Characterization of Exosomes Released from Mycobacterium abscessus-Infected Macrophages.

Extracellular vesicles (EVs), such as exosomes, play a critical role in cell-to-cell communication and regulating cellular processes in recipient cells. Non-tuberculous mycobacteria (NTM), such as Mycobacterium abscessus, are a group of environmental bacteria that can cause severe lung infections in populations with pre-existing lung conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). There is limited knowledge of the engagement of EVs in the host-pathogen interactions in the context of NTM infections. In this study, we found that M. abscessus infection increased the release of a subpopulation of exosomes (CD9, CD63, and/or CD81 positive) by mouse macrophages in cell culture. Proteomic analysis of these vesicles demonstrated that M. abscessus infection affects the enrichment of host proteins in exosomes released by macrophages. When compared to exosomes from uninfected macrophages, exosomes released by M. abscessus-infected macrophages significantly improved M. abscessus growth and downregulated the intracellular level of glutamine in recipient macrophages in cell culture. Increasing glutamine concentration in the medium rescued intracellular glutamine levels and M. abscessus killing in recipient macrophages that were treated with exosomes from M. abscessus-infected macrophages. Taken together, our results indicate that exosomes may serve as extracellular glutamine eliminators that interfere with glutamine-dependent M. abscessus killing in recipient macrophages.

ß2-microglobulin induced apoptosis of tumor cells via the ERK signaling pathway by directly interacting with HFE in HER2-overexpressing breast cancer.

BACKGROUND: Our previous study demonstrated that ß2-microglobulin (ß2M) promoted ER+/HER2- breast cancer survival via the SGK1/Bcl-2 signaling pathway. However, the role of ß2M has not been investigated in ER-/HER2+ breast cancer. Here, we aimed to determine the role of ß2M in ER-/HER2+ breast cancer. METHODS: The interaction between ß2M and HFE was confirmed by co-immunoprecipitation, mass spectrometry, yeast two-hybrid screening, and His pull-down. The knockdown and overexpression of ß2M or HFE were performed in MDA-MB-453 cells, and ERK signaling pathway was subsequently analyzed via western blotting. Apoptotic cells were detected using flow cytometer. ß2M, HFE, and p-ERK1/2 were examined in tumor and paired adjacent tissues via immunohistochemistry. RESULTS: HFE was found to be an interacting protein of ß2M in ER-/HER2+ breast cancer cells MDA-MB-453 by co-immunoprecipitation and mass spectrometry. A yeast two-hybrid system and His-pull down experiments verified that ß2M directly interacted with HFE. ß2M and HFE as a complex were mainly located in the cytoplasm, with some on the cytomembrane of MDA-MB-453 cells. In addition to breast cancer cells BT474, endogenous ß2M directly interacted with HFE in breast cancer cells MDA-MB-453, MDA-MB-231, and MCF-7. ß2M activated the ERK signaling pathway by interacting with HFE and induced apoptosis of MDA-MB-453 cells. The expression of HFE and p-ERK1/2 showed significantly high levels in HER2-overexpressing breast cancer tumor tissue compared with adjacent normal tissue, consistent with the results obtained from the cell experiments. CONCLUSIONS: ß2M induced apoptosis of tumor cells via activation of the ERK signal pathway by directly interacting with HFE in HER2-overexpressing breast cancer.

Mycobacterium abscessus extracellular vesicles increase mycobacterial resistance to clarithromycin in vitro.

Nontuberculous Mycobacteria (NTM) are a group of emerging bacterial pathogens that have been identified in cystic fibrosis (CF) patients with microbial lung infections. The treatment of NTM infection in CF patients is challenging due to the natural resistance of NTM species to many antibiotics. Mycobacterium abscessus is one of the most common NTM species found in the airways of CF patients. In this study, we characterized the extracellular vesicles (EVs) released by drug-sensitive M. abscessus untreated or treated with clarithromycin (CLR), one of the frontline anti-NTM drugs. Our data show that exposure to CLR increases mycobacterial protein trafficking into EVs as well as the secretion of EVs in culture. Additionally, EVs released by CLR-treated M. abscessus increase M. abscessus resistance to CLR when compared to EVs from untreated M. abscessus. Proteomic analysis further indicates that EVs released by CLR-treated M. abscessus carry an increased level of 50S ribosomal subunits, the target of CLR. Taken together, our results suggest that EVs play an important role in M. abscessus resistance to CLR treatment.

