CREB1 promotes cholangiocarcinoma metastasis through transcriptional regulation of the LAYN-mediated TLN1/ß1 integrin axis: CREB1 promotes cholangiocarcinoma metastasis through regulating LAYN/TLN1/ß1 integrin axis.

Background: Layilin (LAYN) plays an important role in tumor progression, invasion, and metastasis; however, its role in cholangiocarcinoma (CHOL) has not been elucidated. Methods: We utilized the GEPIA, STRING, and hTFtarget databases for bioinformatics analysis. Overexpression or knockdown cell lines were constructed by transfecting the cells with different plasmids. Western blot (WB) was performed to detect LAYN, TLN1, and CREB1 expression. Cell proliferation, migration, and invasiveness were assessed using CCK-8 and Transwell assays. Immunofluorescence and WB were used to detect epithelial-mesenchymal transition (EMT) markers. The CHOL metastasis model was established by injecting RBE cells into the tail veins of nude mice. Metastatic lesions were identified using hematoxylin and eosin staining. Co-immunoprecipitation and Chromatin immunoprecipitation were used to validate the interactions. Results: LAYN was highly expressed in the CHOL cells. Knockdown of LAYN significantly inhibited proliferation, migration, invasion, and EMT in both QBC-939 and RBE human CHOL cells, while overexpression of LAYN had the opposite effect. Furthermore, in a CHOL metastasis model using nude mice, knocking down LAYN expression markedly suppressed CHOL liver and lung metastases. LAYN interacts with TLN1, and CREB1 binds to the LAYN promoter, with all three showing a positive correlation. Additionally, bioinformatics analysis revealed high expression of both TLN1 and CREB1 in CHOL. Knockdown of TLN1 or CREB1 in QBC-939 and RBE cells inhibited cell proliferation, migration, invasion, and EMT, reversing the effects of LAYN overexpression. Moreover, knockdown of TLN1 or CREB1 also suppressed the expression of ITGB1 and the phosphorylation levels of c-Jun, p38 MAPK, and ERK, further reversing the effects of LAYN overexpression. Conclusion: Our results suggest that CREB1 promotes CHOL metastasis through transcriptional regulation of the LAYN-mediated TLN1/ß1 integrin axis.

Talin-1 variants associated with spontaneous coronary artery dissection (SCAD) highlight how even subtle changes in multi-functional scaffold proteins can manifest in disease.

Variants of talin-1 (TLN1) have recently been linked with spontaneous coronary artery dissection (SCAD) a condition where a tear can form in the wall of a heart artery necessitating immediate medical care. One talin-1 variant, A2013T, has an extensive familial pedigree of SCAD, which led to the screening for, and identification of, further talin-1 variants in SCAD patients. Here we evaluated these variants with commonly used pathogenicity prediction tools and found it challenging to reliably classify SCAD-associated variants, even A2013T where the evidence of a causal role is strong. Using biochemical and cell biological methods, we show that SCAD-associated variants in talin-1, which would typically be classified as non-pathogenic, still cause a measurable impact on protein structure and cell behaviour, including cell movement and wound healing capacity. Together, this indicates that even subtle variants in central mechanosensitive adapter proteins, can give rise to significant health impacts at the individual level, suggesting the need for a possible re-evaluation of the scoring criteria for pathogenicity prediction for talin variants.

Micro RNA miR-1303 Promotion of Proliferation, Migration and Invasion of Human Liver Cancer Cells Is Enhanced by Low Talin 1 (TLN1) Expression.

