Shengtao Zhu

Integrated Analysis Identifies Molecular Signatures and Specific Prognostic Factors for Different Gastric Cancer Subtypes

BACKGROUND: Gastric cancer (GC) is the fifth leading cause of cancer-related deaths worldwide. As an effective and easily performed method, microscopy-based Lauren classification has been widely accepted by gastrointestinal surgeons and pathologists for GC subtyping, but molecular characteristics of different Lauren subtypes were poorly revealed. METHODS: GSE62254 was used as a derivation cohort, and GSE15459 was used as a validation cohort. The difference between diffuse and intestinal GC on the gene expression level was measured. Gene ontology (GO) enrichment analysis was performed for both subgroups. Hierarchical clustering and heatmap exhibition were also performed. Kaplan-Meier plot and Cox proportional hazards model were used to evaluate survival grouped by the given genes or hierarchical clusters. RESULTS: A total of 4598 genes were found differentially expressed between diffuse and intestinal GC. Immunity- and cell adhesion–related GOs were enriched for diffuse GC, whereas DNA repair– and cell cycle–related GOs were enriched for intestinal GC. We proposed a 40-gene signature (χ2 = 30.71, P < .001) that exhibits better discrimination for prognosis than Lauren classification (χ2 = 12.11, P = .002). FRZB [RR (95% CI) = 1.824 (1.115-2.986), P = .017] and EFEMP1 [RR (95% CI) = 1.537 (0.969-2.437), P = .067] were identified as independent prognostic factors only in diffuse GC but not in intestinal GC patients. KRT23 [RR (95% CI) = 1.616 (0.938-2.785), P = .083] was identified as an independent prognostic factor only in intestinal GC patients but not in diffuse GC patients. Similar results were achieved in the validation cohort. CONCLUSION: We found that GCs with different Lauren classifications had different molecular characteristics and identified FRZB, EFEMP1, and KRT23 as subtype-specific prognostic factors for GC patients.

Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC) is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

Factors associated with gastric adenocarcinoma and dysplasia in patients with chronic gastritis: a population-based study

Objective Gastric cancer (GC) is one of the leading causes of death in China and other Asian countries. Recently, gastric endoscopy has become the main approach for GC screening, but the identification of high-risk individuals remains a challenge in GC screening programs. Methods There were 7,302 patients with chronic gastritis involved in this study. Endoscopic examinations were performed, and their demographic characteristics and lifestyle data were collected. Each possible associated factor of GC/premalignant and precursor lesions was evaluated by univariate and multivariate logistic regressions. Nomograms were used for visualization of those models, and receiver operating characteristic (ROC) curve analysis was used to present the predictive accuracy. Results We detected 8 (0.11%) gastric adenocarcinomas, 17 (0.23%) dysplasia cases, 14 (0.19%) hyperplasia cases, 52 (0.71%) intestinal metaplasia cases, 217 (2.97%) inflammatory lesions, 141 (1.93%) gastric ulcers, 10 (0.14%) atrophic gastritis cases, 1,365 (18.69%) erosive gastritis cases, and 5,957 (81.58%) superficial gastritis cases in 7,302 patients. The age (P<0.001), gender (P=0.086), labor intensity (P=0.018) and leek food intake (P=0.143) were identified as independent predictive factors of GC/premalignant lesions possibility. The corresponding nomogram exhibited an area under the curve (AUC) [95% confidence interval (95% CI)] of 0.82 (0.74–0.89) for the modeling group and 0.80 (0.75–0.85) for the validation group. The age (P=0.002), gender (P=0.024), smoking (P=0.002) and leek food intake (P=0.039) were independent predictive factors of precursor lesions possibility. The corresponding nomogram exhibited an AUC (95% CI) of 0.62 (0.60–0.65) for the modeling group and 0.61 (0.59–0.63) for the validation group. Conclusions We identified several potential associated factors and provided a preclinical nomogram with the potential to predict the possibility of GC/premalignant and precursor lesions.

Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice

Sonic hedgehog (Shh) is crucial for organogenesis in the foregut. This study investigated the function of Shh at the late‐gestational stage; during which, the esophagus continues to differentiate. We established cytokeratin 14 (CK14)‐Cre;Shhfl/fl mice in which the down‐regulation of Shh in the epithelium occurred at approximately the same time as esophageal muscle conversion. Hematoxylin and eosin and immunohistochemical staining, with antibodies against keratin 14, Shh, patched 1 (Ptch1), Gli1, proliferating cell nuclear antigen (PCNA), α‐smooth muscle actin (αSMA), high‐molecular‐weight caldesmon (hCD), myogenin, paired box 7 (Pax7), β3‐tubulin, and protein gene product 9.5 (PGP9.5), was performed to detect specific tissue dysplasia. Organ culture was conducted in vitro, and total mRNA was extracted to determine the transcriptional dysregulation. The esophagus of CK14‐Cre; Shhfl/fl mice developed into an independent tube with an obvious dilatation at postnatal d 0.5. The number of cell layers and the expression of PCNA were decreased in mutant mice, compared with those in wild‐type mice. The expression of hCD declined progressively in the middle, distal, and lower esophageal sphincter levels of the mutant esophagus from embryonic d 17.5, compared with the expression in wild‐type littermates. Pax7 accumulation and myogenin reduction in mutant mice indicated that esophageal skeletal‐myoblast progression was blocked. RNA sequencing analysis revealed a significant down‐regulation of genes involved in proliferation and muscular motivation in CK14‐Cre;Shhfl/fl mice. Thus, loss of Shh at the late‐ gestational stage leads to megaesophagus with reduced proliferation and a muscle development disorder in mice. mice.—Jia, X., Min, L., Zhu, S., Zhang, S., Huang, X. Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice. FASEB J. 32, 5703–5715 (2018). www.fasebj.org

Cadherin Expression Shift Could Well Distinguish Esophageal Squamous Cell Carcinoma from Non- Cancerous Esophageal Tissues

Background: Biomarkers for esophageal squamous cell carcinoma (ESCC) identification with high sensitivity are not well established. Since abnormal expression of cadherins has been widely reported in cancer, we explored its feasibility as an ESCC biomarker. Methods: Expression of E-cadherin and N-cadherin were detected in 150 esophageal tissues by immunohistochemistry. Staining strength and percentage in different subcellular structures of each specimen were evaluated by 2 independent pathologists. A logistic regression-based classifier derived from E-cadherin and N-cadherin staining was generated. Results: E-cadherin exhibited decreased membrane expression in ESCC, while N-cadherin exhibited decreased expression in the nucleus but elevated expression in the cytoplasm. Both E-cadherin and N-cadherin could distinguish ESCC tissues from non-cancerous tissues (area under the curve (AUC) = 0.748, 0.801, respectively). E-cadherin and N-cadherin staining scores could be merged into a cadherin (CDH) logistic index, which showed better discrimination (AUC = 0.909) than E-cadherin or N-cadherin alone. Further investigation indicated that the CDH logistic index was significantly correlated with tumor size and differentiation in ESCC. Conclusion: Both E-cadherin and N-cadherin had a strong expression shift in ESCC compared with non-cancerous tissues. The CDH logistic index, a parameter integrating the expression data of both cadherins, could be used as a marker with high sensitivity and specificity in the identification of ESCC.

Downregulation of NONO induces apoptosis, suppressing growth and invasion in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in China, and is associated with high morbidity and mortality. However, the molecular mechanisms that control ESCC tumorigenicity and metastasis remain unclear. Here, we report that the RNA splicing factor, NONO, is an important regulator of ESCC growth, apoptosis and invasion. NONO protein levels were dramatically upregulated in ESCC when compared with that in adjacent benign esophageal squamous epithelium. Particularly, NONO expression was statistically higher in tumors with greater tumor invasion depth. Using multiple ESCC cell models, we further showed that NONO depletion using siRNA significantly inhibited proliferation, invasion, and promoted apoptosis of ESCC cells. In addition we found that knockdown of NONO could reduce protein levels of phosphorylated Akt and Erk1/2. Our findings suggest that NONO plays a potent role in multiple biological aspects of ESCC through activation of the Akt and Erk1/2 signaling pathways. Taken together, our findings suggest that NONO might play an important role in promoting tumorigenesis of ESCC. It may provide a promising approach to prevent the progress of ESCC.

Gene regulatory network construction identified NFYA as a diffuse subtype-specific prognostic factor in gastric cancer

Lauren classification is a pathology-based gastric cancer (GC) subtyping system, which is widely used in the clinical treatment of patients with GC. However, genome- scale molecular characteristics to distinguish between diffuse (DF) and intestinal (IT) GC remain incompletely characterized, particularly at the transcriptional regulatory level. In the present study, gene regulatory networks were constructed using the Passing Attributes between Networks for Data Assimilation (PANDA) algorithm for DF, IT and mixed GC. The results indicated that >85% of transcription factor (TF)-target edges were shared among all three GC subtypes. In TF enrichment analysis, 13 TFs, including nuclear transcription factor Y subunit α (NFYA) and forkhead box L1, were activated in DF GC, whereas 8 TFs, including RELA proto-oncogene and T-cell leukemia homeobox 1 (TLX1), were activated in IT GC. Out of these identified TFs, NFYA [Hazard ratio (HR) (95% confidence interval, CI)=0.560 (0.349, 0.900), P=0.017] and sex determining region Y [HR (95% CI)=0.603 (0.375, 0.969), P=0.037] were identified as independent prognostic factors in DF GC, but not in IT GC, whereas TLX1 [HR (95% CI)=0.547 (0.321, 0.9325), P=0.027] was identified as an independent prognostic factor in IT GC, but not in DF GC. Verification at the cellular level was also performed; interference of NFYA expression using small interfering RNA in MGC803 cells (DF GC-derived cells) markedly inhibited cell growth and colony formation. Similar effects were also detected in SGC-7901 cells (IT GC-derived cells), but to a lesser extent. In conclusion, identified gene regulatory networks differed between distinct GC subtypes, in which the same TFs had different biological effects. Specifically, NFYA was identified as a DF subtype- specific independent prognostic factor in GC.

