Adjacent Normal Mucosa Tissues Research Articles

Single-Cell Atlas of Infiltrating B Cells and Their Clinical Outcomes in Colorectal Cancer

Background: B cells constitute a major component of infiltrating immune cells in colorectal cancer (CRC). However, the characterization of B cells and their clinical significance remain unclear. Methods: We performed single-cell RNA sequencing to identify the features of B cells in the CRC tumor microenvironment (TME), and validated these findings using flow cytometry and multicolor immunofluorescence staining experiments. B cell-related cellular interactions within the TME were estimated using CellChat and in vitro chemotaxis assay. We then derived gene signatures to construct TME compositions for The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) dataset using the single sample gene set enrichment analysis (ssGSEA) method and unsupervised consensus clustering analysis was employed to identify four colon immune classes (CICs). Finally, we reviewed the landscape of gene mutations across these four CICs. Findings: We identified five distinct subtypes of B cells with their marker genes, distribution patterns, and functional properties in the CRC TME. We found an increased ratio of IgG/IgA plasma cells in tumor sites compared with adjacent normal mucosal tissue. In addition, the CXCL13-producing CD8+ T cells in the tumor tissue could recruit tertiary lymphoid structure (TLS) B cells and CCL28-CCR10 axis are pivotal for IgG plasma cell migration to the intratumor tissue. Finally, we identified four distinct CICs (CIC: A–D) and found that CD20+ B cells within TLS were enriched in one immune-inflamed or hot tumor group (CIC-D). This B cell-rich group, characterized by strong antigen presentation, IgG plasma cell accumulation, microsatellite instability-high (MSI-H) and high tumor mutation burden (TMB-H), as well as immunosuppressive property in particular, might become a potential predictive biomarker for future immunotherapy. Interpretation: Our analysis provides deep insight into infiltrating B cells and their potential clinical implications for immunotherapy in colorectal cancer. Funding Information: This study was financially supported by project grants from the Medical System of Shanghai Minhang District (grant number 2020MWDXK02). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Ethical Committee of The Fifth People’s Hospital of Shanghai, Fudan University. All animal experiment were conducted under the approved protocol Guidelines for the Care and Use of Laboratory Animals.

CircPARD3 drives malignant progression and chemoresistance of laryngeal squamous cell carcinoma by inhibiting autophagy through the PRKCI-Akt-mTOR pathway

BackgroundLaryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumor in head and neck. Autophagy and circular RNAs (circRNAs) play critical roles in cancer progression and chemoresistance. However, the function and mechanism of circRNA in autophagy regulation of LSCC remain unclear.MethodsThe autophagy-suppressive circRNA circPARD3 was identified via RNA sequencing of 107 LSCC tissues and paired adjacent normal mucosal (ANM) tissues and high-content screening. RT-PCR, Sanger sequencing, qPCR and fluorescence in situ hybridization were performed to detect circPARD3 expression and subcellular localization. Biological functions of circPARD3 were assessed by proliferation, migration, invasion, autophagic flux, and chemoresistance assays using in vitro and in vivo models. The mechanism of circPARD3 was investigated by RNA immunoprecipitation, RNA pulldown, luciferase reporter assays, western blotting and immunohistochemical staining.ResultsAutophagy was inhibited in LSCC, and circPARD3 was upregulated in the LSCC tissues (n = 100, p < 0.001). High circPARD3 level was associated with advanced T stages (p < 0.05), N stages (p = 0.001), clinical stages (p < 0.001), poor differentiation degree (p = 0.025), and poor prognosis (p = 0.002) of LSCC patients (n = 100). Functionally, circPARD3 inhibited autophagy and promoted LSCC cell proliferation, migration, invasion and chemoresistance. We further revealed that activation of the PRKCI-Akt-mTOR pathway through sponging miR-145-5p was the main mechanism of circPARD3 inhibited autophagy, promoting LSCC progression and chemoresistance.ConclusionOur study reveals that the novel autophagy-suppressive circPARD3 promotes LSCC progression and chemoresistance through the PRKCI-Akt-mTOR pathway, providing new insights into circRNA-mediated autophagy regulation and potential biomarker and target for LSCC treatment.Graphical abstract

Upregulated CNTN1 is associated with lymph node metastasis and poor prognosis of colorectal cancer.

