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.

The prognostic value of LAYN in HPV-related head and neck squamous cell carcinoma and its influence on immune cell infiltration.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma (HNSCC) exhibits different characteristics from HPV-negative tumors in terms of tumor development, clinical features, treatment response, and prognosis. Layilin (LAYN), which contains homology with C-type lectins, plays a critical role in tumorigenesis and cancer progression. However, the prognostic value of LAYN and the relationship between LAYN and immune infiltration levels in HPV-related HNSCC patients still require a comprehensive understanding. Herein, we aimed to assess the prognostic value of LAYN and to investigate its underlying immunological function in HPV-related HNSCC. METHODS: Through various bioinformatics methods, we analyzed the data from The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) databases to explore the potential underlying oncogenic impression of LAYN, including the relevance of LAYN to survival outcomes, clinicopathological factors, immune cell infiltration, and immune marker sets in HPV-related HNSCC. The expression levels of LAYN and HPV were also verified in HNSCC patient tissues. RESULTS: LAYN was differentially expressed in a variety of tumors. The expression of LAYN in HNSCC was significantly higher than that in adjacent normal tissues (P < 0.0001), and high expression of LAYN was correlated with poor overall survival (OS) in HNSCC patients (Hazard Ratio (HR) = 1.3, P = 0.035). Moreover, LAYN expression level in HPV-positive HNSCC patients was significantly lower than that in HPV-negative patients, with HPV-positive HNSCC patients displaying a trend of favorable prognosis. In addition, the relationship between LAYN expression and immune infiltration levels in HPV-positive HNSCC group was less tightly correlated than that in HPV-negative HNSCC group, and there was a strong relationship between LAYN expression and markers of M2 macrophage (P < 0.001) and exhausted T cells (P < 0.05) in HPV-negative HNSCC. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis suggested that LAYN potentially influenced tumor progression through HPV infection and other cancer-related pathways. CONCLUSIONS: LAYN might contribute to tumorigenesis via its positive correlation with immune checkpoint molecules and tumor-associated macrophages (TAMs). Our study might provide a novel prognostic biomarker and latent therapeutic target for the treatment of HPV-related HNSCC.

Comprehensive landscape of the miRNA-regulated prognostic marker LAYN with immune infiltration and stemness in pan-cancer.

BACKGROUND: COVID-19 has created a significant risk to worldwide public health. According to recent research, C-type lectins may be SARS-CoV-2 receptors. Layilin (LAYN), a broadly expressed integral membrane hyaluronan receptor with a C-type lectin structural domain, is a gene related to cell senescence. There are a few studies on C-type lectins in pan-cancer, and no pan-cancer analysis has been conducted for LAYN. METHODS: The genotype tissue expression (GTEx) portal and the cancer genome map (TCGA) database were used to collect samples from healthy and cancer patients. Bioinformatics methods are used to construct immune landscape, mutation landscape, and stemness landscape of LAYN. The single-cell sequencing data were used from the CancerSEA website to analyze the functions of LAYN. The prognosis potential of LAYN was discussed based on machine learning. RESULTS: LAYN is differentially expressed among cancers. Survival analysis indicated that LAYN was related to a poor overall survival (OS) rate in cancers, like HNSC, MESO, and OV. Mutational landscapes of LAYN in SKCM and STAD were constructed. LAYN was negatively related to Tumor Mutation Burden (TMB) in THCA, PRAD, and UCEC, and with the Microsatellite Instability (MSI) in STAD, LUAD, and UCEC. The immune landscape in pan-cancer suggested that LAYN may be involved in tumor immune escape. LAYN plays a crucial role in the infiltration of immune cells in malignant tumors. LAYN participates in methylation modifications and affects tumor proliferation and metastasis by regulating stemness. Analysis of single-cell sequencing data suggests that LAYN may participate in several biological processes, like stemness, apoptosis, and DNA repair. LAYN transcript was predicted as a liquid-liquid phase separation (LLPS)-related RNA. The results of KIRC were verified in the GEO and ArrayExpress databases. Furthermore, prognostic models based on machine learning of LAYN-related genes were established. Hsa-miR-153-5p and hsa-miR-505-3p may be the upstream miRNAs of LAYN and have a high value for tumor prognosis. CONCLUSION: This study elucidated the functional mechanisms of LAYN from a pan-cancer perspective and provided novel insights into cancer prognosis, metastasis, and immunotherapy. LAYN has the potential to become a new target of mRNA vaccines and molecular therapies in tumors.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

Targeting LAYN inhibits colorectal cancer metastasis and tumor-associated macrophage infiltration induced by hyaluronan oligosaccharides.