BACKGROUND/AIM: Liver cancer is the third-most lethal cancer worldwide. Abnormal expression of microRNAs (miRNAs) modulates gene expression to exert oncogenic or tumor-suppressive effects in liver cancer. However, the biological role of miR-1303 in the progression of liver cancer and its regulatory mechanism has not been elucidated. MATERIALS AND METHODS: The expression levels of miR-1303 were measured in liver-cancer tissues of patients and cell lines by RT-qPCR. Huh-7 and HepG2 liver-cancer cells were co-transfected by TLN1 and miR-1303 constructs. Cell viability was measured by the CCk-8 assay and colony-formation assay. Flow cytometry was used to measure cell apoptosis. Cell migration and invasion were determined by wound-healing and transwell-chamber assays. RT-PCR and western-blotting were used to determine miR-1303 inhibitor-associated marker expression, such as Bax, cleaved-caspase-3 and cleaved-caspase-9. RESULTS: miR-1303 expression was strongly up-regulated in liver-cancer tissues and cells. Knockdown of miR-1303 attenuated cell proliferation, migration and invasion, and induced apoptosis in liver-cancer cells. Talin 1 (TLN1) and miR-1303 expression were negatively correlated, possibly by miR-1303 targeting the TLN1 gene. TLN1 expression enhanced the efficacy of an miR-1303 inhibitor to reduce liver-cancer cell proliferation and invasion. CONCLUSION: miR-1303 plays an important role in liver cancer, which is inhibited by TLN1 expression.

Identification of TLN1 as a prognostic biomarker to effect cell proliferation and differentiation in acute myeloid leukemia.

The protein Talin1 encoded by the TLN1 gene is a focal adhesion-related protein that binds to various cytoskeletal proteins and plays an important role in cell adhesion and movement. Recent studies have shown that it is overexpressed in prostate cancer, liver cancer, and oral squamous cell carcinoma, and is closely related to tumor progression and metastasis. This study integrated bioinformatics and functional analysis to reveal the prognosis and potential functions of TLN1 in AML. The results showed that the expression level of TLN1 was abnormally increased in AML and localized in the cell membrane and cytoplasm, and TLN1 is a significant prognostic indicator of overall survival (OS). Enrichment analysis of related genes showed that TLN1 is related to neutrophil mediated immunity, neutrophil activation and may regulate important signal pathways in hematological tumors including tyrosine kinase receptor, FLT3 and PIK3/AKT. The PPI network shows that TLN1 and MYH9 may be involved in the process of AML tumors together with PIP5K1C, ROCK1, S100A4, MY01A and WAC. Immune infiltration analysis explains that TLN1 is associated with multiple immune cells and may be an important immune marker in AML. Furthermore, molecular biology experiments confirmed that TLN1 is related to the proliferation, differentiation and cycle of AML cells. Silencing TLN1 can inhibit the proliferation of AML cells and promote differentiation through the Talin1/P-AKT/CREB signaling pathway.

Global Proteomics Analysis of Bone Marrow: Establishing Talin-1 and Centrosomal Protein of 55 kDa as Potential Molecular Signatures for Myelodysplastic Syndromes.

Myelodysplastic syndrome (MDS) is a hematological disorder characterized by abnormal stem cell differentiation and a high risk of acute myeloid leukemia transformation. Treatment options for MDS are still limited, making the identification of molecular signatures for MDS progression a vital task. Thus, we evaluated the proteome of bone marrow plasma from patients (n = 28) diagnosed with MDS with ring sideroblasts (MDS-RS) and MDS with blasts in the bone marrow (MDS-EB) using label-free mass spectrometry. This strategy allowed the identification of 1,194 proteins in the bone marrow plasma samples. Polyubiquitin-C (UBC), moesin (MSN), and Talin-1 (TLN1) showed the highest abundances in MDS-EB, and centrosomal protein of 55 kDa (CEP55) showed the highest relative abundance in the bone marrow plasma of MDS-RS patients. In a follow-up, in the second phase of the study, expressions of UBC, MSN, TLN1, and CEP55 genes were evaluated in bone marrow mononuclear cells from 45 patients by using qPCR. This second cohort included only seven patients from the first study. CEP55, MSN, and UBC expressions were similar in mononuclear cells from MDS-RS and MDS-EB individuals. However, TLN1 gene expression was greater in mononuclear cells from MDS-RS (p = 0.049) as compared to MDS-EB patients. Irrespective of the MDS subtype, CEP55 expression was higher (p = 0.045) in MDS patients with abnormal karyotypes, while MSN, UBC, and TALIN1 transcripts were similar in MDS with normal vs. abnormal karyotypes. In conclusion, proteomic and gene expression approaches brought evidence of altered TLN1 and CEP55 expressions in cellular and non-cellular bone marrow compartments of patients with low-risk (MDS-RS) and high-risk (MDS-EB) MDSs and with normal vs. abnormal karyotypes. As MDS is characterized by disrupted apoptosis and chromosomal alterations, leading to mitotic slippage, TLN1 and CEP55 represent potential markers for MDS prognosis and/or targeted therapy.

