CD204-positive M2-like tumor-associated macrophages increase migration of gastric cancer cells by upregulating miR-210 to reduce NTN4 expression.

BACKGROUND: Tumor-associated macrophages (TAMs) are the predominant immune cells in the tumor microenvironment and portend poor prognosis. However, the molecular mechanisms underlying the tumor promotion of TAMs have not been fully elucidated. METHODS: Coculture of gastric cancer cells with U937 cells was performed to investigate the impact of TAMs on cancer cell behavior. MicroRNA (miRNA) microarray and bioinformatics were applied to identify the involved miRNAs and the functional target genes. The regulation of the miRNA on its target gene was studied using anti-miRNA and miRNA mimic. RESULTS: Coculture with CD204+ M2-like TAMs increased proliferation, migration, and epithelial-mesenchymal transition of gastric cancer cells. MiR-210 was the most upregulated miRNA in cancer cells identified by miRNA microarray after coculture. In gastric cancer tissues, miR-210 expression was positively correlated with CD204+ M2-like TAM infiltration. Inactivation of miR-210 by antimir attenuated CD204+ M2-like TAMs-induced cancer cell migration. Using pharmacological inhibitors and neutralizing antibodies, CD204+ M2-like TAMs-secreted TNFα was found to upregulate miR-210 through NF-κB/HIF-1α signaling. Bioinformatics analysis showed netrin-4 (NTN4) as a potential target of miR-210 to suppress gastric cancer cell migration. We also found an inverse expression between miR-210 and NTN4 in cancer cells after coculture or in tumor xenografts. Anti-miR-210 increased NTN4 expression, while miR-210 mimics downregulated NTN4 in cancer cells. Reporter luciferase assays showed that MiR-210 mimics suppressed NTN4 3' untranslated region-driven luciferase activity in cancer cells, but this effect was blocked after mutating miR-210 binding site. CONCLUSIONS: CD204+ M2-like TAMs can utilize the TNF-α/NF-κB/HIF-1α/miR-210/NTN4 pathway to facilitate gastric cancer progression.

eQTL Colocalization Analyses Identify NTN4 as a Candidate Breast Cancer Risk Gene.

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

Key Anti-Fibrosis Associated Long Noncoding RNAs Identified in Human Hepatic Stellate Cell via Transcriptome Sequencing Analysis.

Hepatic fibrosis is the main pathological basis for chronic cirrhosis, and activated hepatic stellate cells (HSCs) are the primary cells involved in liver fibrosis. Our study analyzed anti-fibrosis long noncoding RNAs (lncRNAs) in activated human HSCs (hHSCs). We performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine whether lncRNA expression profile changes between hHSCs activation and quiescence. Eight differentially expressed (DE) lncRNAs and three pairs of co-expression lncRNAs-mRNAs were verified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). A total of 34146 DE lncRNAs were identified in this study. Via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we found several DE lncRNAs regulated hHSC activation by participating in DNA bending/packaging complex, growth factor binding and the Hippo signaling pathway (p < 0.05). With lncRNA-mRNA co-expression analysis, three lncRNAs were identified to be associated with connective tissue growth factor (CTGF), fibroblast growth factor 2 (FGF2) and netrin-4 (NTN4). The quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results of the eight DE lncRNAs and three pairs of co-expression lncRNAs-mRNAs were consistent with the RNA-seq data and previous reports. Several lncRNAs may serve as potential targets to reverse the progression of liver fibrosis. This study provides a first insight into lncRNA expression profile changes associated with activated human HSCs.

EXOSC5 maintains cancer stem cell activity in endometrial cancer by regulating the NTN4/integrin ß1 signalling axis.

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin ß1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin ß1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment.

