Upregulation of circRNA_0023685 promotes gastric cancer progression via a circRNA-miRNA-mRNA interaction network.

Circular RNAs (circRNAs) have been extensively studied for their critical roles as noncoding RNAs (ncRNAs) in gastric cancer (GC). In this study, we focused on the expression, function and molecular mechanism of circRNA_0023685 in gastric cancer (GC) to provide new ways for the diagnosis and treatment of GC. Firstly, a novel differentially expressed circRNA, circRNA_0023685, was identified, and its differential expression in GC plasma, tissue, and cell lines was further verified by RT-qPCR. Next, circRNA_0023685 was verified to promote the proliferation, migration and apoptosis of GC cells in vitro. CircRNA_0023685 was also proved to enhance the growth of GC tumors in xenograft models. Finally, for excavating the mechanism to promote GC, downstream microRNAs (miRNAs) and mRNAs were screened by bioinformatics analyses. After intersecting the target genes and genes enriched in GO analysis, a circRNA competing endogenous RNAs (ceRNAs) network was built. A protein-protein interaction (PPI) network was then constructed to find the candidate gene, APP. Our study confirmed that the highly expressed circRNA_0023685 could promote GC, which provided a new clinical diagnostic biomarker and therapeutic target for GC.

Muse cells decrease the neuroinflammatory response by modulating the proportion of M1 and M2 microglia in vitro.

Neuroinflammation hinders repair of the central nervous system (CNS). Stem cell transplantation is a very promising approach for treatment of CNS injuries. However, it is difficult to select seed cells that can both facilitate nerve regeneration and improve the microenvironment in the CNS. In this study, we isolated multilineage-differentiating stress-enduring (Muse) cells from bone marrow mesenchymal stem cells. We explored the anti-inflammatory effect and mechanism of Muse cells in vitro by coculture of Muse cells with lipopolysaccharide-stimulated microglia. Our results showed that Muse cells effectively reduced the transcription and secretion of tumor necrosis factor α and interleukin-1ß and increased the expression of transforming growth factor-ß and interleukin-10 in microglia. In addition, Muse cells decreased the number of M1 microglia and increased the proportion of M2 microglia in an inflammatory environment more effectively than bone marrow mesenchymal stem cells. We also show that Muse cells inhibited the protein expression of toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and inhibited the expression of the phosphorylated forms of transcription factor p65, nuclear factor (NF)-κB inhibitor alpha, and p38 mitogen-activated protein kinase (MAPK) in microglia. Therefore, we suggest Muse cells cause antineuroinflammatory effects by inhibition of the TLR4/MyD88/NF-κB and p38 MAPK signaling pathways in microglia. Our results shed light on the function of Muse cells in relation to CNS diseases and provide insight into the selection of seed cells.

Never-in-Mitosis A-Related Kinase 8 (NEK8) Regulates Adipogenesis, Glucose Homeostasis, and Obesity.

Background: Adipogenesis is a complex biological process and the leading main cause of obesity. We evaluated the role of never-in-mitosis A-related kinase 8 (NEK8) in adipocyte development and insulin sensitivity in the present study. Methods: NEK8 expression was manipulated using a specific shRNA or the NEK8-full-length expressing recombinant plasmids. The interaction between NEK8 and Tafazzin (TAZ, an oncogenic transcriptional regulator) was examined by Co-immunoprecipitation (Co-IP) and confocal immunofluorescence staining. Western blot assay was performed to determine the protein expression. The in vivo role of NEK8 was explored in a mouse model of high-fat diet- (HFD-) induced insulin resistance. Results: During adipogenesis, the expression of NEK8 was elevated while TAZ was downregulated. Overexpression of NEK8 promoted lipid accumulation and expression of markers for adipocyte differentiation. Mechanically, NEK8 interacted with TAZ and suppressed its expression in adipocytes. Functionally, lentiviral-mediated NEK8 inhibition ameliorates HFD-induced insulin resistance in adipocytes. Conclusion: These findings suggest that NEK8 plays a critical role in adipocyte proliferation, providing novel insight into the link between NEK8 and type 2 diabetes- (T2DM-) related obesity.