Binding blockade between TLN1 and integrin ß1 represses triple-negative breast cancer.

Background: Integrin family are known as key gears in focal adhesion for triple-negative breast cancer (TNBC) metastasis. However, the integrin independent factor TLN1 remains vague in TNBC. Methods: Bioinformatics analysis was performed based on TCGA database and Shengjing Hospital cohort. Western blot and RT-PCR were used to detect the expression of TLN1 and integrin pathway in cells. A small-molecule C67399 was screened for blocking TLN1 and integrin ß1 through a novel computational screening approach by targeting the protein-protein binding interface. Drug pharmacodynamics were determined through xenograft assay. Results: Upregulation of TLN1 in TNBC samples correlates with metastasis and worse prognosis. Silencing TLN1 in TNBC cells significantly attenuated the migration of tumour cells through interfering the dynamic formation of focal adhesion with integrin ß1, thus regulating FAK-AKT signal pathway and epithelial-mesenchymal transformation. Targeting the binding between TLN1 and integrin ß1 by C67399 could repress metastasis of TNBC. Conclusions: TLN1 overexpression contributes to TNBC metastasis and C67399 targeting TLN1 may hold promise for TNBC treatment. Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 81872159, 81902607, 81874301), Liaoning Colleges Innovative Talent Support Program (Name: Cancer Stem Cell Origin and Biological Behaviour), Outstanding Scientific Fund of Shengjing Hospital (201803), and Outstanding Young Scholars of Liaoning Province (2019-YQ-10).

Downregulation of Talin-1 is associated with the increased expression of miR-182-5p and miR-9-5p in coronary artery disease.

BACKGROUND: Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD). METHODS: Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction. RESULTS: Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples. CONCLUSIONS: Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis.

CircRNA_400029 promotes the aggressive behaviors of cervical cancer by regulation of miR-1285-3p/TLN1 axis.

Background: Cervical cancer (CC) is the highest incidence of female malignant tumor in China. Circular RNAs (circRNAs) has been reported to affect CC progression by altering mRNA stability at the transcriptional level or binding to miRNAs to produce competitive endogenous RNA (ceRNA). For this purpose, our study was aimed to investigate the function effects and the potential regulatory mechanism of the circRNA_400029 in CC cells. Materials and methods: The expression levels of circRNA_400029 and miR-1285-3p were detected by real time polymerase chain reaction (RT-PCR). Similarly, the mRNA and protein levels of TLN1 (Talin 1) was detected by RT-PCR and Western blot. Cell-Counting Kit-8 (CCK-8), EdU and Flow cytometry assay were used to detect cell proliferation, cell cycle and apoptosis. Then the Transwell assays were used to test cell migration and invasion. Besides this, the functional targets were confirmed by Dual luciferase reporter assays. Tumor xenograft in nude mice checked the result in vivo. Results: To begin with, circRNA_400029 was upregulated in CC cells and tissue. Knockdown circRNA_400029 inhibited cell proliferation, migration and invasion while induced cell apoptosis. Interestingly, miR-1285-3p targeted circRNA_400029 and down-regulated of miR-1285-3p could reverse the effects of circRNA_400029 weak-expression on progression and apoptosis of CC cells. Moreover, TLN1 was up-regulated in CC cells and identified as a direct target of miR-1285-3p. Meanwhile, we found that miR-1285-3p negatively regulated the function of TLN1. Finally, the circRNA_400029/miR-1285-3p/TLN1 axis could affect tumor growth in vivo. Conclusion: The overexpressed circRNA_400029 promoted CC proliferation, migration and invasion while deduced apoptosis by sponging miR-1285-3p to regulate TLN1. CircRNA_400029 was a potential onco-circRNA in CC, and might be a promised therapy target.

Talin in mechanotransduction and mechanomemory at a glance.