Contactin1 (CNTN1), a member of the immunoglobulin superfamily, is known to correlate with tumor development and progression. Although recent studies have found that elevated CNTN1 has been demonstrated in some types of cancers, the expression and prognosis of CNTN1 in colorectal cancer (CRC) are unclear. Here, we aimed to determine the clinicopathological characteristics and prognostic role of CNTN1 in CRC patients. The protein expression of CNTN1 in tumor tissues was evaluated by immunohistochemistry. In addition, the mRNA and protein expressions of CNTN1 were examined by qRT-PCR and Western blotting analysis in 40 matched adjacent normal mucosa samples. The relationships of CNTN1 with clinicopathological data and prognosis significance were analyzed. Immunohistochemical consequence suggested that the protein level of CNTN1 was obviously raised in CRC compared with adjacent normal mucosa tissues (56.9% vs 10.3%, P< 0.05). In addition, we detected a significant increase in CNTN1 mRNA and protein levels in CRC tissues compared with the matched adjacent normal mucosa tissues. Moreover, increased CNTN1 exprssion was significantly associated with tumor size, lymph node metastasis (LNM), tumor node-metastasis (TNM) stage and carcino-embryonic antigen (CEA) in clinical analysis. Kaplan-Meier analysis suggested that patients with CNTN1 over-expression showed worse overall survival (OS) (P= 0.001). Multivariate analysis indicated that high CNTN1 expression was an independent predictor for poor OS in CRC patients (P= 0.028). Further analysis revealed that patients with high CNTN1 combined with LNM present accurately predicted poorer outcome. Taken together, the findingsindicate that CNTN1 plays a significant role and serve as a potential biomarker for the prediction of adverse prognosis in CRC. Intriguingly, high express of CNTN1 + LNM-present combination may improve the accuracy of prognosis.

The role and clinical significance of long noncoding RNA zinc finger E-box-binding homeobox two antisense RNA 1 in promoting osteosarcoma cancer cell proliferation, inhibiting apoptosis and increasing migration by regulating miR-145.

We aimed to investigate the expression level of long noncoding RNA (lncRNA) zinc finger E-box-binding homeobox two antisense RNA 1 (ZEB2-AS1) in osteosarcoma and explore its possible regulatory mechanisms. Expression of lncRNA ZEB2-AS1 was detected by quantitative real-time PCR in 63 cancerous tissues and 25 adjacent normal mucosal tissues from patients with osteosarcoma. The correlation between the lncRNA ZEB2-AS1 level and clinicopathological characteristics of the osteosarcoma patients were evaluated, and 5-year overall survival (5OS) was also analyzed according to lncRNA ZEB2-AS1 expression. The ZEB2-AS1 and miR-145 recombinant expression vector was used to analyze their relationship in an in vitro cell system. Luciferase reporter gene assays and RNA immunoprecipitation assays were used to verify the interaction between ZEB2-AS1 and miR-145. The proliferation, apoptosis and migration of osteosarcoma cells were determined by Cell counting kit-8 assays, Annexin V-PI assays and transwell assays, respectively. A significantly increased level of lncRNA ZEB2-AS1 with a fold change of 3.86 was found in osteosarcoma tissues compared with control tissues (P < 0.001). The Chi-square test revealed that lncRNA ZEB2-AS1 expression in osteosarcoma was significantly different according to radiology classification (P = 0.018), TNM stage (P = 0.000) and survival status (P = 0.005). The 5OS was 18.4% and 52% in osteosarcoma patients with higher and lower lncRNA ZEB2-AS1 expression, respectively. Significantly increased ZEB2-AS1 expression was found in osteosarcoma cells, while decreased levels of miR-145 were confirmed in osteosarcoma tissues and cell lines compared to controls. Moreover, a negative correlation was found between the expression level of ZEB2-AS1 and miR-145 in osteosarcoma tissues (R2 = 0.71, P < 0.01). ZEB2-AS1 knockdown resulted in decreased osteosarcoma cell proliferation, increased apoptosis and reduced migration. In addition, negative regulation of miR-145 by ZEB2-AS1 in osteosarcoma cells was also observed, and the effects of ZEB2-AS1 on osteosarcoma cells were found to be regulated by miR-145. Significantly upregulated lncRNA ZEB2-AS1 expression in osteosarcoma patients influences the prognosis of patients, and ZEB2-AS1 accelerates tumorigenesis and osteosarcoma development by downregulating miR-145.