Time- and temperature-dependent postmortem ∆9-tetrahydrocannabinol concentration changes in rabbits following controlled inhaled cannabis administration.

ostmortem redistribution (PMR), a well-known phenomenon in forensic toxicology, can result in substantial changes in drug concentrations after death, depending on the chemical characteristics of the drug, blood collection site, storage conditions of the body and postmortem interval (PMI). Limited PMR data are available for ∆9-tetrahydrocannabinol (THC), the primary psychoactive component in Cannabis sativa. PMR was evaluated after controlled cannabis inhalation via a smoking machine and exposure chamber in New Zealand white rabbits. Necropsies were performed on five control rabbits immediately after euthanasia, whereas 27 others were stored at room temperature (21°C) or refrigerated conditions (4°C) until necropsy at 2, 6, 16, 24 or 36 h after death. THC and its Phase I and glucuronidated Phase II metabolites were quantified in blood, vitreous humor, urine, bile and tissues by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Under refrigerated temperature, heart blood THC concentrations significantly increased at PMI 2 h in rabbits, whereas peripheral blood THC concentrations showed a significant increase at PMI 16 h. Central:peripheral blood and liver:peripheral blood ratios for THC ranged from 0.13 to 4.1 and 0.28 to 8.9, respectively. Lung revealed the highest THC concentrations, while brain and liver exhibited the most stable THC concentrations over time. This report contributes much needed data to our understanding of postmortem THC behavior and can aid toxicologists in the interpretation of THC concentrations in medicolegal death investigations.

MicroRNA-9-1 Attenuates Influenza A Virus Replication via Targeting Tankyrase 1.

An unstable influenza genome leads to the virus resistance to antiviral drugs that target viral proteins. Thus, identification of host factors essential for virus replication may pave the way to develop novel antiviral therapies. In this study, we investigated the roles of the poly(ADP-ribose) polymerase enzyme, tankyrase 1 (TNKS1), and the endogenous small noncoding RNA, miR-9-1, in influenza A virus (IAV) infection. Increased expression of TNKS1 was observed in IAV-infected human lung epithelial cells and mouse lungs. TNKS1 knockdown by RNA interference repressed influenza viral replication. A screen using TNKS1 3'-untranslation region (3'-UTR) reporter assays and predicted microRNAs identified that miR-9-1 targeted TNKS1. Overexpression of miR-9-1 reduced influenza viral replication in lung epithelial cells as measured by viral mRNA and protein levels as well as virus production. miR-9-1 induced type I interferon production and enhanced the phosphorylation of STAT1 in cell culture. The ectopic expression of miR-9-1 in the lungs of mice by using an adenoviral viral vector enhanced type I interferon response, inhibited viral replication, and reduced susceptibility to IAV infection. Our results indicate that miR-9-1 is an anti-influenza microRNA that targets TNKS1 and enhances cellular antiviral state.

Axin1: A novel scaffold protein joins the antiviral network of interferon.

Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of ß-Catenin.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced ß-catenin protein, but not mRNA levels. The reduction of ß-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

Revascularization of iatrogenic intraoperative injury to a major artery during hepatobiliary-pancreatic surgery: a single-center experience in China.

BACKGROUND: Although uncommon during hepatobiliary-pancreatic (HBP) surgery, iatrogenic intraoperative injury to a major artery requires prompt and appropriate repair. Here, we outline our surgical experience with the repair of this injury and compare our experience to findings garnered from a selective review of the literature. METHODS: We retrospectively analyzed the clinical diagnoses, surgical methods, sites of arterial injury, operative repair, intra-operative blood loss, blood transfusion requirements, postoperative management and outcome of 17 consecutive patients who sustained iatrogenic intra-operative injuries to major arteries during HBP surgery between January 2008 and December 2013. RESULTS: Depending on the location and extent of injury, suture repair, primary end-to-end anastomosis, artery transposition, interposition grafting, or arterio-portal shunting were used. Postoperative morbidity occurred in three cases and there was only one case of in-hospital mortality (5.9%). No arterial thrombosis or other repair-related complications were found after the operation with a follow-up duration of 6 months. CONCLUSIONS: The use of an optimal repair method for injured arteries based on their location and extent resulted in a satisfactory outcome.