Talins are cytoskeletal linker proteins that consist of an N-terminal head domain, a flexible neck region and a C-terminal rod domain made of 13 helical bundles. The head domain binds integrin ß-subunit cytoplasmic tails, which triggers integrin conformational activation to increase affinity for extracellular matrix proteins. The rod domain links to actin filaments inside the cell to transmit mechanical loads and serves as a mechanosensitive signalling hub for the recruitment of many other proteins. The α-helical bundles function as force-dependent switches - proteins that interact with folded bundles are displaced when force induces unfolding, exposing previously cryptic binding sites for other ligands. This leads to the notion of a talin code. In this Cell Science at a Glance article and the accompanying poster, we propose that the multiple switches within the talin rod function to process and store time- and force-dependent mechanical and chemical information.

The driving role of the Cdk5/Tln1/FAKS732 axis in cancer cell extravasation dissected by human vascularized microfluidic models.

BACKGROUND: Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans-endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis. METHODS: Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis. RESULTS: Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression. CONCLUSIONS: These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Novel targetable FGFR2 and FGFR3 alterations in glioblastoma associate with aggressive phenotype and distinct gene expression programs.

Prognostic molecular subgrouping of glioblastoma is an ongoing effort and the current classification includes IDH-wild-type and IDH-mutant entities, the latter showing significantly better prognosis. We performed a comparative integrated analysis of the FGFR glioblastoma subgroup consisting of 5 cases from a prospective 101-patient-cohort. FGFR alterations included FGFR2-TACC2 and FGFR2 amplifications arising in a multifocal IDH-mutant glioblastoma with unexpected 2.5-month patient survival, novel FGFR3 carboxy-terminal duplication and FGFR3-TLN1 fusion, and two previously described FGFR3-TACC3 fusions. The FGFR2 tumors showed additional mutations in SERPINE1/PAI-1 and MMP16, as part of extensive extracellular matrix remodeling programs. Whole transcriptomic analysis revealed common proliferation but distinct morphogenetic gene expression programs that correlated with tumor histology. The kinase program revealed EPHA3, LTK and ALK receptor tyrosine kinase overexpression in individual FGFR tumors. Paradoxically, all FGFR-fused glioblastomas shared strong PI3K and MAPK pathway suppression effected by SPRY, DUSP and AKAP12 inhibitors, whereas the FGFR2-TACC2 tumor elicited also EGFR suppression by ERRFI1 upregulation. This integrated analysis outlined the proliferation and morphogenetic expression programs in FGFR glioblastoma, and identified four novel, clinically targetable FGFR2 and FGFR3 alterations that confer aggressive phenotype and trigger canonical pathway feedback inhibition, with important therapeutic implications.

Tanshinone IIA Suppresses Glioma Cell Proliferation, Migration and Invasion Both in vitro and in vivo Partially Through miR-16-5p/Talin-1 (TLN1) Axis.

BACKGROUND: Tanshinone IIA (TIIA) is one of the active constituents derived from the rhizome of Danshen, a traditional Chinese herbal. Recently, microRNAs (miRNAs) have been suggested to be associated with the anticancer role of TIIA. However, it remains vague of the interaction between miRNAs and TIIA in glioma, a common aggressive brain tumor in humans. METHODS: Expression of miRNA (miR)-16-5p and talin-1 (TLN1) was detected using reverse transcription-quantitative polymerase chain reaction and Western blotting. Cell proliferation, migration and invasion were assessed with cell viability assay, transwell assay, Western blotting, and xenograft tumor experiment. The target binding between miR-16-5p and TLN1 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: TIIA treatment inhibited cell viability, migration and invasion, and decreased Cyclin D1, matrix metalloproteinase (MMP)-9 and Vimentin expression in glioma T98G and A172 cells both in vitro and in vivo. Thus, TIIA induced anti-glioma role, wherein miR-16-5p was upregulated and TLN1 was downregulated. Moreover, silencing miR-16-5p could abate TIIA-mediated suppression on glioma cell proliferation, migration and invasion in vitro and in vivo. TLN1 overexpression also exerted tumor-promoting effect in TIIA-treated T98G and A172 cells. Mechanically, miR-16-5p could regulate TLN1 expression via target binding, and depleting TLN1 could counteract the inhibitory effect of miR-16-5p knockdown on the curative effect of TIIA in T98G and A172 cells. CONCLUSION: TIIA exerted the anti-proliferation, anti-migration and anti-invasion role in glioma cells both in vitro and in vivo partially through regulating miR-16-5p/TLN1 axis.

