Small Extracellular Vesicle piR-hsa-30937 Derived from Pancreatic Neuroendocrine Neoplasms Upregulates CD276 in Macrophages to Promote Immune Evasion.

The role of PIWI-interacting RNAs (piRNA) in small extracellular vesicles (sEV) derived from pancreatic neuroendocrine neoplasms (PNEN) in the tumor microenvironment (TME) remains unexplored. We used multiplex IHC to analyze the expression of CD68, CD276 (B7H3), and CD3 on PNEN. CD276+ tumor-associated macrophages (TAM) were more abundant in tumor tissues than nontumor tissues and negatively correlated with T-cell infiltration. Serum sEV piRNA sequencing was performed to identify piRNAs enriched in patients with PNEN. We then investigated the function and mechanism of sEV piR-hsa-30937 in the cross-talk between tumor cells and macrophages in the PNEN TME. PNEN-derived sEV piR-hsa-30937 targeted PTEN to activate the AKT pathway and drive CD276 expression. CD276+ macrophages inhibited T-cell proliferation and IFNγ production. piR-hsa-30937 knockdown and anti-CD276 treatment suppressed progression and metastasis in a preclinical model of PNEN by enhancing T-cell immunity. Thus, our data show that PNEN-derived sEV piR-hsa-30937 promotes CD276 expression in macrophages through the PTEN/AKT pathway and that CD276+ TAMs suppress T-cell antitumor immunity. sEV piR-hsa-30937 and CD276 are potential therapeutic targets for immunotherapy of PNEN.

Prognostic Evaluation of Patients with Rectal Neuroendocrine Neoplasms and Hepatic Metastases: A SEER Database Analysis.

Background: This study is aimed at investigating the clinical characteristics and prognosis-affecting factors of patients with rectal neuroendocrine neoplasms (r-NENs) and hepatic metastases and offering a scientific-theoretical basis for selective use of an optimized treatment method for r-NENs. Methods: This study was retrospectively evaluated based on the analysis of the data from Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2016. Results: A total of 4,723 r-NEN patients were enrolled in this study, including 168 patients with hepatic metastases (3.56%). Kaplan-Meier analysis revealed that the overall survival (OS) of patients with hepatic metastases receiving primary tumor excision was obviously greater than that of patients without receiving primary tumor excision (OS: nonsurgical patients vs. patients undergoing local resection: P < 0.0001 and nonsurgical patients vs. patients undergoing radical resection: P < 0.0001); the patients with hepatic metastases in the chemotherapy group had a significantly worse prognosis compared with those in the nonchemotherapy group (OS: P = 0.021). Multivariate cox regression analysis revealed that independent affecting factors of overall and tumor-related prognoses in patients with hepatic metastases included tumor grade (G3 and G4), surgical treatment, and chemotherapy. Conclusion: Among r-NEN patients with hepatic metastases, those undergoing radical excision of lower-grade tumors and chemotherapy will have a better prognosis.

Trends of incidence and prognosis of gastric neuroendocrine neoplasms: a study based on SEER and our multicenter research.

BACKGROUND: To investigate the recent epidemiological trends of gastric neuroendocrine neoplasms (GNENs) and establish a new tool to estimate the prognosis of gastric neuroendocrine carcinoma (GNEC) and gastric neuroendocrine tumor (GNET). METHODS: Nomograms were established based on a retrospective study on patients diagnosed with GNENs from 1975 to 2016 in Surveillance, Epidemiology and End Results database. External validation was performed among 246 GNENs patients in Jiangsu province to verify the discrimination and calibration of the nomograms. RESULTS: The age-adjusted incidence of GNENs has increased from 0.309 to 6.149 per 1,000,000 persons in the past 4 decades. Multivariate analysis indicated independent prognostic factors for both GNEC and GNET including age, distant metastasis and surgical intervention (P < 0.05). In addition, T, N staging and grade were significantly associated with survival of GNEC, while size was a predictor for GNET (P < 0.05). The C-indexes of the nomograms were 0.840 for GNEC and 0.718 for GNET, which were higher than those of the 8th AJCC staging system (0.773 and 0.599). Excellent discrimination was observed in the validation cohorts (C-index of nomogram vs AJCC staging for GNEC: 0.743 vs 0.714; GNET: 0.945 vs 0.927). Survival rates predicted by nomograms were close to the actual survival rates in the calibration plots in both training and validation sets. CONCLUSIONS: The incidence of the GNENs is increasing steadily in the past 40 years. We established more excellent nomograms to predict the prognosis of GNENs than traditional staging system, helping clinicians to make tailored decisions.