Circular RNA circCORO1C promotes laryngeal squamous cell carcinoma progression by modulating the let-7c-5p/PBX3 axis

BackgroundLaryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of the head and neck. LSCC patients have seriously impaired vocal, respiratory, and swallowing functions with poor prognosis. Circular RNA (circRNA) has attracted great attention in cancer research. However, the expression patterns and roles of circRNAs in LSCC remain largely unknown.MethodsRNA sequencing was performed on 57 pairs of LSCC and matched adjacent normal mucosa tissues to construct circRNA, miRNA, and mRNA expression profiles. RT-PCR, qPCR, Sanger sequencing, and FISH were undertaken to study the expression, localization, and clinical significance of circCORO1C in LSCC tissues and cells. The functions of circCORO1C in LSCC were investigated by RNAi-mediated knockdown, proliferation analysis, EdU staining, colony formation assay, Transwell assay, and apoptosis analysis. The regulatory mechanisms among circCORO1C, let-7c-5p, and PBX3 were investigated by luciferase assay, RNA immunoprecipitation, western blotting, and immunohistochemistry.ResultscircCORO1C was highly expressed in LSCC tissues and cells, and this high expression was closely associated with the malignant progression and poor prognosis of LSCC. Knockdown of circCORO1C inhibited the proliferation, migration, invasion, and in vivo tumorigenesis of LSCC cells. Mechanistic studies revealed that circCORO1C competitively bound to let-7c-5p and prevented it from decreasing the level of PBX3, which promoted the epithelial–mesenchymal transition and finally facilitated the malignant progression of LSCC.ConclusionscircCORO1C has an oncogenic role in LSCC progression and may serve as a novel target for LSCC therapy. circCORO1C expression has the potential to serve as a novel diagnostic and prognostic biomarker for LSCC detection.

Expression, Location, Clinical Implication, and Bioinformatics Analysis of RNASET2 in Gastric Adenocarcinoma.

Background: In addition to exploiting its ribonuclease capacity, Ribonuclease T2 (RNASET2) has been reported to exert anti-angiogenic and anti-tumorigenic effects in several tumors. However, the role of RNASET2 in gastric adenocarcinoma (GAC) remains unclear. The purpose of this study was to explore the expression, location, and clinical implications of RNASET2 in GAC.Methods: Data of RNASET2 mRNA expression in GAC and normal gastric mucosa tissues were extracted from three GSE series and 388 TCGA samples and reanalyzed. Genome-wide CRISPR/Cas9 proliferation screening datasets were used to investigate cell growth changes after RNASET2 knockout in 19 GAC cell lines. The biological processes involved in RNASET2 were studied by the bioinformatics analysis. Furthermore, the corresponding experiments including immunohistochemical staining, clinicopathological features analysis, survival curve, microvessel density detection, cell viability assay, and colony formation assay were performed to validate the expression and function of RNASET2 in GAC.Results: An abundance of RNASET2 was present in the fundus glands and pylorus glands of the normal gastric mucosa. RNASET2 mRNA and protein were down-regulated in GAC compared with adjacent non-cancerous or normal gastric mucosa tissues. The expression of RNASET2 mRNA and protein in early GAC was higher than that in advanced GAC. 79/134 gene sets involved in the early GAC pathway were enriched in the RNASET2 mRNA high expression group. Genome-wide shRNA and CRISPR/Cas9 proliferation screening showed that knockdown or knockout of RNASET2 could not significantly promote GAC cell growth. AlamarBlue cell viability assay and colony formation assay in AGS cells further validated these results. Clinicopathologic features and survival analysis demonstrated that RNASET2 protein was significantly correlated with tumor cell differentiation, Lauren's classification, and TM4SF1 protein expression, but not correlated with lymph nodal metastasis and patient's prognosis. Microvessel density detection indicated that no significant correlation was found between the expression of RNASET2 protein and the angiogenesis of GAC.Conclusions: Down-regulation of RNASET2 in GAC was only the consequence of the GAC, instead of the driver. The expression of RNASET2 could be regarded as a good biomarker for identifying the early stage of GAC.

Elevated Levels of circRUNX1 in Colorectal Cancer Promote Cell Growth and Metastasis via miR-145-5p/IGF1 Signalling.

