Reclassification of an FBN1 variant emphasizes the importance of segregation analysis, information sharing, and multidisciplinary teamwork in understanding genetic variants in health and disease.

Marfan syndrome (MFS) is a complex connective tissue disorder characterized by considerable clinical variability. The diagnosis of MFS is based on the Ghent criteria, which require the presence of both clinical and genetic features. MFS is primarily caused by pathogenic alterations in FBN1, which encodes the fibrillin-1 protein. Fibrillin-1 comprises multiple domains rich in cysteine residues, with disulfide bonds formed between these residues. It has long been recognized that variants that alter or introduce cysteine residues damage protein function, leading to the development of MFS. In this study, we report a cysteine-introducing variant: FBN1 variant, c.6724C>T (p.[Arg2242Cys]). We have observed this variant in several individuals without MFS, challenging our previous understanding of the underlying mechanism of MFS. This finding emphasizes the importance of revisiting and reevaluating our current knowledge in light of new and unexpected observations. Moreover, our study highlights the significance of incorporating local and national data on allele frequencies, as well as employing multidisciplinary phenotyping approaches, in the classification of genetic variants. By considering a wide range of information, we can enhance the accuracy and reliability of variant classification, ultimately improving the diagnosis and management of individuals with genetic disorders like MFS.

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/ß-catenin signaling.

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/ß-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/ß-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/ß-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/ß-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/ß-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

Analysis of tumor microenvironment alterations in partially responsive rectal cancer patients treated with neoadjuvant chemoradiotherapy.

PURPOSE: Achieving a pathologic complete response (pCR) after neoadjuvant chemoradiotherapy (NCRT) remains a challenge for most patients with rectal cancer. Exploring the potential of combining NCRT with immunotherapy or targeted therapy for those achieving a partial response (PR) offers a promising avenue to enhance treatment efficacy. This study investigated the impact of NCRT on the tumor microenvironment in locally advanced rectal cancer (LARC) patients who exhibited a PR. METHODS: This was a retrospective, observational study. Five patients demonstrating a PR after neoadjuvant treatment for LARC were enrolled in the study. Biopsy samples before treatment and resected specimens after treatment were stained with a panel of 26 antibodies targeting various immune and tumor-related markers, each labeled with distinct metal tags. The labeled samples were then analyzed using the Hyperion imaging system. RESULTS: Heterogeneity within the tumor microenvironment was observed both before and after NCRT. Notably, tumor-associated macrophages, CD4 + T cells, CD8 + T cells, CD56 + natural killer cells, tumor-associated neutrophils, cytokeratin, and E-cadherin exhibited slight increase in abundance within the tumor microenvironment following treatment (change ratios = 0.78, 0.2, 0.27, 0.32, 0.17, 0.46, 0.32, respectively). Conversely, the number of CD14 + monocytes, CD19 + B cells, CD45 + CD4 + T cells, collagen I, α-smooth muscle actin, vimentin, and ß-catenin proteins displayed significant decreases post-treatment (change ratios = 1.73, 1.92, 1.52, 1.25, 1.52, 1.12, 2.66, respectively). Meanwhile, Foxp3 + regulatory cells demonstrated no significant change (change ratio = 0.001). CONCLUSIONS: NCRT has diverse effects on various components of the tumor microenvironment in LARC patients who achieve a PR after treatment. Leveraging combination therapies may optimize treatment outcomes in this patient population.

Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection.

We have recently reported that transcription factor Runx3 is required for pulmonary generation of CD8+ cytotoxic T lymphocytes (CTLs) that play a crucial role in the clearance of influenza A virus (IAV). To understand the underlying mechanisms, we determined the effects of Runx3 knockout (KO) on CD8+ T cell local expansion and phenotypes using an inducible general Runx3 KO mouse model. We found that in contrast to the lungs, Runx3 general KO promoted enlargement of lung-draining mediastinal lymph node (mLN) and enhanced CD8+ and CD4+ T cell expansion during H1N1 IAV infection. We further found that Runx3 deficiency greatly inhibited core 2 O-glycosylation of selectin ligand CD43 on activated CD8+ T cells but minimally affected the cell surface expression of CD43, activation markers (CD44 and CD69) and cell adhesion molecules (CD11a and CD54). Runx3 KO had a minor effect on lung effector CD8+ T cell death by IAV infection. Our findings indicate that Runx3 differently regulates CD8+ T cell expansion in mLNs and lungs by H1N1 IAV infection. Runx3 is required for CD43 core 2 O-glycosylation on activated CD8+ T cells, and the involved Runx3 signal pathway may mediate CD8+ T cell phenotype for pulmonary generation of CTLs.

Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant.

BACKGROUND: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype. METHODS: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells. RESULTS: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability. CONCLUSIONS: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Hsa_circRNA_001676 accelerates the proliferation, migration and stemness in colorectal cancer through regulating miR-556-3p/G3BP2 axis.

Circular RNAs (circRNAs) play key roles in colorectal cancer (CRC) progression, but little is known about the biological functions of hsa_circRNA_001676 in CRC. Therefore, we explored the potential role of hsa_circRNA_001676 in CRC development. RT-qPCR was performed to determine hsa_circRNA_001676, miR-556-3p and Ras-GTPase-activating SH3 domain-binding-proteins 2 (G3BP2) levels in CRC tissues. Meanwhile, to evaluate the roles of hsa_circRNA_001676, miR-556-3p and G3BP2 on CRC, functional analysis of cell proliferation, migration and stemness were then performed. Our results showed that compared to normal tissues, hsa_circRNA_001676 and G3BP2 level was elevated, but miR-556-3p level was reduced in CRC tissues. Additionally, luciferase reporter results showed that hsa_circRNA_001676 was shown to target miR-556-3p, and G3BP2 was targeted by miR-556-3p. Hsa_circRNA_001676 or G3BP2 overexpression promoted CRC cell proliferation and migration. Conversely, miR-556-3p overexpression suppressed CRC cell proliferation and migration. Moreover, deficiency of hsa_circRNA_001676 or G3BP2 repressed the CRC cell proliferation, migration and stemness. Meanwhile, hsa_circRNA_001676 deficiency obviously reduced tumor growth and stemness in a CRC mouse xenograft model. Furthermore, hsa_circRNA_001676 deficiency notably reduced G3BP2 level, but elevated miR-556-3p level in tumor tissues from tumor-bearing mice. Mechanistically, hsa_circRNA_001676 targeted miR-556-3p to increase G3BP2 level, contributing to the progression of CRC. Collectively, hsa_circRNA_001676 was able to accelerate proliferation, migration and stemness in CRC through regulating miR-556-3p/G3BP2 axis, suggesting that hsa_circRNA_001676 may become a potential therapeutic target in treating CRC.

A Novel CDC42 Variant with Impaired Thymopoiesis, IL-7R Signaling, PAK1 Binding, and TCR Repertoire Diversity.

Genetic variants in cell division cycle 42 (CDC42) can manifest with dysmorphic features, autoinflammation, hemophagocytic lymphohistiocytosis, and thrombocytopenia, whereas defective thymopoiesis is a rare disease manifestation. We report a novel CDC42 missense variant (c.46A > G, p.Lys16Glu) resulting in infection and HPV-driven carcinogenesis in the mosaic mother and impaired thymopoiesis and profound T cell lymphopenia in the heterozygous daughter identified through newborn screening for SCID. We found that surface expression of IL-7Rα (CD127) was decreased, consistent with reduced IL-7-induced STAT5 phosphorylation and accelerated apoptotic T cell death. Consistent with the vital role of IL-7 in regulating thymopoiesis, both patients displayed reduced T cell receptor CDR3 repertoires. Moreover, the CDC42 variant prevented binding to the downstream effector, p21-activated kinase (PAK)1, suggesting this impaired interaction to underlie reduced IL-7Rα expression and signaling. Here, we provide the first report of severely compromised thymopoiesis and perturbed IL-7Rα signaling caused by a novel CDC42 variant and presenting with diverging clinical and immunological phenotypes in patients.

Validation of the BOADICEA model for predicting the likelihood of carrying pathogenic variants in eight breast and ovarian cancer susceptibility genes.