MicroRNA-206 inhibits influenza A virus replication by targeting tankyrase 2.

Due to the frequent mutations, influenza A virus (IAV) becomes resistant to anti-viral drugs targeting influenza viral proteins. There are increasing interests in anti-viral agents that target host cellular proteins required for virus replication. Tankyrase (TNKS) has poly (ADP-ribose) polymerase activity and is a negative regulator of many host proteins. The objectives of this study are to study the role of TNKS2 in IAV infection, identify the microRNAs targeting TNKS2, and to understand the mechanisms involved. We found that TNKS2 expression was elevated in human lung epithelial cells and mouse lungs during IAV infection. Knock-down of TNKS2 by RNA interference reduced viral replication. Using a computation approach and 3'-untranslation regions (3'-UTR) reporter assay, we identified miR-206 as the microRNA that targeted TNKS2. Overexpression of miR-206 reduced viral protein levels and virus production in cell culture. The effect of miR-206 on IAV replication was strain-independent. miR-206 activated JNK/c-Jun signalling, induced type I interferon expression and enhanced Stat signalling. Finally, the delivery of an adenovirus expressing miR-206 into the lung of mice challenged with IAV increased type I interferon response, suppressed viral load in the lungs and increased survival. Our results indicate that miR-206 has anti-influenza activity by targeting TNKS2 and subsequently activating the anti-viral state.

miR-29a is a negative regulator of influenza virus infection through targeting of the frizzled 5 receptor.

Influenza A virus (IAV) infections result in a large number of deaths and substantial economic losses each year. MicroRNAs repress gene expression and are involved in virus-host interactions. miR-29a is known to have anti-tumor and anti-fibrotic effects. However, the role of miR-29a in IAV infection is unclear. In the present study, we investigated the effect of miR-29a on IAV infection and the mechanisms by which it functions. IAV infection was found to cause decreased miR-29a expression in lung epithelial A549 cells and mouse lungs. Overexpression of miR-29a reduced IAV mRNA and protein levels and progeny virus production in HEK293 and A549 cells. Inhibition of IAV infection by miR-29a was observed with different strains of IAV, including A/PR/8/34, A/WSN/1933, and clinical isolates A/OK/3052/09 and A/OK/309/06 H3N2. Knockout of miR-29a using CRISPR/Cas9 resulted in an increase in viral mRNA and protein levels, confirming that miR-29a suppresses IAV infection. A 3' untranslated region (3'-UTR) reporter assay showed that miR-29a had binding sites in the 3'-UTR of the Wnt-Ca2+ signaling receptor frizzled 5 gene, and overexpression of miR-29a reduced the level of the endogenous frizzled 5 protein. Wnt5a treatment of HEK293 and A549 cells enhanced IAV infection. Our results suggest that miR-29a inhibits IAV infection, probably via the frizzled 5 receptor.

Long non-coding RNA FENDRR regulates IFNγ-induced M1 phenotype in macrophages.

Macrophages play an essential role in host defense and display remarkable plasticity in switching between classically (pro-inflammatory-M1) and alternatively activated (anti-inflammatory-M2) phenotypes. The molecular mechanisms of macrophage polarization are not fully understood. Long non-coding RNAs (lncRNAs) with a length of > 200 nucleotides have been shown to play diverse roles in biological processes. Aberrant expression of lncRNAs is associated with a variety of pathophysiological conditions such as cancer, diabetes, cardiovascular, pulmonary diseases, and tissue fibrosis. In this study, we investigated the role of lncRNA FENDRR in human and mouse macrophage polarization. Human THP-1 monocytes were activated with phorbol-12-myristate-13-acetate (PMA) and differentiated into M1 macrophages with IFNγ or M2 macrophages with IL4. Real-time PCR analysis revealed that FENDRR was expressed 80-fold higher in M1 macrophages than that in M2 macrophages. Overexpression of FENDRR in PMA-activated THP-1 cells increased the IFNγ-induced expression of M1 markers, including IL1ß and TNFα at both mRNA and protein levels. Knockdown of FENDRR had an opposite effect. Similarly, FENDRR overexpression in primary mouse bone marrow-derived macrophages increased mRNA expression of M1 markers. FENDRR overexpression increased, while FENDRR knock-down decreased, the IFNγ-induced phosphorylation of STAT1 in PMA-activated THP-1 cells. Our studies suggest that FENDRR enhances IFNγ-induced M1 macrophage polarization via the STAT1 pathway.

