogeneous clinical spectrum of MDC1A and presents two novel LAMA2 mutations to expand the mutation spectrum of MDC1A.

MiR-539 inhibits proliferation and migration of triple-negative breast cancer cells by down-regulating LAMA4 expression

BackgroundRecent studies have shown that laminin subunit alpha 4 (LAMA4) plays an important role in carcinogenesis. However, its molecular biological function in triple-negative breast cancer (TNBC) has not been entirely clarified. This study investigated the expression of LAMA4 in TNBC and its effect on cell proliferation, migration and invasion. Furthermore, we also identified the potential miRNA directly targeting LAMA4.MethodsWestern blot, Real-time quantitative PCR (qPCR) and immunohistochemical staining (IHC) were used to detect the expression of LAMA4 in TNBC. The effects of LAMA4 on TNBC cell proliferation, migration and invasion were also explored in vitro. The potential miRNA that targets LAMA4 was determined by dual luciferase reporter assay and verified by qPCR and western blot analysis.ResultsOur study showed LAMA4 mRNA (p = 0.001) and protein (p = 0.005) expression in TNBC tissue samples were elevated compared with adjacent normal tissue samples, and LAMA4 was mainly expressed in the cytoplasm of breast carcinoma cells. Knockdown of LAMA4 inhibited TNBC cell proliferation, migration and invasion in vitro. Moreover, further study revealed that LAMA4 was a putative target of miR-539, and miR-539 negatively regulated LAMA4 expression by directly targeting its 3′-UTR.ConclusionsOur study suggested that miR-539 suppressed the expression of LAMA4. LAMA4 plays an important role in tumor progression and may be an important target in treatment of TNBC.