BOADICEA is a comprehensive risk prediction model for breast and/or ovarian cancer (BC/OC) and for carrying pathogenic variants (PVs) in cancer susceptibility genes. In addition to BRCA1 and BRCA2, BOADICEA version 6 includes PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D. To validate its predictions for these genes, we conducted a retrospective study including 2033 individuals counselled at clinical genetics departments in Denmark. All counselees underwent comprehensive genetic testing by next generation sequencing on suspicion of hereditary susceptibility to BC/OC. Likelihoods of PVs were predicted from information about diagnosis, family history and tumour pathology. Calibration was examined using the observed-to-expected ratio (O/E) and discrimination using the area under the receiver operating characteristics curve (AUC). The O/E was 1.11 (95% CI 0.97-1.26) for all genes combined. At sub-categories of predicted likelihood, the model performed well with limited misestimation at the extremes of predicted likelihood. Discrimination was acceptable with an AUC of 0.70 (95% CI 0.66-0.74), although discrimination was better for BRCA1 and BRCA2 than for the other genes in the model. This suggests that BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing for hereditary susceptibility to BC/OC despite suboptimal calibration for individual genes in this population.

An effective tool for tracking steroids and their metabolites at the watershed level: Combining fugacity modeling and a chemical indicator.

Steroids have attracted concern worldwide because of their potential carcinogenicity and severe adverse effects on aquatic organisms. However, the contamination status of various steroids, particularly their metabolites, at the watershed level remains unknown. This was the first study to employ field investigations to elucidate the spatiotemporal patterns, riverine fluxes, and mass inventories, and conduct a risk assessment of 22 steroids and their metabolites. This study also developed an effective tool for predicting the target steroids and their metabolites in a typical watershed based on the fugacity model combined with a chemical indicator. Thirteen steroids in the river water and seven steroids in sediments were identified with total concentrations of 1.0-76 ng/L and

Inducible general knockout of Runx3 profoundly reduces pulmonary cytotoxic CD8+ T cells with minimal effect on outcomes in mice following influenza infection.

Respiratory viruses pose a continuing and substantive threat to human health globally. Host innate and adaptive immune responses are the critical antiviral defense mechanisms to control viral replication and spread. The present study is designed to determine the role of transcription factor Runx3 in the host immune response to influenza A virus (IAV) infection. As Runx3 is required for embryonic development, we generated an inducible Runx3 global knockout (KO) mouse model and found that Runx3 KO in adult C57BL/6 mice minimally affected thymic function under normal conditions and survival was at least 250 days post Runx3 deletion. We applied the mouse model to IAV infection and found that Runx3 KO resulted in a huge reduction (>85%) in numbers of total and antigen-specific pulmonary CD8+ cytotoxic T cells during IAV infection, while it had a minor effect on pulmonary generation of CD4+ T cells. To our surprise, this general KO of Runx3 did not significantly alter viral clearance and animal survival following IAV infection. Interestingly, we found that Runx3 KO significantly increased the numbers of pulmonary innate immune cells such as macrophages and neutrophils and the production of pro-inflammatory cytokines during IAV infection. We further found that Runx3 was strongly detected in CCR2+ immune cells in IAV-infected mouse lungs and was induced in activated macrophages and dendritic cells (DCs). As pulmonary CD8+ cytotoxic T cells play a central role in the clearance of IAV, our findings suggest that Runx3 KO may enhance host innate immunity to compensate for the loss of pulmonary CD8+ cytotoxic T cells during IAV infection.

Interferon-γ Preferentially Promotes Necroptosis of Lung Epithelial Cells by Upregulating MLKL.

Necroptosis, a form of programmed lytic cell death, has emerged as a driving factor in the pathogenesis of acute lung injury (ALI). As ALI is often associated with a cytokine storm, we determined whether pro-inflammatory cytokines modulate the susceptibility of lung cells to necroptosis and which mediators dominate to control necroptosis. In this study, we pretreated/primed mouse primary lung epithelial and endothelial cells with various inflammatory mediators and assessed cell type-dependent responses to different necroptosis inducers and their underlying mechanisms. We found that interferon-γ (IFNγ) as low as 1 ng/mL preferentially promoted necroptosis and accelerated the release of damage-associated molecular patterns from primary alveolar and airway epithelial cells but not lung microvascular endothelial cells. Type-I IFNα was about fifty-fold less effective than IFNγ. Conversely, TNFα or agonists of Toll-like receptor-3 (TLR3), TLR4, TLR7 and TLR9 had a minor effect. The enhanced necroptosis in IFNγ-activated lung epithelial cells was dependent on IFNγ signaling and receptor-interacting protein kinase-3. We further showed that necroptosis effector mixed lineage kinase domain-like protein (MLKL) was predominantly induced by IFNγ, contributing to the enhanced necroptosis in lung epithelial cells. Collectively, our findings indicate that IFNγ is a potent enhancer of lung epithelial cell susceptibility to necroptosis.