LncNEN885 inhibits epithelial-mesenchymal transition by partially regulation of Wnt/ß-catenin signalling in gastroenteropancreatic neuroendocrine neoplasms.

It has been shown that long noncoding RNAs (lncRNAs) are involved in the carcinogenesis of multiple cancers. However, the roles of lncRNAs in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) remain elusive. In the present study, we found that lncNEN885 was markedly decreased in human gastric NEN samples compared to adjacent normal tissues by transcriptome sequencing. Functionally, silencing or overexpression of lncNEN885 could not obviously affect cell proliferation or apoptosis in BON-1 or LCC-18 cells but could affect cell migration and invasion as well as wound-healing rates. Furthermore, dysregulation of lncNEN885 affected these biological functions by activating epithelial-mesenchymal transition through increased expression of Snail, vimentin, and N-cadherin as well as decreased E-cadherin levels in BON-1 and LCC-18 cells. Silencing of lncNEN885 could dramatically increase the phosphorylation of glycogen synthase kinase-3ß and decrease the expression of adenomatous polyposis coli and Axin, with the subsequent accumulation of ß-catenin. Taken together, dysregulation of lncNEN885 can regulate cell migration and invasion by activating epithelial-mesenchymal transition process partially through canonical Wnt/ß-catenin signaling in GEP-NEN cells, which may be a novel biomarker for the metastasis of GEP-NENs.

Anticancer Effects of Baicalein in Pancreatic Neuroendocrine Tumors In Vitro and In Vivo.

OBJECTIVES: Baicalein is a Chinese traditional medicine that inhibits tumor migration and growth. Pancreatic neuroendocrine tumors (pNETs) have a high incidence in China, but there are still no effective treatments. The aim of our study was to investigate whether baicalein could inhibit pNETs. METHODS: In vitro, we used BON1-a cell line of pNETs-to analyze the apoptosis and migration and invasion after baicalein treatment via flow cytometry and Western blot. In vivo, we used a xenograft tumors model to evaluate the size of tumors after baicalein treatment. Western blot was used to analyze the expression of apoptosis and migration-related protein. RESULTS: In vitro, the Cell Counting Kit 8 assay showed that baicalein decreased BON1 viability, and flow cytometry demonstrated that baicalein induced BON1 apoptosis and protein changes. In addition, baicalein inhibited BON1 migration and invasion as shown via a Transwell assay. In vivo, baicalein inhibited tumor growth and migration and also increased apoptosis-related protein expression. CONCLUSIONS: Baicalein could increase caspase-3 and Bax expression and decrease survivin and Bcl-2 to induce apoptosis. It inhibits migration and invasion by decreasing expression of vascular endothelial growth factor and matrix metalloproteinases 2 and 9.

Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease.

OBJECTIVE: To investigate the role of chromosome region maintenance-1 (CRM1) in Crohn's disease (CD) and its potential pathological mechanisms. METHODS: The expression and distribution of CRM1 in mucosal biopsies from patients with active CD and normal controls were detected by immunohistochemistry (IHC). We established a murine model of acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Western blot was performed to investigate the expression levels of CRM1, apoptotic markers (active caspase-3 and cleaved PARP), p27kip1 and p-p27ser10. IHC was performed to evaluate the distribution of CRM1, and double immunofluorescence (IF) was performed to evaluate the co-localization of CRM1 and active capase-3. Cells of the human intestinal epithelial cell line HT-29 were incubated with tumor necrosis factor-α (TNF-α) to establish an apoptotic in vitro model. Western blot was performed to determine the expression levels of CRM1, active caspase-3, cleaved PARP and p-p27ser10. Cytoplasmic and nuclear extracts were assessed to examine the translocation of CRM1. The interaction between CRM1 and p27kip1 was assessed by co-immunoprecipitation (co-IP) assays. Furthermore, we used small interfering RNA (siRNA) to knock down the protein expression of CRM1 in HT-29 cells and then measured the expression of active caspase-3, cleaved PARP and p-p27ser10. Flow cytometry was used to determine the effect of CRM1 on intestinal epithelial cell (IEC) apoptosis. RESULTS: We observed up-regulation of CRM1 accompanied by elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and p-p27ser10 in IECs of patients with active CD and in TNBS-induced colitis model cells. However, the expression of p27kip1 was negatively correlated with the expression patterns of CRM1, p-p27ser10 and apoptotic biochemical markers. Co-localization of CRM1 and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of CRM1 in IEC apoptosis. By employing TNF-α-treated HT-29 cells as an in vitro IEC apoptosis model, we found that the expression levels of CRM1 and p-p27ser10 were in accordance with active caspase-3 and cleaved PARP. In addition, immunoprecipitation confirmed the physical interaction between CRM1 and p27kip1. siRNA knockdown of CRM1 significantly inhibited the phosphorylation of p27kip1 and the expression of active caspase-3 and cleaved PARP. In addition, flow cytometry analysis also showed that silencing CRM1 by siRNA inhibited TNF-α-induced cellular apoptosis in HT-29 cells. CONCLUSIONS: Up-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.