3'UTR variants of TNS3, PHLDB1, NTN4, and GNG2 genes are associated with IgA nephropathy risk in Chinese Han population.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis and is characterized by mesangial cell proliferation and agglomeration of the mesangial matrix. METHODS: In this study, we aimed to explore the role of TNS3, PHLDB1, NTN4, and GNG2 3'untranslated region (3'UTR) polymorphisms with the risk of IgAN in a Chinese Han cohort. A logistic recession model was used to calculate the effects of candidate single nucleotide polymorphism (SNP) on IgAN risk after adjusting age and gender difference. In silico prediction was conducted to identify potential functions of SNPs. RESULTS: The analysis revealed a significant relationship between the homozygotic genotype for NTN4 rs1362970 A/A and higher risk of IgAN (p = 0.003). Statistically significant associations were found when the sample was stratified by gender and Lee's grade. As a result, NTN4 rs1362970 A/A and GNG2 rs3204008 G/G genotypes were associated with enhanced IgAN risk in males (p = 0.006, p = 0.023, respectively), and the association between the PHLDB1 rs7389 G/T genotype and higher IgAN risk was found in females (p = 0.008). In the Lee's grade III-V subgroup, the rs1369270 in NTN4 was significantly correlated with the risk of IgAN (p = 0.004). Bioinformatics prediction suggested that rs1362970 within NTN4 3'UTR was located in the potential target sequence of miR-483-5p. CONCLUSIONS: Our research confirmed that NTN4, GNG2, and PHLDB1 gene polymorphisms were implicated in IgAN susceptibility in Chinese Han population. Further research should be conducted to investigate and validate the mechanism by which the above-mentioned polymorphisms affect IgAN.

miR-17-5p promotes migration and invasion in breast cancer cells by repressing netrin 4.

Netrin 4 (NTN4) is downregulated in breast cancer (BC) and can inhibit the migration of BC cells. miRNAs dysregulation plays prominent roles in BC tumorigenesis. However, the function of miR-17-5p, its relationship with NTN4 and its underlying functional mechanism in BC are unclear and were investigated in the current study. Compared with normal breast samples, miR-17-5p was upregulated in BC specimens in The Cancer Genome Atlas (TCGA). A clinical analysis based on TCGA showed that miR-17-5p expression correlated with BC tumor stage, lymph node status, estrogen receptor, and progesterone receptor status. A wound-healing assay and Transwell assay implied that miR-17-5p upregulation promotes BC cell migration and invasion. Reverse transcription-quantitative PCR and ELISA showed that NTN4 mRNA and protein were both downregulated after miR-17-5p was overexpressed in Hs578T cells, whereas miR-17-5p inhibition had the opposite effect in MCF-7 cells. We also performed a dual-fluorescent reporter assay, the results of which demonstrated that miR-17-5p represses NTN4 expression by directly targeting the 3' untranslated region of NTN4 mRNA. In summary, miR-17-5p considerably promotes BC cell migration by suppressing NTN4 expression, and may therefore offer a potential therapeutic target for BC.

Investigation of SNP rs2060546 Immediately Upstream to NTN4 in a Danish Gilles de la Tourette Syndrome Cohort.

Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder characterized by multiple motor and vocal tics. GTS is a complex disorder, with environmental factors and several genes involved. Although variations within a few genes such as AADAC, NRXN1, SLITRK1, HDC, and IMMP2L have been tentatively associated with GTS (in a small number of patients), the causative genes underlying GTS pathophysiology remain unknown. In a previous genome-wide association study (GWAS) a single nucleotide polymorphism (SNP, rs2060546) near the Netrin-4 (NTN4 - MIM 610401) gene was shown to be associated with GTS [odds ratio (OR) = 1.7; p-value = 5.8 × 10-7] thus warranting further investigations. As NTN4 is one of the axon guidance molecules expressed in the central nervous system and it interacts with the encoded proteins of SLIT and WNT genes guiding the growth cone toward its target, it is an attractive candidate susceptibility gene for GTS. In this study we attempted to replicate the association of rs2060546 with GTS by genotyping a Danish cohort of 240 GTS patients and 1006 healthy controls. Our results did not reveal an association (OR = 1.363; p-value = 0.3329) in the Danish cohort alone, which may be due to the small sample size. However, a meta-analysis including the present cohort and a total of 1316 GTS patients and 5023 controls from the GTS GWAS Replication Initiative (GGRI) and the first GTS-GWAS yielded a significant signal (OR = 3.74; p-value = 0.00018) and same direction of effect in the three cohorts. Thus, our study strengthens the evidence of the possible involvement of NTN4 in GTS etiology, suggesting that further studies in even larger samples and functional studies are warranted to investigate the role of this region in GTS pathogenesis.