Influence of miR-101 on proliferation of liver cancer cells through the MAPK/ERK signaling pathway.

The expression of miR-101 in carcinoma and para-carcinoma tissues of patients with liver cancer was studied. The carcinoma and para-carcinoma tissues of 67 patients with liver cancer treated in Chinese PLA General Hospital were collected, and the expression of miR-101 in carcinoma and para-carcinoma tissues was detected via reverse transcription-polymerase chain reaction (RT-PCR). The liver cancer HepG2 cell line was transfected with miR-101 mimics. Moreover, the influence of miR-101 overexpression on the proliferation of liver cancer cells was detected via Cell Counting Kit-8 assay and colony formation assay. The proportion of Ki67-positive cells in the control group (NC group) and miR-101 overexpression group (miR-101 mimics group) was detected via Ki67 staining. The proportions of cells were detected via flow cytometry, and the predicted target gene Zeste2 enhancer (EZH2) was further verified via luciferase reporter gene assay and western blotting. The miR-101 overexpression significantly inhibited the colony formation and proliferation ability of liver cancer cells (P<0.05). The proportion of Ki67-positive cells in liver cancer cells was lower in miR-101 mimics group (P<0.05). The proportion of cells in G0/G1 phase was increased in miR-101 mimics group compared with that in NC group (P<0.05). The extracellular signal-regulated kinase (ERK)1/2 phosphorylation level in liver cancer cells was obviously suppressed in miR-101 mimics group (P<0.05). Therefore, the expression level of miR-101 declines in liver cancer tissues, and the miR-101 overexpression can inhibit the proliferation of liver cancer cells. The inhibitory effect of miR-101 on the proliferation of liver cancer cells may be related to its inhibition on the mitogen-activated protein kinase (MAPK)/ERK signaling pathway, and the inhibition on the MAPK/ERK may be mediated by the targeted inhibition of miR-101 on EZH2.

Long Noncoding RNA FENDRR Exhibits Antifibrotic Activity in Pulmonary Fibrosis.

Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element-binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-ß1 (transforming growth factor-ß1)-SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

Repeated Exposure to Streptococcus pneumoniae Exacerbates Chronic Obstructive Pulmonary Disease.

Streptococcus pneumoniae is commonly found in patients with chronic obstructive pulmonary disease (COPD) and is linked to acute exacerbation of COPD. However, current clinical therapy neglects asymptomatic insidious S. pneumoniae colonization. We studied the roles of repeated exposure to S. pneumoniae in COPD progression using a mouse model. C57BL/6J mice were intranasally inoculated with S. pneumoniae ST262 every 4 weeks with or without cigarette smoke (CS) exposure up to 20 weeks to maintain persistent S. pneumoniae presence in the lower airways. Streptococcus pneumoniae enhanced CS-induced inflammatory cell infiltration at 12 to 20 weeks of exposure. Streptococcus pneumoniae also increased CS-induced release of inflammatory cytokines, including IL-1ß, tumor necrosis factor-α, IL-12 (p70), and IL-5 at 20 weeks of exposure. Moreover, a combination of CS and S. pneumoniae caused alveolar epithelial injury, a decline in lung function, and an increased expression of platelet-activating factor receptor and bacterial load. Our results suggest that repeated exposure to S. pneumoniae in lower airways exacerbates CS-induced COPD.

miR-193b represses influenza A virus infection by inhibiting Wnt/ß-catenin signalling.