Investigation of the underlying genes and mechanism of familial hypercholesterolemia through bioinformatics analysis.

BACKGROUND: Familial hypercholesterolemia (FH) is one of the commonest inherited metabolic disorders. Abnormally high level of low-density lipoprotein cholesterol (LDL-C) in blood leads to premature atherosclerosis onset and a high risk of cardiovascular disease (CVD). However, the specific mechanisms of the progression process are still unclear. Our study aimed to investigate the potential differently expressed genes (DEGs) and mechanism of FH using various bioinformatic tools. METHODS: GSE13985 and GSE6054 were downloaded from the Gene Expression Omnibus (GEO) database for bioinformatic analysis in this study. First, limma package of R was used to identify DEGs between blood samples of patients with FH and those from healthy individuals. Then, the functional annotation of DEGs was carried out by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) analysis. Based on Search Tool for the Retrieval of Interacting Genes (STRING) tool, we constructed the Protein-Protein Interactions (PPIs) network among DEGs and mined the core genes as well. RESULTS: A total of 102 communal DEGs (49 up-regulated and 53 down-regulated) are identified in FH samples compared with control samples. The functional changes of DEGs are mainly associated with the focal adhere and glucagon signaling pathway. Ten genes (ITGAL, TLN1, POLR2A, CD69, GZMA, VASP, HNRNPUL1, SF1, SRRM2, ITGAV) were identified as core genes. Bioinformatic analysis showed that the core genes are mainly enriched in numerous processes related to cell adhesion, integrin-mediated signaling pathway and cell-matrix adhesion. In the transcription factor (TF) target regulating network, 219 nodes were detected, including 214 DEGs and 5 TFs (SP1, EGR3, CREB, SEF1, HOX13). In conclusion, the DEGs and hub genes identified in this study may help us understand the potential etiology of the occurrence and development of AS. CONCLUSION: Up-regulated ITGAL, TLN1, POLR2A, VASP, HNRNPUL1, SF1, SRRM2, and down-regulated CD69, GZMA and ITGAV performed important promotional effects for the formation of atherosclerotic plaques those suffering from FH. Moreover, SP1, EGR3, CREB, SEF1 and HOX13 were the potential transcription factors for DEGs and could serve as underlying targets for AS rupture prevention. These findings provide a theoretical basis for us to understand the potential etiology of the occurrence and development of AS in FH patients and we may be able to find potential diagnostic and therapeutic targets.

ALCAM (CD166) as a gene expression marker for human mesenchymal stromal cell characterisation.

BACKGROUND: Human mesenchymal stromal cells (MSCs) phenotypically share their positive expression of the International Society for Cell and Gene Therapy (ISCT) markers CD73, CD90 and CD105 with fibroblasts. Fibroblasts are often co-isolated as an unwanted by-product from biopsy and they can rapidly overgrow the MSCs in culture. Indeed, many other surface markers have been proposed, though no unique MSC specific marker has been identified yet. Quantitative PCR (qPCR) is a precise, efficient and rapid method for gene expression analysis. To identify a marker suitable for accurate MSC characterisation, qPCR was exploited. METHODS AND RESULTS: Two commercially obtained bone marrow (BM) derived MSCs and an hTERT immortalised BM-MSC line (MSC-TERT) have been cultured for different days and at different oxygen levels before RNA extraction. Together with RNA samples previous extracted from umbilical cord derived MSCs and MSC-TERT cells cultured in 2D or 3D, this heterogeneous sample set was quantitatively analysed for the expression levels of 18 candidate MSC marker genes. The expression levels in MSCs were compared with the expression levels in fibroblasts to verify the differentiation capability of these genes between MSCs and fibroblasts. None of the ISCT markers could differentiate between fibroblasts and MSCs. A total of six other genes (ALCAM, CLIC1, EDIL3, EPHA2, NECTIN2, and TMEM47) were identified as possible biomarkers for accurate identification of MSCs. CONCLUSION: Justified by considerations on expression level, reliability and specificity, Activated-Leukocyte Cell Adhesion Molecule (ALCAM) was the best candidate for improving the biomarker set of MSC identification.

miR-429 suppresses cell proliferation, migration and invasion in nasopharyngeal carcinoma by downregulation of TLN1.