BackgroundEmerging evidence suggests that circular RNAs (circRNAs) are vital regulators in a range of cancers. “miRNA sponge” is the most reported role played by circRNAs in many tumors. The insulin-like growth factor (IGF) 1 pathway plays a key role in the development and progression of many cancers, including colorectal cancer (CRC). The aim of the study is to establish the potential clinical value and driving molecular mechanisms of circRNAs in CRC.Materials and MethodsReal-time quantitative RT-PCR (qRT-PCR) was performed to measure the circRUNX1 expression in 52 tissue samples from CRC patients. We verified the tumor promotor role of circRUNX1 in cell-based in vitro and in vivo assays. Human growth factor array was used to identify circRUNX1-regulated signaling pathways. We then used a double luciferase reporter assay and RNA fluorescence in situ hybridization to identify the downstream miR-145-5p of circRUNX1. Furthermore, we performed Western blotting and biological function assays to demonstrate if the circRUNX1/miR-145-5p/IGF1 axis is responsible for the proliferation of CRC cells and promotes CRC development.ResultsBy performing qRT-PCR from CRC tissues and paired adjacent normal mucosa tissues, we identified that circRUNX1 expression was significantly upregulated in CRC tissues and positively related with lymph node metastasis, distant metastasis and advanced tumor-node-metastasis tumor stage in patients. Functionally, circRUNX1 knockdown inhibited cell proliferation and migration and promoted apoptosis, whereas its overexpression exerted opposite effects. In vivo, circRUNX1 promoted tumor growth and metastasis. Mechanically, circRUNX1 shared miRNA response elements with IGF1. circRUNX1 competitively bound to miR-145-5p and prevented miR-145-5p from decreasing the expression of IGF1, which facilitated tumor growth.ConclusionOur studies verified that circRUNX1 functions as a tumor promotor in CRC cells by targeting the miR-145-5p/IGF1 signaling pathway and may have potential use as a prognostic indicator and therapeutic target in CRC patients.

Construction and evaluation of a prognosis lncRNA model for hepatocellular carcinoma.

Current studies indicate that long non-coding RNA (lncRNA) is often abnormally expressed in hepatocellular carcinoma (HCC). We intend to generate a multi-lncRNA signal to improve the prognosis of HCC. By analyzing 12 pairs of HCC and adjacent normal mucosal tissues, 3900 differentially expressed lncrnas were identified as candidate biomarkers for the prognosis of HCC. Then, the 12-lncrna signature was constructed using the LASSO Cox regression method and verified in the TCGA training dataset. Finally, we established a novel 12-lncrna signature that was significantly associated with overall survival (OS) in the training data set. With the use of 12-lncrna markers, patients in the training cohort were divided into high-risk and low-risk groups with significant OV differences (P < .0001). Similar results were consistent in the TCGA verification dataset (P = .046). Multivariate Cox model was used to analyze and construct the risk scores of selected key lncRNA and AJCC stages. The results showed that, compared with AJCC stages, lncRNA-based risk scores were another important factor affecting the OS of patients. We found that risk scores based on lncRNA have a stronger prediction ability than the AJCC stage alone on 4-year OS. For 4-year survival rates, prediction combined with the lncRNA risk score and AJCC stage, model effectiveness (sensitivity and specificity) has reached to 0.750. To further explore the biological processes involved in prognostic lncRNA, all HCC samples in TCGA are divided into two groups according to the median lncRNA risk score, and analyzed the gene enrichment of high expression genes and low expression genes in KEGG data using goana in limma. The results suggest that the genes associated with tumor pathways, such as PI3K-Akt and ECM-receptor interaction, are highly expressed in the high risk group.

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer

BackgroundThe human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking.ResultsWe found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation.ConclusionsOur study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.

Expression Patterns of Xenobiotic-Metabolizing Enzymes in Tumor and Adjacent Normal Mucosa Tissues among Patients with Colorectal Cancer: The ColoCare Study.

Xenobiotic-metabolizing enzymes (XME) play a critical role in the activation and detoxification of several carcinogens. However, the role of XMEs in colorectal carcinogenesis is unclear. We investigated the expression of XMEs in human colorectal tissues among patients with stage I-IV colorectal cancer (n = 71) from the ColoCare Study. Transcriptomic profiling using paired colorectal tumor and adjacent normal mucosa tissues of XMEs (GSTM1, GSTA1, UGT1A8, UGT1A10, CYP3A4, CYP2C9, GSTP1, and CYP2W1) by RNA microarray was compared using Wilcoxon rank-sum tests. We assessed associations between clinicopathologic, dietary, and lifestyle factors and XME expression with linear regression models. GSTM1, GSTA1, UGT1A8, UGT1A10, and CYP3A4 were all statistically significantly downregulated in colorectal tumor relative to normal mucosa tissues (all P ≤ 0.03). Women had significantly higher expression of GSTM1 in normal tissues compared with men (β = 0.37, P = 0.02). By tumor site, CYP2C9 expression was lower in normal mucosa among patients with rectal cancer versus colon cancer cases (β = -0.21, P = 0.0005). Smokers demonstrated higher CYP2C9 expression levels in normal mucosa (β = 0.17, P = 0.02) when compared with nonsmokers. Individuals who used NSAIDs had higher GSTP1 tumor expression compared with non-NSAID users (β = 0.17, P = 0.03). Higher consumption of cooked vegetables (>1×/week) was associated with higher CYP3A4 expression in colorectal tumor tissues (β = 0.14, P = 0.007). XMEs have lower expression in colorectal tumor relative to normal mucosa tissues and may modify colorectal carcinogenesis via associations with clinicopathologic, lifestyle, and dietary factors. Better understanding into the role of drug-metabolizing enzymes in colorectal cancer may reveal biological differences that contribute to cancer development, as well as treatment response, leading to clinical implications in colorectal cancer prevention and management.

Overexpression of ATAD2 indicates Poor Prognosis in Oral Squamous Cell Carcinoma.

ATPase family AAA domain-containing protein 2 (ATAD2) is highly expressed in a variety of malignancies and can promote the proliferation of tumor cells and inhibit their differentiation. However, the expression of ATAD2 and its related mechanism in oral squamous cell carcinoma (OSCC) are still unknown. Immunohistochemical staining of ATAD2, cancer stem cells (CSCs) markers and immune checkpoint molecules was conducted on human OSCC specimens to determine the expression levels of these proteins and their correlations with the clinicopathological characteristics of ATAD2 in OSCC. Moreover, the role of ATAD2 in cell proliferation, apoptosis, migration and epithelial-mesenchymal transition (EMT) were assessed by silencing ATAD2 in vitro. Immunohistochemical analysis revealed that ATAD2 expression in OSCC tissues was markedly higher than that in adjacent dysplastic tissues and normal mucosal tissues. Overexpression of ATAD2 was related to poor overall survival in OSCC patients. In addition, the protein expression of ATAD2 was notably correlated with the expression of B7-H4, PD-L1, CMTM6, Slug and ALDH1 in human OSCC. ATAD2 knockdown arrested the cell cycle, promoted the apoptosis, and inhibited the proliferation, migration, and EMT of OSCC cells. In conclusion, these findings revealed that ATAD2 is highly expressed in OSCC and can act as a poor prognostic indicator.

Identification and Verification of the Main Differentially Expressed Proteins in Gastric Cancer via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.

Background To find the potential intersections between the differentially expressed proteins and abnormally expressed genes in gastric cancer (GC) patients. Methods Gastric cancer tissue and adjacent normal mucosa tissue were used for iTRAQ analysis. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) analysis were used to evaluate gene function. Western blotting and immunohistochemistry (IHC) were applied to verify the protein expression. Results A total of 2770 proteins were identified, of which 147 proteins were upregulated and 159 proteins were downregulated. GO analysis revealed that the differentially expressed genes were mainly enriched for the terms “cellular process,” “binding,” and “cell.” The results of the KEGG analysis showed that the most abundantly enriched proteins were involved in the “focal adhesion” pathway. The results of the PPI analysis showed that VCAM1 was located at the center of the PPI network. Western blotting and IHC analysis demonstrated that VCAM1, FLNA, VASP, CAV1, PICK1, and COL4A2 were differentially expressed in GC and adjacent normal tissues, which was consistent with the results of the iTRAQ analysis. Conclusion In conclusion, 6 highly differentially expressed proteins were identified as novel differentially expressed proteins in human GC. This exploratory research may provide useful information for the treatment of gastric cancer in the clinic.

Identification of genes and miRNAs associated with angiogenesis, metastasis, and apoptosis in colorectal cancer

Colorectal cancer (CRC) is the main cause of cancer-related mortalities, worldwide and is the most prevalent cancer of the gastrointestinal tract. Different techniques exist to diagnose CRC including colonoscopy, sigmoidoscopy, and plasma miRNA test. This study evaluates the important miRNAs and main genes playing role in metastasis, angiogenesis and apoptosis that can be considered as prognostic and progression markers of CRC. We have measured the expression levels of the miRNAs, and multiple genes related to angiogenesis, metastasis and apoptosis in 45 paired samples of CRC tissue specimens and adjacent normal mucosa tissues obtained from 45 patients with CRC utilizing Real-time PCR. We have also analyzed the relationship between the expression of these genes and clinic pathological features. Results showed that the expression levels of angiogenesis genes (PGE-2, VEGF, Vimentin, CD105, CXCL-1, and IGFBP-7), metastatic genes (IGF-1R, Snail-1, and MMP-7) and apoptotic genes (Bcl-2, Bcl-xl, and NF-κβ1) were significantly higher in cancer tissues. But the expression levels of E-cadherin, AdipoR1, Claudin-7, Bax, Puma, and Noxa, were notably lower in cancer tissues. Additionally, it was revealed that miRNAs (miR-21, miR-19a, miR-18a, and miR-92a) expression levels were in higher levels in cancer tissues when compared to the normal tissues. In contrast, the expression levels of miR-31, and miR-139-5p were significantly lower in cancer tissues. It is hoped that, by measuring the metastasis, angiogenesis and apoptosis resistance associated miRNAs expression, better treatment results would be achieved in CRC treatment.

Evaluation of MGP gene expression in colorectal cancer

PurposeMatrix Gla protein (MGP) is a vitamin K-dependent, γ-carboxylated protein that was initially found to be a physiological inhibitor of ectopic calcifications affecting mainly cartilage and the vascular system. Mutations in the MGP gene were found to be responsible for a human pathology, the Keutel syndrome, characterized by abnormal calcifications in cartilage, lungs, brain and vascular system. MGP was recently implicated in tumorigenic processes such as angiogenesis and shown to be abnormally regulated in several tumors, including cervical, ovarian, urogenital and breast. This fact has triggered our interest in analyzing the expression of MGP and of its regulator, the transcription factor runt related transcription factor 2 (RUNX2), in colorectal cancer (CRC). MethodsMGP and RUNX2 expression were analyzed in cancer and non-tumor biopsies samples from 33 CRC patients and 9 healthy controls by RT-qPCR. Consequently, statistical analyses were performed to evaluate the clinical-pathological significance of MGP and RUNX2 in CRC. MGP protein was also detected by immunohistochemical analysis. ResultsShowed an overall overexpression of MGP in the tumor mucosa of patients at mRNA level when compared to adjacent normal mucosa and healthy control tissues. In addition, analysis of the expression of RUNX2 mRNA demonstrated an overexpression in CRC tissue samples and a positive correlation with MGP expression (Pearson correlation coefficient 0.636; p ≤ 0.01) in tumor mucosa. However correlations between MGP gene expression and clinical-pathological characteristics, such as gender, age and pathology classification did not provide relevant information that may shed light towards the differences of MGP expression observed between normal and malignant tissue. ConclusionsWe were able to associate the high levels of MGP mRNA expression with a worse prognosis and survival rate lower than five years.These results contributed to improve our understanding of the molecular mechanism underlying MGP deregulation in cancer.

MIR100HG: a credible prognostic biomarker and an oncogenic lncRNA in gastric cancer.

The MIR100HG expression was observed to be up-regulated or down-regulated in human cancer tissues depending on tumor types. However, there was no report about the role of MIR100HG in gastric cancer. In our study, we first found levels of MIR100HG expression were increased in gastric cancer cell lines and tissue samples compared with normal gastric epithelial cell line and adjacent normal gastric mucosa tissue samples, respectively. Moreover, high MIR100HG expression was positively associated with clinical stage, tumor invasion, lymph node metastasis, and distant metastasis in gastric cancer patients. Survival analysis showed MIR100HG expression was negative correlated with clinical outcome in gastric cancer patients from The Cancer Genome Atlas (TCGA) database or our study, and high MIR100HG expression served as an independent poor prognostic factor for gastric cancer patient’s overall survival. The study in vitro suggested down-regulation of MIR100HG expression inhibits cell proliferation, migration, and invasion in gastric cancer. In conclusion, MIR100HG is a credible prognostic biomarker and functions as an oncogenic lncRNA in gastric cancer.

MicroRNA-1224 Inhibits Tumor Metastasis in Intestinal-Type Gastric Cancer by Directly Targeting FAK.

Intestinal-type gastric cancer (GC) of the Lauren classification system has specific epidemiological characteristics and carcinogenesis patterns. MicroRNAs (miRNAs) have prognostic significance, and some can be used as prognostic biomarkers in GC. In this study, we identified miR-1224 as a potential survival-related miRNA in intestinal-type GC patients by The Cancer Genome Atlas (TCGA) analysis. Using quantitative real-time PCR (qRT-PCR), we showed that the relative expression of miR-1224 was significantly decreased in intestinal-type GC tissues compared to matched adjacent normal mucosa tissues (p < 0.01). We found that high miR-1224 expression was associated with no lymph-node metastasis (p < 0.05) and good prognosis (p = 0.028) in 90 intestinal-type GC tissues. Transfection of intestinal-type GC cells with miR-1224 mimics showed that miR-1224 suppressed cell migration in vitro (wound healing assay and Transwell migration assay), whereas the transfection of cells with miR-1224 inhibitor promoted cell migration in vitro. miR-1224 also suppressed intestinal-type GC cell metastasis in a xenograft mouse model. Furthermore, bioinformatics, luciferase reporter, Western blotting, and immunohistochemistry (IHC) studies demonstrated that miR-1224 directly bound to the focal adhesion kinase (FAK) gene, and downregulated its expression, which decreased STAT3 and NF-κB signaling and subsequent the epithelial-to-mesenchymal transition (EMT). Repression of FAK is required for the miR-1224-mediated inhibition of cell migration in intestinal-type GC. The present study demonstrated that miR-1224 is downregulated in intestinal-type GC. miR-1224 inhibits the metastasis of intestinal-type GC by suppressing FAK-mediated activation of the STAT3 and NF-κB pathways, and subsequent EMT. miR-1224 could represent an important prognostic factor in intestinal-type GC.

PRSS23 knockdown inhibits gastric tumorigenesis through EIF2 signaling

PRSS23 is a newly discovered serine protease that has been associated with tumor progression in various types of cancers. Our previous study showed PRSS23 is down-regulated obviously in Hedgehog pathway blocked gastric cancer cells. However, the correlation between PRSS23 and tumor progression of gastric cancer remains unclear. Here, the role and mechanism of PRSS23 in tumor progression of gastric cancer were determined. PRSS23 protein levels were significantly increased in gastric cancer tissues compared with the paired adjacent normal gastric mucosa tissues. The high expression of PRSS23 correlated strongly with both poor differentiated histology and cancer region of sinus ventriculi. Gastric cancer patients with low PRSS23 expression displayed a better prognosis. In gastric cancer cells, PRSS23 knockdown inhibited cell proliferation and induced apoptosis. In xenograft tumor model, PRSS23 knockdown led to dramatic decreases of the average tumor volume and the average tumor weight. In addition, PRSS23 knockdown suppressed gastric cancer growth through inhibiting EIF2 signaling using gene expression microarray analysis. Taken together, our results suggest PRSS23 is highly associated with human gastric tumorigenesis and progression. PRSS23 knockdown could suppress tumor growth of gastric cancer in vitro and in vivo through inhibiting EIF2 signaling, and EIF4E maybe a potential target of PRSS23. PRSS23 could serve as a potential target for gastric cancer therapy, and also a biomarker for the prediction of prognosis of gastric cancer.

Overexpression of periostin is positively associated with gastric cancer metastasis through promoting tumor metastasis and invasion.

Gastric tumors generally have a poor prognosis and molecular markers to improve early detection and predict outcomes are greatly needed. The present study reports that periostin (POSTN), a secretory protein that can alter the remodeling of the extracellular matrix, is highly expressed in gastric tumors. Gastric tissues were collected from patients at the Department of Thoracic Surgery/Huiqiao Medical Center, Nanfang Hospital, Southern Medical University. These patients provided an informed consent and were approved by the institute. Normal, cancer, and metastatic gastric tissues from lymph nodes and tissues adjacent to the tumor were collected from patients diagnosed with gastric cancer. Periostin expression gradually increased as the risk grade of the NIH classification increased, and this was closely correlated with disease-free survival and overall survival. Compared with adjacent normal gastric mucosa tissues, protein expression of POSTN in gastric cancer tissues and metastases was significantly higher by immunohistochemistry and Western blot analysis. In addition, POSTN was upregulated in advanced gastric cancer tissues than in early gastric cancer tissues. Moreover, the ectopic expression of POSTN in the immortalized human gastric cell line could increase the metastasis and invasion of gastric cancer cells. The present results could establish the significance of POSTN in driving oncogenesis and metastasis in gastric tumors, with implications for its potential use as a diagnostic or prognostic biomarker, and as a candidate therapeutic target.