Scalable Biosynthetic Production of Knotted Peptides Enables ADME and Thermodynamic Folding Studies.

Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.

Mucin 1 promotes tumor progression through activating WNT/ß-catenin signaling pathway in intrahepatic cholangiocarcinoma.

Background: Current treatment options for intrahepatic cholangiocarcinoma (ICC) are limited by the lack of understanding of the disease pathogenesis. It has been known that mucin 1 (MUC1) is a cell surface mucin that highly expressed in various cancer tissues. However, its role in ICC has not been well studied. The purpose of this study was to investigate the clinical significance and biological function of MUC1 in ICC. Methods: qRT-PCR and western blot assays were performed to examine MUC1 expression. RNA-Seq (RNA Sequencing) s conducted to explore the RNA expression. A tissue microarray study including 214 ICC cases was also conducted to evaluate the clinical relevance and prognostic significance of MUC1. The role and underlying mechanisms of MUC1 in regulating cell growth and invasion were further explored both in vitro and in vivo models. Results: The mRNA and protein levels of MUC1 were significantly up-regulated in ICC compared to paired non-tumor tissues. Depletion of MUC1 in HCCC9810 cells significantly inhibited cell proliferation, migration and invasion in vitro and overexpression of MUC1 in RBE cells resulted in increased cell proliferation, migration and invasion. Both univariate and multivariate analysis revealed that the protein expression of MUC1 was associated with overall survival and relapse-free survival after tumor resection. Clinically, high MUC1 expression was more commonly observed in aggressive tumors. Further studies indicated that MUC1 exerted its function through activating Wnt/ ß-catenin pathway. Conclusions: Our data suggests that MUC1 promoted ICC progression via activating Wnt / ß-catenin pathway. This study not only deciphered the role of MUC in ICC pathogenesis, but also shed light upon identifying novel potential therapeutic targets.

MicroRNA-24-3p Inhibits Microglia Inflammation by Regulating MK2 Following Spinal Cord Injury.

Spinal cord injury (SCI) is a functional impairment of the spinal cord caused by external forces, accompanied by limb movement disorders and permanent paralysis, which seriously lowers the life quality of SCI patients. Secondary injury caused by inflammation attenuated the therapeutic effects of SCI. Therefore, the exploration of biomarkers associated with the inflammatory response following SCI might provide novel therapy strategy against SCI.SCI rat model was established as previously reported and evaluated by BBB score. The expression of microRNA-24-3p (miR-24-3p) and MAPK-activated protein kinase 2 (MK2) in spinal cord tissues of SCI rats and HAPI cells was analyzed by qRT-PCR. Protein expression of MK2, ionized calcium-binding adapter molecule-1 (Iba-1), tumor necrosis factor-alpha (TNF-α), and interleukin-1ß (IL-1ß) was assessed by western blot assay. The release of inflammatory cytokines TNF-α and IL-1ß was measured by enzyme-linked immunosorbent assay (ELISA). The interaction between miR-24-3p and MK2 was examined by the luciferase reporter system. Basso-Beattie-Bresnahan (BBB) score dramatically reduced in rats following SCI compared with sham rats. Moreover, the expression of miR-24-3p was down-regulated, while MK2 was up-regulated in the spinal cord tissues of SCI rats and LPS-induced microglia cells compared with the corresponding control group. Luciferase reporter system confirmed the interaction between miR-24-3p and MK2. In addition, miR-24-3p upregulation or MK2 knockdown attenuated LPS induced activation of microglial cells and expression of inflammatory cytokine TNF-α and IL-1ß. Besides, we discovered that miR-24-3p regulated inflammation of highly aggressively proliferating immortalized (HAPI) cells by targeting MK2.In our study, we clarified that miR-24-3p repressed inflammation of microglia cells following SCI by regulating MK2, thereby providing promising biomarkers for SCI therapy.

Enhanced RIPK3 kinase activity-dependent lytic cell death in M1 but not M2 macrophages.

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

hsa_circRNA_000166 Facilitated Cell Growth and Limited Apoptosis through Targeting miR-326/LASP1 Axis in Colorectal Cancer.