Fecal Signatures of Streptococcus anginosus and Streptococcus constellatus for Noninvasive Screening and Early Warning of Gastric Cancer.

BACKGROUND & AIMS: Most patients with gastric cancer (GCa) are diagnosed at an advanced stage. We aimed to investigate novel fecal signatures for clinical application in early diagnosis of GCa. METHODS: This was an observational study that included 1043 patients from 10 hospitals in China. In the discovery cohort, 16S ribosomal RNA gene analysis was performed in paired samples (tissues and feces) from patients with GCa and chronic gastritis (ChG) to determine differential abundant microbes. Their relative abundances were detected using quantitative real-time polymerase chain reaction to test them as bacterial candidates in the training cohort. Their diagnostic efficacy was validated in the validation cohort. RESULTS: Significant enrichments of Streptococcus anginosus (Sa) and Streptococcus constellatus (Sc) in GCa tumor tissues (P < .05) and feces (P < .0001) were observed in patients with intraepithelial neoplasia, early and advanced GCa. Either the signature parallel test Sa∪Sc or single signature Sa/Sc demonstrated superior sensitivity (Sa: 75.6% vs 72.1%, P < .05; Sc: 84.4% vs 64.0%, P < .001; and Sa∪Sc: 91.1% vs 81.4%, P < .01) in detecting early GCa compared with advanced GCa (specificity: Sa: 84.0% vs 83.9%, Sc: 70.4% vs 82.3%, and Sa∪Sc: 64.0% vs 73.4%). Fecal signature Sa∪Sc outperformed Sa∪CEA/Sc∪CEA in the discrimination of advanced GCa (sensitivity: 81.4% vs 74.2% and 81.4% vs 72.3%, P < .01; specificity: 73.4% vs 81.0 % and 73.4% vs 81.0%). The performance of Sa∪Sc in the diagnosis of both early and advanced GCa was verified in the validation cohort. CONCLUSION: Fecal Sa and Sc are noninvasive, accurate, and sensitive signatures for early warning in GCa. (ClinicalTrials.gov, Number: NCT04638959).

Cigarette Smoking and Mortality in Patients With Pancreatic Cancer: A Systematic Review and Meta-analysis.

Current evidence on cigarette smoking associated with pancreatic cancer mortality is limited. We searched MEDLINE, Web of Science, and Embase databases to identify relevant studies published through January 31, 2018. A random-effects model was used to estimate summary hazard ratios (HRs) and 95% confidence intervals (CIs). A total of 20 studies were retrieved, involving 2,517,623 participants. Of these, more than 15,341 patients with pancreatic cancer died. Compared with never smokers, current (summary HR, 1.56; 95% CI, 1.34-1.83) and former (summary HR, 1.15; 95% CI, 1.06-1.26) smokers had elevated risk of total mortality in patients diagnosed with pancreatic cancer. This effect of cigarette smoking is observed both in the Western regions and the Asia-Pacific regions. This effect of smoking is independent of alcohol use, body mass index, and history of diabetes but is modified by tumor stage and study settings. Dose-response associations between smoking and pancreatic cancer mortality were revealed for smoking intensity, cumulative amount of cigarettes smoked, and duration of smoking. Cigarette smoking was associated with an increase in total mortality for patients with pancreatic cancer. Future studies should further clarify the role of smoking as an effect modifier in treatment trials of pancreatic cancer.

Oridonin inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/ß-catenin signaling pathway.