BACKGROUND: miR-429 and TLN1 have been shown to affect the biological behaviours of many carcinomas. However, their effects in nasopharyngeal carcinoma (NPC) are not yet clear. Here, we investigated their regulatory relationships and effects on NPC cells. METHODS: TargetScan was used to predict the regulatory relationships of miR-429 and TLN1 in NPC cells. Then, Western blotting and quantitative real-time PCR (qPCR) were performed to examine TLN1 levels, and qPCR was used to determine miR-429 levels in NPC cell lines with different metastatic characteristics (5-8F, CNE-2, CNE-1, 6-10B and NP69), to investigate whether TLN1 and miR-429 are correlated with the metastatic characteristics of these cells. Next, we upregulated or downregulated miR-429 in 5-8F and 6-10B cells, which have different tumourigenicity and transferability, and examined TLN1 expression by western blotting and qPCR after transfection. QPCR was also performed to confirm successful transfection of miR-429 mimic into 5-8F and 6-10B cells. Dual luciferase reporter gene assay was performed to investigate whether miR-429 regulates TLN1 by binding to its 3'UTR. After transfection, Cell Counting Kit-8 (CCK8) and IncuCyte were used to examine the proliferation of these cells, and wound-healing assay, Transwell migration assay, and invasion assays were performed to investigate the changes in migration and invasion after transfection. RESULTS: Western blotting and qPCR analyses showed that the protein level of TLN1 was negatively correlated with miR-429 in NPC cell lines (P < 0.05), while the mRNA level showed no relation with miR429 expression (P > 0.05). In addition, cells with high transferability showed high TLN1 expression at the protein level, while miR429 expression showed the opposite trend (P < 0.05), but there were no differences at the mRNA level between the different cell lines. Overexpression of miR429 in 5-8F and 6-10B cells was accompanied by downregulation of TLN1 at the protein level (P < 0.05), while there were no significant differences at the mRNA level (P > 0.05). In addition, transferability, proliferation, and invasion were downregulated by miR429 overexpression (P < 0.05). However, dual-luciferase reporter gene assay indicated that TLN1 was not a direct target of miR-429. CONCLUSION: This study showed that miR-429 functions as a tumour suppressor in NPC by downregulation of TLN1, although the relationship is not direct.

microRNA-96-5p induces the epithelial-mesenchymal transition to promote the metastasis of hepatocellular carcinoma by post-transcriptionally downregulating Talin 1.

Numerous microRNAs (miRNAs) have been shown to play an important regulatory role in the progression of hepatocellular carcinoma (HCC). miR-96-5p, a cancer-related microRNA, was previously reported to inhibit cell apoptosis in HCC, but the function and underlying mechanism of miR-96-5p's involvement in HCC metastasis and progression still remain unknown. In this study, we showed that a significant up-regulation of miR-96-5p in HCC tissues and cell lines, and its increased expression, are associated with microvascular invasion and with the TNM stages of HCC patients. Gain-of-function assays revealed that miR-96-5p induced the epithelial-mesenchymal transition (EMT) to promote the migration and invasion of HCC in vitro. The expression of TLN1 (Talin 1) is significantly decreased in HCC tissues and is inversely correlated to miR-96-5p levels. Notably, through a luciferase reporter assay and a Western blot analysis, TLN1 was confirmed to be a direct target gene of miR-96-5p. Furthermore, results of cell functional assays revealed that the over-expression of TLN1 partially reverses the promotive effects of miR-96-5p overexpression on the migration, invasion, and EMT of HCC. Overall, data from the present study demonstrate that miR-96-5p induces EMT to promote the migration and invasion of HCC by post-transcriptionally downregulating TLN1, indicating that the miR-96-5p/TLN1 axis might provide a potential therapeutic target for the treatment of HCC.

Inhibition of AZIN2-sv induces neovascularization and improves prognosis after myocardial infarction by blocking ubiquitin-dependent talin1 degradation and activating the Akt pathway.

BACKGROUND: We previously found that loss of lncRNA-AZIN2 splice variant (AZIN2-sv) increases cardiomyocyte (CM) proliferation and attenuates adverse ventricular remodelling post-myocardial infarction (MI). However, whether inhibition of AZIN2-sv can simultaneously induce angiogenesis and thus improve prognosis after MI is unclear. METHODS: We used in situ hybridization and quantitative PCR to determine AZIN2-sv expression in endothelial cells. Knockdown and overexpression were performed to detect the role of AZIN2-sv in endothelial cell function, angiogenesis and prognosis after MI. RNA pulldown, RNA immunoprecipitation and luciferase reporter assays were used to determine the interaction with talin1 (Tln1) protein and miRNA-214 (miR-214). DNA pulldown and chromatin immunoprecipitation (ChIP) assays were used to study AZIN2-sv binding to upstream transcription factors. FINDINGS: AZIN2-sv was enriched in cardiac endothelial cells. The loss of AZIN2-sv reduced endothelial cell apoptosis and promoted endothelial sprouting and capillary network formation in vitro. Moreover, in vivo, the loss of AZIN2-sv induced angiogenesis and improved cardiac function after MI. Mechanistically, AZIN2-sv reduced Tln1 and integrin ß1 (ITGB1) protein levels to inhibit neovascularization. AZIN2-sv activated the ubiquitination-dependent degradation of Tln1 mediated by proteasome 26S subunit ATPase 5 (PSMC5). In addition, AZIN2-sv could bind to miR-214 and suppress the phosphatase and tensin homologue (PTEN)/Akt pathway to inhibit angiogenesis. With regard to the upstream mechanism, Bach1, a negative regulator of angiogenesis, bound to the promoter of AZIN2-sv and increased its expression. INTERPRETATION: Bach1-activated AZIN2-sv could participate in angiogenesis by promoting the PSMC5-mediated ubiquitination-dependent degradation of Tln1 and blocking the miR-214/PTEN/Akt pathway. Inhibition of AZIN2-sv induced angiogenesis and myocardial regeneration simultaneously, thus, AZIN2-sv could be an ideal therapeutic target for improving myocardial repair after MI. FUND: National Natural Science Foundations of China.

Hepatitis B Virus Protein X Induces Degradation of Talin-1.

In the infected human hepatocyte, expression of the hepatitis B virus (HBV) accessory protein X (HBx) is essential to maintain viral replication in vivo. HBx critically interacts with the host damaged DNA binding protein 1 (DDB1) and the associated ubiquitin ligase machinery, suggesting that HBx functions by inducing the degradation of host proteins. To identify such host proteins, we systematically analyzed the HBx interactome. One HBx interacting protein, talin-1 (TLN1), was proteasomally degraded upon HBx expression. Further analysis showed that TLN1 levels indeed modulate HBV transcriptional activity in an HBx-dependent manner. This indicates that HBx-mediated TLN1 degradation is essential and sufficient to stimulate HBV replication. Our data show that TLN1 can act as a viral restriction factor that suppresses HBV replication, and suggest that the HBx relieves this restriction by inducing TLN1 degradation.

Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Talin (tln) binds and activates integrins to couple extracellular matrix-bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1(fl02k) mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin ß1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgß1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.

Novel serum autoantibodies against talin1 in multiple sclerosis: Possible pathogenetic roles of the antibodies.

In the pathogenesis of multiple sclerosis (MS), B cell/antibody-related mechanisms have recently received attention. To investigate the role of autoantibody in MS, we performed SEREX which can identify autoantibody cyclopedically. We identified serum antibodies against cytoskeletal protein talin1, and the levels of whom were remarkably higher in 39 MS than 43 normal controls (P < 0.01) and 35 disease controls (P = 0.06), and in MS patients without oligoclonal bands than ones with them. Moreover, we found negative-correlations between serum anti-talin1 antibody and IgG index in MS (P = 0.03). Anti-talin1 antibody exists in MS patients' sera, which may have some protective factor.