Circular RNAs (circRNAs) belong to noncoding RNAs and are widely expressed in a variety of cell species, including cancers. However, the function and mechanism of circRNAs in colorectal cancer (CRC) has not been well investigated. Here, we firstly downloaded and analyzed the circRNA expression profile of CRC from the Gene Expression Omnibus (GEO) database. And we identified 181 differentially expressed circRNAs between 10 pairs of CRC and adjacent normal tissues. Interestingly, we observed that the expression of hsa_circRNA_000166 was the top increased among these circRNAs. Then, we confirmed an upregulation of hsa_circRNA_000166 in CRC tissues and cell lines and observed that higher expression of hsa_circRNA_000166 was associated with poor 5-year survival rate of patients with CRC. Next, we investigated the function of hsa_circRNA_000166 during CRC progression by knocking down its expression. Cell growth and apoptosis assay revealed that hsa_circRNA_000166 regulated the cell growth and apoptosis in CRC cell lines. Furthermore, we identified that hsa_circRNA_000166 targeted the miR-326/LASP1 pathway using bioinformatics analysis and luciferase reporter assay. Finally, suppression of miR-326 or overexpression of LASP1 could sufficiently rescue the aberrant cell growth and apoptosis in CRC cell lines. Taken together, our results indicated that downregulation of hsa_circRNA_000166 inhibited the cell growth and facilitated apoptosis during CRC development by sponging the miR-326/LASP1 pathway.

Identification of the hub genes RUNX2 and FN1 in gastric cancer.

BACKGROUND: This study identified key genes in gastric cancer (GC) based on the mRNA microarray GSE19826 from the Gene Expression Omnibus (GEO) database and preliminarily explored the relationships among the key genes. METHODS: Differentially expressed genes (DEGs) were obtained using the GEO2R tool. The functions and pathway enrichment of the DEGs were analyzed using the Enrichr database. Protein-protein interactions (PPIs) were established by STRING. A lentiviral vector was constructed to silence RUNX2 expression in MGC-803 cells. The expression levels of RUNX2 and FN1 were measured. The influences of RUNX2 and FN1 on overall survival (OS) were determined using the Kaplan-Meier plotter online tool. RESULTS: In total, 69 upregulated and 65 downregulated genes were identified. Based on the PPI network of the DEGs, 20 genes were considered hub genes. RUNX2 silencing significantly downregulated the FN1 expression in MGC-803 cells. High expression of RUNX2 and low expression of FN1 were associated with long survival time in diffuse, poorly differentiated, and lymph node-positive GC. CONCLUSION: High RUNX2 and FN1 expression were associated with poor OS in patients with GC. RUNX2 can negatively regulate the secretion of FN1, and both genes may serve as promising targets for GC treatment.

Serum PIWI-Interacting RNAs piR-020619 and piR-020450 Are Promising Novel Biomarkers for Early Detection of Colorectal Cancer.

BACKGROUND: Early diagnosis can significantly reduce colorectal cancer deaths. We sought to identify serum PIWI-interacting RNAs (piRNAs) that could serve as sensitive and specific noninvasive biomarkers for early colorectal cancer detection. METHODS: We screened the piRNA expression profile in sera from 7 patients with colorectal cancer and 7 normal controls using small RNA sequencing. Differentially expressed piRNAs were measured in a training cohort of 140 patients with colorectal cancer and 140 normal controls using reverse transcription quantitative PCR. The identified piRNAs were evaluated in two independent validation cohorts of 180 patients with colorectal cancer and 180 normal controls. Finally, the diagnostic value of the identified piRNAs for colorectal adenoma (CRA) was assessed, and their expression was measured in 50 patients with lung cancer, 50 with breast cancer, and 50 with gastric cancer. RESULTS: The piRNAs piR-020619 and piR-020450 were consistently elevated in sera of patients with colorectal cancer as compared with controls. A predicative panel based on the two piRNAs was established that displayed high diagnostic accuracy for colorectal cancer detection. The two-piRNA panel could detect small-size and early-stage colorectal cancer with an area under the ROC curve of 0.863 and 0.839, respectively. Combined use of the two piRNAs could effectively distinguish CRA from controls. Aberrant elevation of the two piRNAs was not observed in sera of patients with lung, breast, and gastric cancer. CONCLUSIONS: Serum piR-020619 and piR-020450 show a strong potential as colorectal cancer-specific early detection biomarkers. IMPACT: The field of circulating piRNAs could allow for novel tumor biomarker development.