BACKGROUND: Oridonin (ORI) can inhibit proliferation and migration in various types of cancer cell lines. However, the exact mechanism remains unclear. We investigated the migration inhibitory effect of ORI on human pancreatic cancer SW1990 cells and dissected the possible molecular mechanism(s). METHODS: CCK-8 assay was used to observe the cell viability. Wound healing assay, transwell assay and spontaneous metastasis model were used to observe the migration activities. Real-time PCR, immunofluorescence, western blot analysis and immunohistochemistry methods were used to observe the expression of genes or proteins. RESULTS: ORI inhibited the migration of SW1990 cells. Real-time PCR and immuno-fluorescence analyses of epithelial-to-mesenchymal transition (EMT) markers were compared between control group and ORI group. The expression of mesenchymal molecular markers, such as vimentin, snail and slug decreased. The expression of epithelial-related marker E-cadherin increased. Wnt/ß-catenin signalling was inhibited by ORI using luciferase reporter assay. ORI can decrease the ß-catenin protein level not only in the nucleus, but also in the cytoplasm and the whole cell after the treatment with ORI and glycogen synthase kinase 3ß (GSK3ß) was increased in the ORI-treated group. CHIR could attenuate the effects of ORI in SW1990 cells. We established a mice model by injecting 1 × 10(6) SW1990 cells into nude mice intraperitoneally to test whether ORI affects tumour metastasis. Metastatic formation was inhibited by ORI (5 and 10 mg/kg) compared with the control group. Tumour sections stained with anti-E-cadherin, anti-vimentin and anti-ß-catenin antibodies revealed that ORI inhibited EMT, as well as the Wnt/ß-catenin pathway in vivo. CONCLUSIONS: ORI can inhibit pancreatic cancer cell SW1990 migration and EMT by down-regulating Wnt/ß-catenin signal transduction in vitro and in vivo. Therefore, it can be potentially and effectively used in the clinical management of pancreatic cancer.

Oridonin derivative ameliorates experimental colitis by inhibiting activated T-cells and translocation of nuclear factor-kappa B.

OBJECTIVE: To confirm the potential therapeutic efficacy of HAO472 against inflammatory bowel disease (IBD), we investigated the modulatory functions of HAO472 in a mouse model of trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: Colitis was induced via an intrarectal injection of TNBS in mice. HAO472 (5.0 mg/kg or 7.5 mg/kg) or 1 mg/kg dexamethasone (DX) was injected intraperitoneally into the mice after the TNBS administration. Behavioral and weight changes, macroscopic and histological assessments of colon, the expressions of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-17A, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) in the colonic tissues were evaluated. The effect of HAO472 on NF-κB signaling pathway in lymphocytes was also invesigated. RESULTS: HAO472 significantly ameliorated the clinical symptoms, reduced the severity of the inflammation and decreased mortality in the mouse model. HAO472 also reduced TNF-α, IFN-γ, IL-17A, iNOS/COX-2 and lymphocyte proliferation. These changes were associated with a significant decrease in NF-κB p65 expression and activity. CONCLUSION: HAO472 has positive effects on TNBS-induced colitis by modulating the subsets and functions of lymphocytes, suppressing inflammation and inhibiting the nuclear translocation of NF-κB p65 subunits.

Tumor suppressor XIAP-Associated factor 1 (XAF1) cooperates with tumor necrosis factor-related apoptosis-inducing ligand to suppress colon cancer growth and trigger tumor regression.

BACKGROUND: XIAP-associated factor 1 (XAF1) antagonizes the anticaspase activity of XIAP (X-linked inhibitor of apoptosis) and functions as a tumor suppressor in colon cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known as a potential anticancer agent. In this study, the synergistic effect of XAF1 and TRAIL on colon cancer growth was investigated. METHODS: Adeno-XAF1 virus was generated and purified. Cell apoptosis was detected by flow-cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Protein expression of the different genes was determined by Western blot analysis. Tumorigenesis and tumor growth were assessed in subcutaneous nude mouse xenograft experiments. RESULTS: Stable overexpression of XAF1-sensitized colon cancer cells to TRAIL-induced apoptosis significantly increased the activity of caspase 3, 7, 8, and 9; released cytochrome c; and down-regulated XIAP, survivin, and c-IAP-2. The restoration of XAF1 expression mediated by adenovirus (adeno-XAF1) directly induced apoptosis, and synergized TRAIL-induced apoptosis in colon cancer cells. Ex vivo transduction of adeno-XAF1 suppressed colon cancer formation in vivo. Furthermore, adeno-XAF1 treatment of mice significantly inhibited tumor growth, strongly enhanced TRAIL-induced apoptosis and antitumor activity in colon cancer xenograft models in vivo, and markedly prolonged the survival. Notably, the combined treatment with adeno-XAF1 and TRAIL completely eradicated the established tumors without detectable toxicity in normal tissue. CONCLUSIONS: The combined restoration of XAF1 expression and TRAIL treatment may be a potent strategy for colon cancer therapy.

XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer.

BACKGROUND: XIAP-associated factor 1 (XAF1) negatively regulates the function of the X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family that exerts antiapoptotic effects. The extracellular signal-regulated kinase (ERK) pathway is thought to increase cell proliferation and to protect cells from apoptosis. The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer. METHODS: Four human colon cancer cell lines, HCT1116 and Lovo (wildtype p53), DLD1 and SW1116 (mutant p53), were used. Lovo stable transfectants with XAF1 sense and antisense were established. The effects of dominant-negative MEK1 (DN-MEK1) and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined. The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay. Cell proliferation was measured by MTT assay. Apoptosis was determined by Hoechst 33258 staining. RESULTS: U0126 increased the expression of XAF1 in a time- and dose-dependent manner. A similar result was obtained in cells transfected with DN-MEK1 treatment. Conversely, the expression of XIAP was down-regulated. Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection. rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression. Overexpression of XAF1 was more sensitive to U0126-induced apoptosis, whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation. CONCLUSIONS: XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation, which required de novo protein synthesis. The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers.

BACKGROUND & AIMS: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression. METHODS: XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis. RESULTS: Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers. CONCLUSIONS: A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at -2nd, -1st, and +3rd positions are functionally more important in its transcriptional regulation.

All-trans retinoic acid induces XAF1 expression through an interferon regulatory factor-1 element in colon cancer.

BACKGROUND & AIMS: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is a novel tumor suppressor and interferon (IFN)-stimulated gene. All-trans retinoic acid (ATRA) exerts an antiproliferative effect on tumor cells through up-regulation of IFN regulatory factor 1 (IRF-1) and the downstream IFN-stimulated genes. The aim of this study was to determine the effect and mechanism of ATRA on XAF1 expression and the role of XAF1 in ATRA-induced growth inhibition in colon cancer. METHODS: Gene expression is detected by reverse-transcription polymerase chain reaction and immunoblotting. The transcription activity of XAF1 promoter is examined by luciferase reporter assay. The activity of IFN regulatory factor binding element (IRF-E) is assessed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Cell growth is evaluated by both in vitro and in vivo in nude mice xenografts. RESULTS: IFN-alfa stimulates XAF1 promoter activity in the colon cancer cells Lovo and SW1116 dose-dependently. An IRF-1 binding element (IRF-E-XAF1) is found in the -30 to -38 nucleotide region upstream of the ATG initiator codon of the XAF1 gene. Site-directed mutagenesis of IRF-E-XAF1 abrogates native and IFN-induced promoter activity and binding capacity. ATRA induces XAF1 expression both in vitro and in vivo through interaction with IRF-E-XAF1. Overexpression of XAF1 increases cell susceptibility to ATRA-induced growth suppression both in vitro and in vivo. Furthermore, the effect of ATRA on XAF1 expression is independent of the promoter methylation and the subcellular distribution of XIAP. CONCLUSIONS: XAF1 participates in ATRA-induced growth suppression through IRF-1-mediated transcriptional regulation.

Induction of apoptosis and cell cycle arrest by a specific c-Jun NH2-terminal kinase (JNK) inhibitor, SP-600125, in gastrointestinal cancers.