RNA sequencing reveals the expression profiles of circRNA and indicates that circDDX17 acts as a tumor suppressor in colorectal cancer

BackgroundCircular RNA (circRNA) is a novel class of noncoding RNAs with functions in various pathophysiological activities. However, the expression profiles and functions of circRNAs in colorectal cancer (CRC) remain largely unknown.MethodsHigh-throughput RNA sequencing (RNA-seq) was performed to assess circRNA expression profiles in 4 paired CRC tissues, and significantly dysregulated circRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the potential functions of dysregulated circRNAs. Target miRNAs of circRNAs were predicted using miRanda software, and were further analyzed combining DIANA-miRPath v.3 platform (Reverse Search module) with KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer (Entry: map05210 and map05206). CircRNA-miRNA interaction networks were constructed using Cytoscape software. Expression levels of a significantly down-regulated circRNA, circDDX17 (hsa_circ_0002211), was detected by qRT-PCR in 60 paired CRC tissues. CircDDX17 was knockdown by siRNA, and the biological functions of circDDX17 were examined in CRC cell lines.ResultsTotally 448 differentially expressed circRNAs were identified, including 394 up-regulated and 54 down-regulated circRNAs. qRT-PCR validation confirmed the reliability of the RNA-Seq data. GO and KEGG analyses revealed that these dysregulated circRNAs were potentially implicated in CRC pathogenesis. Analyses by combining miRanda and miRPath softwares with KEGG pathways suggested that the miRNAs targeted by the top 10 dysregulated circRNAs were associated with the KEGG pathways of COLORECTAL CANCER and MicroRNAs in cancer, indicating that circRNA-miRNA interactions might play important functional roles in the initiation and progression of CRC. The results of qRT-PCR for circDDX17 in 60 paired CRC tissues showed that circDDX17 was significantly down-regulated in CRC tissues and associated with unfavorable clinicopathological parameters. In vitro experiments showed that silencing of circDDX17 promoted CRC cell proliferation, migration, invasion, and inhibited apoptosis.ConclusionsIn conclusion, we have identified numerous circRNAs that are dysregulated in CRC tissues compared with adjacent normal mucosa tissues. Bioinformatic analyses suggested that these dysregulated circRNAs might play important functional roles in CRC tumorigenesis. CircDDX17 functions as a tumor suppressor and could serve as a potential biomarker and a therapeutic target for CRC.

Upregulation of 24(R/S),25-epoxycholesterol and 27-hydroxycholesterol suppresses the proliferation and migration of gastric cancer cells

Gastric cancer (GC) is one of the most common cancers and is the second-leading cause of cancer-associated morbidity worldwide. Oxysterols are oxidized derivatives of cholesterol that may be important in many biological processes, but the levels and roles of oxysterols in gastric tumours remain to be elucidated. The levels of cholesterol, oxysterols and sulfated oxysterols in human gastric tumour tissues, adjacent normal mucosal tissues, cancerous gastric juice and gastric juice obtained from healthy subjects were detected by LC-MS. It was found that the levels of 24(R/S),25-EC and 27HC in human gastric tumour tissues and cancerous gastric juice were significantly increased compared with those of adjacent normal mucosal tissues and gastric juice from healthy subjects. Compared with normal gastric mucosal tissue, the levels of sulfated 25-hydroxycholesterol (25HC3S) and the ratio of 25HC3S/25HC were decreased in human gastric tumour tissues, which might be related to the dramatically decreased SULT2A1 expression in gastric tumour tissue. Both 24(R/S),25-EC and 27HC suppressed gastric cancer proliferation, which was not altered by LXRα-siRNA treatment. The suppression of cell proliferation induced by 27HC was attenuated by LXRβ-siRNA, but the suppression of cell proliferation induced by 24(R/S),25-EC was intensified by LXRβ-siRNA. Both 24(R/S),25-EC and 27HC dramatically inhibited HGC-27 cell migration, which was attenuated by the co-transfection of cells with LXRα-siRNA and LXRβ-siRNA, but not LXRα-siRNA or LXRβ-siRNA alone. In conclusion, the accumulated 24(R/S),25-EC and 27HC in human gastric tumour tissues might play important roles in gastric cancer development.