Transcriptome and methylome profiling in a rat model of irritable bowel syndrome induced by stress

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is associated with psychological stress. However, the full landscape of IBS-related epigenetic factors remains unveiled and needs to be elucidated. The water-avoidance stress (WAS) method was used to induce a rat IBS model. Each rat was monitored, and its defecation and behavior were recorded. Total colon RNA was isolated and subjected to Affymetrix GeneChip analysis. Reduced Representation Bisulfate Sequencing (RRBS) was applied to determine the genome-wide methylation pattern in both IBS and control rats. Rats with IBS egested a significantly increased amount of dry and loose stools compared with the control animals, without significant changes in body weight. Compared with the control group, 309 genes were upregulated and 224 genes were downregulated in the colon of the IBS rats. Notch signaling and focal adhesion were increased in the differentially expressed genes (DEGs). A total of 541 genes had significant lower methylation level and 626 genes had significantly higher methylation level in their promoter regions. Adherens junction and leukocyte transendothelial migration were enriched in the differentially methylated genes (DMGs). Few genes were identified in common in both DEGs and DMGs, suggesting that gene expression was not altered by promoter methylation. Reverse transcription-quantitative polymerase chain reaction validation revealed that the mRNA levels of SSX2IP, PARD3 and VCL were significantly downregulated in the IBS group, in accordance with hypermethylation of their promoters. In summary, the present study used a WAS-induced IBS rat model to provide transcriptome and methylome profiling. Most DEGs were associated with Notch signaling and focal adhesion, and only a few were altered by promoter methylation. The present results demonstrated that psychological stress could influence the integrity of the intestinal mucosa barrier and regulate inflammatory response.

Efficacy and Safety of Wei Bi Mei, a Chinese Herb Compound, as an Alternative to Bismuth for Eradication of Helicobacter pylori

Bismuth-containing quadruple therapy has been recommended as the first line of treatment in areas of high clarithromycin or metronidazole resistance. However, safety concerns of bismuth agents have long been raised. We first assessed the efficacy and safety of Wei Bi Mei granules, which are bismuth compounds consisting of three synthetic drugs and five medicinal herbs, compared to bismuth aluminate and colloidal bismuth subcitrate (CBS) in H. pylori-infected mouse model. We then used atomic fluorescence spectroscopy and autometallography to measure the accumulation of three bismuth agents in the brain, heart, liver, and kidneys in adult Sprague-Dawley rats. We also evaluated the safety of bismuth agents by conducting clinical biochemistry tests in blood samples of experimental animals. Wei Bi Mei granules exhibited the highest efficacy of anti-H. pylori activity and yielded the lowest bismuth accumulation when compared to CBS and bismuth aluminate. Our findings show that Wei Bi Mei granules are a safe Chinese medicinal herb with potent anti-H. pylori activity and can be considered as an alternative to current bismuth compounds. Thus, Wei Bi Mei granules merit further evaluation, particularly with regard to efficacy and safety when they are combined with other H. pylori eradication medications in the clinical setting.

Helicobacter pylori infection and inflammatory bowel disease: a crosstalk between upper and lower digestive tract

Helicobacter pylori has coexisted with humans for approximately 60,000 years and greater than 50% of the global population is infected with H. pylori. H. pylori was successfully cultured in vitro in 1983 and studies of H. pylori have achieved substantial advances over the last 35 years. Since then, H. pylori has been characterized as the primary pathogenic factor for chronic gastritis, peptic ulcer, and gastric malignancy. Numerous patients have received H. pylori eradication treatment, but only 1–2% of H. pylori-infected individuals ultimately develop gastric cancer. Recently, numerous epidemiological and basic experimental studies suggested a role for chronic H. pylori infection in protecting against inflammatory bowel disease (IBD) by inducing systematic immune tolerance and suppressing inflammatory responses. Here we summarize the current research progress on the association between H. pylori and IBD, and further describe the detailed molecular mechanism underlying H. pylori-induced dendritic cells (DCs) with the tolerogenic phenotype and immunosuppressive regulatory T cells (Tregs). Based on the potential protective role of H. pylori infection on IBD, we suggest that the interaction between H. pylori and the host is complicated, and H. pylori eradication treatment should be administered with caution, especially for children and young adults.