The accumulation of hyaluronan oligosaccharides (oHA) in colorectal cancer (CRC) is closely related to tumor metastasis, but the underlying mechanism remains unclear. In this study, we first described that LAYN, a novel HA receptor, was upregulated in CRC tissue. Aberrant LAYN expression correlated with CRC metastasis and poor prognosis and positively correlated with tumor-associated macrophage (TAM) infiltration and M2 macrophage polarization in the tumor environment. Both in vitro and in vivo studies demonstrated that LAYN is activated by oHA and subsequently induces CRC metastasis and macrophage infiltration. Mechanistic studies demonstrated that oHA activates LAYN by binding to the 60-68th amino acid region of the extracellular segment. oHA-induced LAYN activation promoted metastasis and CCL20 secretion through the NF-kB pathway in CRC cells. Furthermore, targeting LAYN using a blocking antibody prevented oHA-mediated tumor metastasis, TAM infiltration and M2 polarization. This study revealed the LAYN activation mechanism and identified a potential target for the treatment of CRC tumor exhibiting high oHA levels.

Low-dose anti-VEGFR2 therapy promotes anti-tumor immunity in lung adenocarcinoma by down-regulating the expression of layilin on tumor-infiltrating CD8+T cells.

PURPOSE: Our study intended to explore how low-dose anti-angiogenic drugs affected anti-tumor immunity of tumor-infiltrating exhausted CD8+T cells and achieved better clinical response when combined with immunotherapy. We set out to find potential targets or predictive biomarker on CD8+T cells for immunotherapy. METHODS: We tested different doses of anti-VEGFR2 antibody combined with anti-PD1 antibody to treat LUAD in vivo and analyzed tumor-infiltrating CD8+T cells by flow cytometry. CD8+T cells overexpressing LAYN were co-cultured with LA795 cell lines to identify the function of LAYN in CD8+T cells. We also analyzed clinical samples from advanced LUAD patients treated with anti-angiogenesis therapy combined with immunotherapy. RESULTS: Low-dose anti-VEGFR2 antibody combined with anti-PD1 antibody treatment delayed tumor growth and prolonged the survival time of tumor-bearing mice. The number of tumor-infiltrating CD8+T cells was reduced and the expression of LAYN was down-regulated in tumor-infiltrating CD8+T cells in the low-dose anti-VEGFR2 combination group. It was found that LAYN inhibited the killing function of CD8+T cells. In patients with advanced LUAD who received anti-angiogenesis therapy combined with immunotherapy, the LAYN+CD8+T cell subpopulation in good responders was significantly higher than that in poor responders. Furthermore, we demonstrated the expression of LAYN was regulated by upstream transcription factor NR4A1. CONCLUSION: Low-dose anti-VEGFR2 antibody combined with anti-PD1 antibody therapy promoted anti-tumor immunity and the downregulation of LAYN in tumor-infiltrating CD8+T cells played an important role in this process. These findings had implications for improving the efficacy of immune checkpoint blockade therapy and further optimized clinical treatment guidelines in advanced LUAD.

LAYN Is a Prognostic Biomarker and Correlated With Immune Infiltrates in Gastric and Colon Cancers.

Background: Layilin (LAYN) is a critical gene that regulates T cell function. However, the correlations of LAYN to prognosis and tumor-infiltrating lymphocytes in different cancers remain unclear. Methods: LAYN expression was analyzed via the Oncomine database and Tumor Immune Estimation Resource (TIMER) site. We evaluated the influence of LAYN on clinical prognosis using Kaplan-Meier plotter, the PrognoScan database and Gene Expression Profiling Interactive Analysis (GEPIA). The correlations between LAYN and cancer immune infiltrates was investigated via TIMER. In addition, correlations between LAYN expression and gene marker sets of immune infiltrates were analyzed by TIMER and GEPIA. Results: A cohort (GSE17536) of colorectal cancer patients showed that high LAYN expression was associated with poorer overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). In addition, high LAYN expression was significantly correlated with poor OS and progression-free survival (PFS) in gastric cancers (OS HR = 1.97, P = 3.6e-10; PFS HR = 2.12, P = 2.3e-10). Moreover, LAYN significantly impacts the prognosis of diverse cancers via The Cancer Genome Atlas (TCGA). Specifically, high LAYN expression was correlated with worse OS and PFS in stage 2 to 4 but not stage 1 and stage N0 gastric cancer patients (P = 0.28, 0.34; P = 0.073, 0.092). LAYN expression was positively correlated with infiltrating levels of CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells (DCs) in colon adenocarcinoma (COAD) and stomach adenocarcinoma (STAD). LAYN expression showed strong correlations with diverse immune marker sets in COAD and STAD. Conclusions: These findings suggest that LAYN is correlated with prognosis and immune infiltrating levels of, including those of CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and DCs in multiple cancers, especially in colon and gastric cancer patients. In addition, LAYN expression potentially contributes to regulation of tumor-associated macrophages (TAMs), DCs, T cell exhaustion and Tregs in colon and gastric cancer. These findings suggest that LAYN can be used as a prognostic biomarker for determining prognosis and immune infiltration in gastric and colon cancers.

Transcriptome profiling of human FoxP3+ regulatory T cells.

The major goal of this study was to perform an in depth characterization of the "gene signature" of human FoxP3(+) T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs.