Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. CONCLUSION: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

Bioinformatics Analysis and Identification of Potential Genes Associated with Pathogenesis and Prognosis of Gastric Cancer.

OBJECTIVE: Gastric cancer (GC) is a deadly cancer and a challenging public health problem globally. This study aimed to analyze potential genes associated with pathogenesis and prognosis of gastric cancer. METHODS: This work selected the overlapping differentially expressed genes (DEGs) in GC from four datasets, the GSE29272, GSE29998, GSE54129 and GSE118916 Gene Expression Omnibus databases. These DEGs were used to carry out comprehensive bioinformatic analysis to analyze the related functions and pathways enriched, the relative expression levels and immune infiltrates, the prognostic characteristics and the interaction network. RESULTS: In total, 55 DEGs increased while 98 decreased in their expression levels. For those DEGs with increased expression, they were mostly concentrated on "focal adhesion" and "ECM-receptor interaction", whereas DEGs with decreased expression were mostly associated with "gastric acid secretion" and "drug metabolism cytochrome P450". MCODE and ClueGO results were then integrated to screen 10 hub genes, which were FN1, COL1A1, COL3A1, BGN, TIMP1, COL1A2, LUM, VCAN, COL5A2 and SPP1. Survival analysis revealed that higher expression of the ten hub genes significantly predicted lower overall survival of GC patients. TIMP1 was most significantly related to neutrophils, CD8+ T cells, as well as dendritic cells, while LUM was most significantly related to macrophages. CONCLUSION: Immunohistochemistry results and functional testing showed that the expression of COL5A2 was elevated in GC and that it might be a key gene in GC tumorigenesis.

Overexpression of eRF3a Promotes Cell Proliferation and Migration in Liver Cancer.

OBJECTIVE: The eukaryotic release factor 3a (eRF3a), a member of the eukaryotic peptide chain release factor family, is overexpressed in several types of cancer. This study aims to investigate the biological role and mechanism of eRF3a in the progression of liver cancer. METHODS: Western blotting and RT-qPCR were used to detect the expression level of eRF3a in normal liver cells and liver cancer cells. The cell transfection experiments were performed to overexpress eRF3a levels in liver cancer cells HCCLM9 and Huh7, and then cell cycle and apoptosis experiments, Cell Counting Kit-8 (CCK8), plate cloning, and Transwell experiments were done to evaluate the function of eRF3a in the progression of liver cancer. The Western blotting was done to explore the mechanism of eRF3a promoting the development of liver cancer. Western blotting and RT-qPCR were used to detect the expression level of eRF3a in normal liver cells and liver cancer cells. The cell transfection experiments were performed to overexpress eRF3a levels in liver cancer cells HCCLM9 and Huh7, and then cell cycle and apoptosis experiments, Cell Counting Kit-8 (CCK8), plate cloning, and Transwell experiments were done to evaluate the function of eRF3a in the progression of liver cancer. The Western blotting was done to explore the mechanism of eRF3a promoting the development of liver cancer. RESULTS: eRF3a was significantly highly expressed in liver cancer cells, and its expression level was negatively correlated with the clinical prognosis of patients. In addition, in vitro experiments showed that eRF3a could promote the proliferation and migration of liver cancer cells through the ERK and JNK signaling pathways. CONCLUSION: This study suggests that eRF3a may be a potential prognostic marker for liver cancer and act as an oncogene by activating JNK and ERK signaling; therefore, eRF3a may be a new target for the treatment of liver cancer.

Identification of Prognostic Genes for Colon Cancer through Gene Co-expression Network Analysis.

OBJECTIVE: The present study was aimed to identify novel key genes, prognostic biomarkers and molecular pathways implicated in tumorigenesis of colon cancer. METHODS: The microarray data GSE41328 containing 10 colon cancer samples and 10 adjacent normal tissues was analyzed to identify 4763 differentially expressed genes. Meanwhile, another microarray data GSE17536 was performed for weighted gene co-expression network analysis (WGCNA). RESULTS: In present study, 12 co-expressed gene modules associated with tumor progression were identified for further studies. The red module showed the highest association with pathological stage by Pearson's correlation analysis. Functional enrichment analysis revealed that genes in red module focused on cell division, cell proliferation, cell cycle and metabolic related pathway. Then, a total of 26 key hub genes were identified, and GEPIA database was subsequently selected for validation. Holliday junction-recognizing protein (HJURP) and cell division cycle 25 homolog C (CDC25C) were identified as effective prognosis biomarkers, which were all detrimental to prognosis. Gene set enrichment analyses (GSEA) found the two hub genes were enriched in "oocyte meiosis", "oocyte maturation that are progesterone-mediated", "p53 signaling pathway", and "cell cycle". Furthermore, the immunohistochemistry and western blotting showed that HJURP was highly expressed in colon cancer tissue. CONCLUSION: HJURP was identified as a key gene associated with colon cancer progression and prognosis by WGCNA, which might influence the prognosis by regulating cell cycle pathways.

