MIR31HG promotes cell proliferation and invasion by activating the Wnt/ß-catenin signaling pathway in non-small cell lung cancer.

Long non-coding RNAs (lncRNAs) have recently been demonstrated to serve crucial roles in various diseases including tumor initiation and progression. However, the role of the lncRNA MIR31HG in non-small cell lung cancer (NSCLC) was not well established. The present study demonstrated that MIR31HG was significantly increased in tumor tissues compared with adjacent normal tissues, and increased MIR31HG expression levels were associated with histological differentiation grade, lymph node metastasis and Tumor-node metastasis (TNM) stage in patients with NSCLC. Patients who had a higher MIR31HG expression level, were predicted a shorter over survival (OS) time. Using in vitro assays, the present study demonstrated that the downregulation of MIR31HG expression significantly inhibited cell proliferation and cell invasion abilities. Furthermore, it was identified that knockdown of MIR31HG expression suppressed the cell epithelial-mesenchymal transition (EMT) phenotype by reducing the expression levels of Twist1 and Vimentin, but also increased the expression level of E-cadherin in NSCLC cells. Furthermore, the results of the present study demonstrated that downregulated MIR31HG inhibited the Wnt/ß-catenin signaling pathway by decreasing the expression of glycogen synthase kinase 3ß (GSK3ß) and ß-catenin, but increasing the phosphorylated (p)-GSK3ß expression in NSCLC cells. Together, these data demonstrated that MIR31HG could be identified as a poor prognostic biomarker and a novel therapeutic target for patients with NSCLC.

Downregulation of miR-138 predicts poor prognosis in patients with esophageal squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) have been proven to be critical players in many different types of tumors including esophageal squamous cell carcinoma (ESCC). OBJECTIVE: This study aimed at investigating the correlation of miR-138 expression and clinical outcome of patients with ESCC. METHODS: A total of 168 serum samples and 128 fresh cancer tissues as well as their corresponding adjacent non-cancerous tissues were collected. Real-time PCR was performed to evaluate the clinical value of miR-138 in ESCC. RESULTS: Our results showed that tissue and serum miR-138 levels were both significantly reduced in ESCC compared to their respective controls. Tissue miR-138 levels were highly correlated with serum miR-138 levels. Serum miR-138 differentiated patients with ESCC from healthy controls with high accuracy. In addition, reduced tissue/serum miR-138 levels were correlated with unfavorable clinicopathological parameters including T stage, lymph node metastasis and TNM stage. ESCC patients with lower tissue/serum miR-138 levels had shorter five year overall survival compared with those with higher tissue/serum miR-138 levels. Finally, downregulation of miR-138 was demonstrated to be an independent prognostic risk factor for ESCC. CONCLUSIONS: In conclusion, both tissue and serum miR-138 levels are reduced in ESCC, and might be promising prognostic biomarkers for ESCC.

CD8+ T lymphocyte response against extrahepatic biliary epithelium is activated by epitopes within NSP4 in experimental biliary atresia.

Interferon (IFN)-γ-driven and CD8+ T cell-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen that causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4(157-170), NSP4(144-152), NSP4(93-110), NSP4(24-32)) identified by computer analysis as candidate CD8+ T cell epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN-γ release and CD8+ T cell response against EHBE. The results revealed, at 7 days postinjection, inoculation of glutathione S-transferase (GST)-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 days postinject, inoculation of GST-NSP4, NSP4(144-152), or NSP4(157-170) increased IFN-γ release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naive EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-γ. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4(144-152), or NSP4(157-170) decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4(144-152) or NSP4(157-170) is effective in protecting neonates from developing RRV-related BA.

HMGB1-promoted and TLR2/4-dependent NK cell maturation and activation take part in rotavirus-induced murine biliary atresia.

Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.

Silencing of the rotavirus NSP4 protein decreases the incidence of biliary atresia in murine model.

Biliary atresia is a common disease in neonates which causes obstructive jaundice and progressive hepatic fibrosis. Our previous studies indicate that rotavirus infection is an initiator in the pathogenesis of experimental biliary atresia (BA) through the induction of increased nuclear factor-kappaB and abnormal activation of the osteopontin inflammation pathway. In the setting of rotavirus infection, rotavirus nonstructural protein 4 (NSP4) serves as an important immunogen, viral protein 7 (VP7) is necessary in rotavirus maturity and viral protein 4 (VP4) is a virulence determiner. The purpose of the current study is to clarify the roles of NSP4, VP7 and VP4 in the pathogenesis of experimental BA. Primary cultured extrahepatic biliary epithelia were infected with Rotavirus (mmu18006). Small interfering RNA targeting NSP4, VP7 or VP4 was transfected before rotavirus infection both in vitro and in vivo. We analyzed the incidence of BA, morphological change, morphogenesis of viral particles and viral mRNA and protein expression. The in vitro experiments showed NSP4 silencing decreased the levels of VP7 and VP4, reduced viral particles and decreased cytopathic effect. NSP4-positive cells had strongly positive expression of integrin subunit α2. Silencing of VP7 or VP4 partially decreased epithelial injury. Animal experiments indicated after NSP4 silencing, mouse pups had lower incidence of BA than after VP7 or VP4 silencing. However, 33.3% of VP4-silenced pups (N = 6) suffered BA and 50% of pups (N = 6) suffered biliary injury after VP7 silencing. Hepatic injury was decreased after NSP4 or VP4 silencing. Neither VP4 nor VP7 were detected in the biliary ducts after NSP4. All together, NSP4 silencing down-regulates VP7 and VP4, resulting in decreased incidence of BA.

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice.

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts' epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO(2). The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5-6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrane, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.