The c-Jun NH(2)-terminal kinase (JNK) is activated in several tumor cell lines. The aim of this study was to determine the effects of SP-600125, a specific JNK inhibitor, on the viability, apoptosis, cell cycle distribution of gastrointestinal cancer cells, and the potential anti-tumor mechanisms. Three gastric cancer cell lines, AGS, BCG-823 and MKN-45, and three colorectal cancer cell lines, SW1116, COLO205 and HT-29, were used. Cells were treated with SP-600125, and cell viability, apoptosis and cell cycle distribution, caspase-3 activity, expression of JNK and apoptosis related proteins were detected. SP-600125 inhibited cell proliferation by 10-80% for the different cell lines, and increased apoptosis by 1.5-4.5 folds for COLO205, BCG-823, MKN-45, AGS cells. Caspase-8 and caspase-3 were involved in the induction of apoptosis. SP-600125 caused G2/M cell cycle arrest and elevation of cyclin B1 and p27(kip). The differential response in cells to SP-600125 was associated with the basal level of phosphorylated JNK2. It is concluded that SP-600125 inhibits proliferation, induces apoptosis and causes cell cycle arrest in gastrointestinal cancer cells, indicating that JNK inhibitors have an anti-tumor effect and are potential therapeutic agents for cancers.

[The study of regulation of connective tissue growth factor gene promoter by transforming growth factor beta1 in pancreatic stellate cells].

OBJECTIVE: To investigate the molecular mechanism for transforming growth factor beta(1) (TGF-beta(1)) on regulation of connective tissue growth factor (CTGF) gene promoter in pancreatic stellate cells (PSCs). METHODS: We tried to transfect the passaged 2 approximately 5 PSCs with pCTGF-luc plasmids, which were composed of CTGF promoter and PGL3 vector. And different concentrations of TGF-beta(1) or stimulation time were used to observe the reaction of luciferase activities by the measurement of dual-luciferase assay system. RESULTS: TGF-beta(1) could enhance the activities of pCTGF-luc in PSCs by means of time and dose dependently. TGF-beta(1) could stimulate the expression of CTGF gene promoter at the high level in a short time. CONCLUSION: TGF-beta(1) could enhance the activities of CTGF promoter in PSCs.

[The effects of octreotide on portal hemodynamics in patients with liver cirrhosis].

OBJECTIVE: To compare the portal hemodynamic effects of different doses of somatostatin analogue octreotide (sandostatin) with beta-blocker (propranolol) on prevention of early variceal re-bleeding in patients with cirrhotic portal hypertension. METHODS: Thirty patients of cirrhosis with moderate to severe esophageal varices were randomly allocated into three groups. 10 patients in group A were given propranolol 10-20 mg orally three times a day for 7 days; 10 patients in group B were administrated octreotide 0.05 mg subcutaneously two times a day for 3 days, while 10 patients in group C received octreotide 0.1 mg subcutaneously two times a day for 3 days. The postprandial vessel diameter, maximal and average flow velocity (Vmax/V) and flow volume of portal vein (PV), splenic vein (SV) and superior mesenteric vein (SMV) were measured before and after therapy by using Echo-Doppler technology. RESULTS: There was a trend of decrease of the vessel diameter in all the three groups, but with no significant difference. However, the average velocity and flow volume of PV were significantly diminished in the propranolol group (P < 0.05), while there was no statistical change of them in SV and SMV groups. In octreotide 0.05 mg and 0.1 mg group, the average velocity and flow volume of PV, SV and SMV were significantly decreased (P > 0.05). Besides, octreotide 0.1 mg group but not 0.05 mg group was more active in decreasing flow volume of PV as compared with the propranolol group, while both octreotide groups were superior to the propranolol group in decreasing flow volume of SV and SMV. CONCLUSIONS: Octreotide may have potential prophylactic effect in prevention of early variceal re-bleeding in cirrhotic patients with portal hypertension.