Microtubule-associated protein 1S-related autophagy inhibits apoptosis of intestinal epithelial cells via Wnt/ß-catenin signaling in Crohn's disease.

Many autophagy-related genes, to our knowledge, have been identified as Crohn's disease (CD) polymorphic sites by genomic wide studies. As a novel member of the microtubule-associated protein 1 (MAP1) family, MAP1S is a microtubule-binding proteins involved in autophagy. However, its expression and potential functions in CD have not been understood. For the first time, we discovered the up-regulated MAP1S and autophagy level (indicated by LC3-Ⅱ/LC3-Ⅰ) in inflamed epithelium among CD patients. Similarly, in TNBS-induced murine colitis model, MAP1S expression was obviously increased. Meanwhile, we found the co-location of MAP1S and active-caspase 3 which acted as "apoptotic executor" which might indicate the basis of their co-efficient. At the cellular level, MAP1S silencing inhibited starvation-induced over-expression of active-caspase 3 partially via Wnt/ß-catenin signaling activation in HCT-116 cells. Finally, we demonstrated that IWP-2, an inhibitor of the Wnt/ß-catenin signaling, reversed the down-regulation of active-caspase 3 induced by MAP1S siRNA in HCT-116 cells. Taken together, our results suggested that MAP1S were up-regulated among CD patients and MAP1S-related autophagy inhibits apoptosis of intestinal epithelial cells (IECs) through Wnt/ß-catenin signaling pathway which might play a vital role in the protection of intestinal mucosal barrier and inhibition the progression of CD.

A retrospective study of NENs and miR-224 promotes apoptosis of BON-1 cells by targeting PCSK9 inhibition.

Neuroendocrine neoplasms (NENs) represent relatively rare tumors. The lack of diagnostic, therapeutic method and prognostic factors makes them a challenge to us. We retrospectively reviewed the data of 205 NENs patients among which 157 cases were followed-up. Proprotein convertase subtilisin/kexin 9 (PCSK9), a regulator of low density lipoprotein cholesterol (LDL-C), was confirmed as a target gene of microRNA-224. We found an increased incidence of NENs from 2012 to 2015. Women were usually diagnosed at earlier stages than men (P < 0.05). Tumor grading was associated with primary tumor site, especially esophagus and cardia NENs all at G3 (P <0.001). Age, tumor grading and LDL-C levels were independent risk factors of digestive NENs. Low LDL-C level was significantly correlated with survival rate and median overall survival (OS, P < 0.05). MicroRNA-224 agomir and PCSK9 siRNA could promote apoptosis and suppress proliferation, invasion of BON-1 cells (P < 0.05), but increase the level of glucocorticoid (GC, P < 0.05). Taken together, age, tumor grading and LDL-C level are independent risk factors of NENs. The miR-224/PCSK9/GC axis binds to tumorigenesis and prognosis of pancreatic NENs (p-NENs).

GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis.

Glycinamide ribonucleotide formyltransferase (GART) has been established as a pivotal enzyme in de novo purine synthesis, and mediates cellular apoptosis in many diseases. We aimed to investigate the role of GART in the pathogenesis of Crohn's disease (CD). In our study, we demonstrated for the first time that GART expression is up-regulated in patients with active CD and in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model. Moreover, the inhibition of GART induced cellular apoptosis and suppressed the migration of IECs through the activation of the MEKK3-MKK3-p38 mitogen-activated protein kinase (MAPK) pathway, following with the dys-regulation of p53 and p53 up-regulated modulator of apoptosis (PUMA). Taken together, GART plays a critical role in the protection of cellular apoptosis and migration of intestinal epithelial cells to maintain the integrity of the epithelial barrier, thus providing a new potential approach in designing a novel therapy for CD.

SGK1 inhibits cellular apoptosis and promotes proliferation via the MEK/ERK/p53 pathway in colitis.

AIM: To investigate the role of serum-and-glucocorticoid-inducible-kinase-1 (SGK1) in colitis and its potential pathological mechanisms. METHODS: SGK1 expression in mucosal biopsies from patients with active Crohn's disease (CD) and normal controls was detected by immunohistochemistry. We established an acute colitis model in mice induced by 2,4,6-trinitrobenzene sulfonicacid, and demonstrated the presence of colitis using the disease activity index, the histologic activity index and hematoxylin and eosin staining. The cellular events and potential mechanisms were implemented with small interference RNA and an inhibitor of signaling molecule (i.e., U0126) in intestinal epithelial cells (IECs). The interaction between SGK1 and the signaling molecule was assessed by co-immunoprecipitation. RESULTS: SGK1 expression was significantly increased in the inflamed epithelia of patients with active CD and TNBS-induced colitis model (0.58 ± 0.055 vs 0.85 ± 0.06, P < 0.01). At the cellular level, silencing of SGK1 by small interference RNA (siSGK1) significantly inhibited the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1) and the downstream molecule extracellular signal regulated protein kinase (ERK) 1/2, which induced the upregulation of p53 and Bcl-2-associated X protein, mediating the subsequent cellular apoptosis and proliferation in IECs. Cells treated with MEK1 inhibitor (i.e., U0126) before siSGK1 transfection showed a reversal of the siSGK1-induced cellular apoptosis. CONCLUSION: Our data suggested that SGK1 may protect IECs in colitis from tumor necrosis factor-α-induced apoptosis partly by triggering MEK/ERK activation.

Pyruvate kinase M2 regulates apoptosis of intestinal epithelial cells in Crohn's disease.

BACKGROUND: Pyruvate kinase M2 (PKM2), a key glycolytic enzyme, is involved in multiple cellular processes including apoptosis. Recently increased fecal PKM2 has been found in Crohn's disease (CD), but little is known regarding its function in the pathophysiology of the disease. AIM: The intestinal expression of PKM2 and its involvement in CD was investigated. METHODS: Pyruvate kinase M2 expression in mucosal biopsies from patients with CD and normal controls was detected by immunohistochemistry. A murine model of colitis induced by trinitrobenzenesulphonic acid (TNBS) was established and expression of PKM2, B cell lymphoma-extra large (Bcl-xl), active caspase-3 as well as cleaved poly (ADP-ribose) polymerase (PARP) was examined for association of PKM2 with intestinal epithelial cell (IEC) apoptosis. Furthermore, we treated human IEC line HT-29 by tumor necrosis factor-α (TNF-α) and used RNA interference to analyze the role of PKM2 in IEC apoptosis. RESULTS: Intestinal expression of PKM2 was higher in patients with CD compared with normal controls mainly locating in IECs. In TNBS-induced colitis, up-regulation of PKM2 was accompanied by the elevated expression of Bcl-xl, active caspase-3, and cleaved PARP. PKM2 was co-localized with active caspase-3 in IECs marked by E-cadherin, suggesting its role in IEC apoptosis. Expression of PKM2 and Bcl-xl in TNF-α-induced HT-29 cells was increased, while TNF-α had no effect on cellular localization of PKM2. Furthermore, knockdown of PKM2 by siRNA could inhibit expression of Bcl-xl but enhance apoptosis in TNF-α-treated HT-29 cells. CONCLUSION: The up-regulation of PKM2 might protect IECs against apoptosis possibly through Bcl-xl in CD, indicating its important role in the pathophysiology of CD.

Role of far upstream element binding protein 1 in colonic epithelial disruption during dextran sulphate sodium-induced murine colitis.

AIM: Intestinal epithelial barrier is essential for maintaining normal intestinal homeostasis; its breakdown leads to chronic inflammatory pathologies, such as inflammatory bowel diseases. Far upstream element binding protein 1 (FBP1) has been reported to play an important role in cell apoptosis and proliferation. We aimed to investigate the expression and the role of FBP1 in dextran sodium sulphate (DSS)-induced experimental colitis. METHODS: Mice experimental colitis model was established by administration of DSS, and the expression and localization of FBP1 was examined using Western blot and immunohistochemistry. Colon epithelial cell line HT-29 was used to determine the role of FBP1. In vitro study, the expression of FBP1 was determined in HT-29 cells stimulated with tumor necrosis factor α (TNF-α). HT-29 cells were transfected with FBP1 siRNA and then measured for viability. RESULTS: Significant decreasing of FBP1 expression was found in mice colitis. In addition, FBP1 was cleaved and translocated from nucleus to cytoplasm during apoptosis. Downregulated expression of FBP1 induced cell cycle arrest. CONCLUSIONS: We demonstrate that apoptosis-mediated cleavage of FBP1 and its decreased expression in epithelial cells induces cell cycle arrest, which may play an important role in colonic epithelial disruption.