The miR-370/UQCRC2 axis facilitates tumorigenesis by regulating epithelial-mesenchymal transition in Gastric Cancer.

Ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) is an important mitochondrial complex III subunit. This study investigated the role of UQCRC2 in gastric cancer (GC) and its upstream regulatory microRNAs (miRNAs). UQCRC2 expression levels were lower in GC tissues than non-carcinoma tissues. Furthermore, UQCRC2 levels were negatively correlated with lymph node metastasis, relapse, and tumor grade. Bioinformatics analysis predicted UQCRC2 as the target gene for miR-370, and this was verified in luciferase reporter assays. MiR-370 levels were inversely correlated with UQCRC2 levels in GC. UQCRC2 overexpression suppressed GC cell migration and invasion in vitro and in vivo, whereas up-regulating miR-370 reversed these effects. Western blotting analysis showed that miR-370 targeted UQCRC2 and positively regulated the epithelial-mesenchymal transition (EMT) signaling pathway in GC cells. Therefore, the miR-370/UQCRC2 axis may regulate EMT signaling pathways to affect tumor proliferation and metastasis and is, thus, a potential target for GC treatment.

Expression of Stanniocalcin 2 in Breast Cancer and Its Clinical Significance.

This study aims to explore the expression of stanniocalcin 2 (STC2) gene in breast cancer and its clinical significance. Female patients with breast cancer from Zhongnan Hospital of Wuhan University admitted during March 2014 to October 2014 were enrolled in this study. All the tissues used in this experiment included 50 cases of breast cancer tissues and corresponding 50 cases of paracancer normal breast tissues with complete patients' information. The real-time quantitative polymerase chain reaction (qPCR) was applied to detect the expression of STC2 gene in 50 cases of breast cancer and paracancer normal breast tissues. The results showed that the expression level of STC2 gene in 50 cases of breast cancer tissues was significantly higher than that in paracancer normal breast tissues (P<0.001). The expression of STC2 gene was correlated with lymph node metastasis, distant metastasis, TNM stage and histological grade (P<0.001). The expression level of STC2 gene was significantly higher in breast cancer tissues with higher expression of Ki-67 (P<0.001). The expression level of STC2 gene was significantly higher in estrogen receptor (ER) positive breast cancer tissues than in ER negative ones (P<0.001). However, different groups of age, pathological type, tumor size, PR expression and human epidermal growth factor receptor-2 (HER2) expression did not show significant differences in STC2 expression (P>0.05). In conclusion, the abnormal overexpression of STC2 gene may play a role in the development and progression of breast cancer, and it can be used as an independent metastasis and prognostic factor of breast cancer. In addition, STC2 gene probably promotes the development and metastasis of breast cancer by interacting with estrogen and ER, and it may become a new direction for breast cancer endocrine therapy.

Tumor necrosis factor receptor-associated factor (TRAF) 6 inhibition mitigates the pro-inflammatory roles and proliferation of rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) play a crucial role in RA through producing inflammatory cytokines and proteases which could cause cartilage destruction. We showed previously that elevated expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in RA synovium correlated significantly with the severity of synovitis and the number of infiltrated inflammatory cells. The aims of this study are to detect the roles of TRAF6 in RA-FLSs. METHODS: Synovium were collected by closed needle biopsy from inflamed knees of active RA patients, and FLSs were isolated by modified tissue culture method. Expression of TRAF6 and CD55 in RA synivium was tested by double immunofluorescence (IF) staining. TRAF6 in RA-FLSs was inhibited using Lentiviral-TRAF6-shRNA transfection. Real-time PCR and ELISA were used to detect the mRNA expression and secretion of matrix metalloproteinase (MMP) and pro-inflammatory cytokines. Cell Counting Kit-8 was used to detect cell proliferation, flow cytometry was used to detect cell cycle, and Annexin V assay was used to detect cell apoptosis. RESULTS: We showed that in the intimal and subintimal area of RA synovium, TRAF6 was expressed obviously not only in CD55+ cells, but also in some other CD55- cells. TRAF6 expression in RA-FLSs was suppressed effectively by Lentiviral-TRAF6-shRNA transfection. Inhibition of TRAF6 in RA-FLSs mitigated the mRNA levels and secretion of pro-inflammatory cytokines and MMPs, such as IL-1ß, IL-8, IL-6, TNF-α, MMP-13, and MMP-3. In addition, it decreased the proliferation of RA-FLSs, blocked RA-FLSs in G0/G1-phase, and inhibited the cells to go into S-phase and G2/M-phase, but not facilitated apoptosis of RA-FLSs. CONCLUSION: TRAF6 plays direct roles in the pro-inflammatory effects and proliferation of RA-FLSs. TRAF6 may serve as a potential treatment target in RA.

Do not resuscitate orders for patients with intracerebral hemorrhage: experience from a Chinese tertiary care center.

AIM: This study aimed to determine the frequency and determinant factors of do not resuscitate (DNR) orders in patients with intracerebral hemorrhage (ICH) at a university hospital in China. METHODS: Data collected from June 2010 to December 2012 for patients with ICH were retrospectively reviewed. The characteristics and care of patients with and without DNR orders and those with early (≤24 h) and late (>24 h) DNR establishment were compared. RESULTS: Formal DNR orders were filed during hospitalization for 64/759 (8.4%) patients with complete medical records enrolled in this study. Patients with DNR orders were older on average (73.1 ± 10.1 vs. 56.0 ± 13.2 years; p < 0.001) and a larger proportion had pre-ICH comorbidity impacting dependency (87.5 vs. 17.0%; p < 0.001) than did those with no DNR order. Patients with DNR orders were in worse clinical condition on arrival than those without a DNR order, as judged by Glasgow Coma Scale scores, and more frequently had large hematoma volumes (78.1 vs. 39.7%; p < 0.001). CONCLUSIONS: DNR orders were not used commonly for patients with ICH in this Chinese sample. No relationship between ICH severity and DNR decision making was observed.

Gastrointestinal bleeding after intracerebral hemorrhage: a retrospective review of 808 cases.

BACKGROUND: This study examined the incidence and risk factors for gastrointestinal (GI) bleeding after spontaneous intracerebral hemorrhage (ICH). METHODS: The available medical records of patients with ICH admitted from June 2008 to December 2009 for any episode of GI bleeding, possible precipitating factors and administration of ulcer prophylaxis were reviewed. RESULTS: The prevalence of GI bleeding was 26.7%, including 3 cases of severe GI bleeding (0.35%). Patients with GI bleeding had significantly longer hospital stay and higher in-hospital mortality compared with patients without GI bleeding. Multivariate logistic regression analyses showed that age, Glasgow Coma Scale scores, sepsis and ICH volume were independent predictors of GI bleeding. About 63.4% of patients with ICH received stress ulcer prophylaxis. CONCLUSIONS: GI bleeding occurred frequently after ICH, but severe events were rare. Age, Glasgow Coma Scale score, sepsis and ICH volume were independent predictors of GI bleeding occurring after ICH.

Magnolol attenuates sepsis-induced gastrointestinal dysmotility in rats by modulating inflammatory mediators.

AIM: To investigate the protective effects of magnolol on sepsis-induced inflammation and intestinal dysmotility. METHODS: Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS). Male Wistar rats were randomly assigned to one of three treatment groups: magnolol prior to LPS injection (LPS/Mag group); vehicle prior to LPS injection (LPS/Veh group); vehicle prior to injection of saline (Control/Veh). Intestinal transit and circular muscle mechanical activity were assessed 12 h after LPS injection. Tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS) mRNA in rat ileum were studied by RT-PCR 2 h after LPS injection. Nuclear factor-kappaB (NF-kappaB) activity in the intestine was also investigated at this time using electrophoretic mobility shift assay. In addition, antioxidant activity was determined by measuring malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in the intestine 2 h after LPS injection. RESULTS: Magnolol significantly increased intestinal transit and circular muscle mechanical activity in LPS-treated animals. TNF-alpha, MCP-1 and iNOS mRNA expression in the small intestine were significantly reduced after magnolol treatment in LPS-induced septic animals, compared with untreated septic animals. Additionally, magnolol significantly increased IL-10 mRNA expression in septic rat ileum. Magnolol also significantly suppressed NF-kappaB activity in septic rat intestine. In addition, magnolol significantly decreased MDA concentration and increased SOD activity in rat ileum. CONCLUSION: Magnolol prevents sepsis-induced suppression of intestinal motility in rats. The potential mechanism of this benefit of magnolol appears to be modulation of self-amplified inflammatory events and block of oxidative stress in the intestine.

[Clinical application of fistulation of artery and vein with self-blood vessel transplantation].

OBJECTIVE: To investigate the clinical application of fistulation of artery and vein with self-blood vessel transplantation. METHODS: Seven patients with renal failure were given antebrachial fistulation of artery and vein with great saphenous veins of themselves. The ortho- and pachy-great saphenous vein was chosed after it was cut. The great saphenous vein was passed bridge inside forearm in straight line or morpha-U. The method was anastomosis of the radial artery or brachial artery and cephalic vein, basilic vein or median cubital vein. RESULTS: The fistulations of artery and vein were successful and all patients were in hemodialysis regularly. CONCLUSION: The fistulation of artery and vein with self-blood vessel transplantation is a convenient, easy, cheap operation. It can coincide with the clinical demand and be used to make up the failure of fistulation or the fistulation that there is no blood vessel in the forearm.