Due to an increasing emergence of new and drug-resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/ß-catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/ß-catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up-regulated and 20 miRNAs that down-regulated the Wnt/ß-catenin signalling pathway. Fifteen miRNAs were validated to up-regulate and five miRNAs to down-regulate the pathway. Overexpression of four selected miRNAs (miR-193b, miR-548f-1, miR-1-1, and miR-509-1) that down-regulated the Wnt/ß-catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34-infected HEK293 cells, and progeny virus production. Overexpression of miR-193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR-193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that ß-catenin was a target of miR-193b, and ß-catenin rescued miR-193b-mediated suppression of IAV infection. miR-193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus-mediated gene transfer of miR-193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR-193b represses IAV infection by inhibiting Wnt/ß-catenin signalling.

Serum amyloid A promotes LPS clearance and suppresses LPS-induced inflammation and tissue injury.

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram-negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins, but the relationship between SAA expression and LPS-induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα-induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1-LPS interaction with a SAA1-derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute-phase SAA provides innate feedback protection against LPS-induced inflammation and tissue injury.

A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection.

Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation.

Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-ß-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-ß receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis.

[Development of laparoscopic technology in biliary surgery in the past 23 years: a single-center experience].

OBJECTIVE: To summarize the 23-year experience of laparoscopic biliary surgery in General Hospital of PLA and evaluate the application of laparoscopic surgery in the treatment of biliary diseases. METHODS: We retrospectively analyzed the clinical data of 11 419 consecutive patients with biliary diseases undergoing laparoscopic surgery from April, 1992 and December, 2014. The disease spectrum was compared between patients treated before December 31, 2003 and those treated after the time point. RESULTS: The 11419 patients receiving laparoscopic surgery accounted for 56.3% of the total patients undergoing biliary surgeries during the 23 years, including 4701 male and 6718 female patients with a mean age of 50.9∓13.2 years (6-93 years). Most (80.83%) of the patients received laparoscopic surgery for gallbladder stones, and 12.53% patients had the operation for gallbladder polyps. The laparoscopic operation rate was 84.81% in patients with gallbladder stones and 34.91% in patients with extrahepatic bile duct stones, but remained low in patients with biliary carcinoma. In laparoscopic operations, laparoscopic cholecystectomy was the most frequent (96.18%) followed by operations for extrahepatic bile duct stones, in which primary suture accounted for 1.38%, traditional T tube drainage for 0.90% and laparoscopic transcystic duct exploration for 0.72%. For malignant tumors, laparoscopic technique was used mainly for the purpose of exploration (0.34%). The application of laparoscopic technique in biliary surgery tended to increase after the year 2004, especially for benign gallbladder diseases and extrahepatic bile duct stones (P<0.05). CONCLUSION: Laparoscopic technique in biliary surgery is gradually replacing the traditional open operation and becomes the gold standard for the treatment of benign biliary diseases.

EZH2 enhances the differentiation of fibroblasts into myofibroblasts in idiopathic pulmonary fibrosis.

The accumulation of fibroblasts/myofibroblasts in fibrotic foci is one of the characteristics of idiopathic pulmonary fibrosis (IPF). Enhancer of zeste homolog 2 (EZH2) is the catalytic component of a multiprotein complex, polycomb repressive complex 2, which is involved in the trimethylation of histone H3 at lysine 27. In this study, we investigated the role and mechanisms of EZH2 in the differentiation of fibroblasts into myofibroblasts. We found that EZH2 was upregulated in the lungs of patients with IPF and in mice with bleomycin-induced lung fibrosis. The upregulation of EZH2 occurred in myofibroblasts. The inhibition of EZH2 by its inhibitor 3-deazaneplanocin A (DZNep) or an shRNA reduced the TGF-ß1-induced differentiation of human lung fibroblasts into myofibroblasts, as demonstrated by the expression of the myofibroblast markers α-smooth muscle actin and fibronectin, and contractility. DZNep inhibited Smad2/3 nuclear translocation without affecting Smad2/3 phosphorylation. DZNep treatment attenuated bleomycin-induced pulmonary fibrosis in mice. We conclude that EZH2 induces the differentiation of fibroblasts to myofibroblasts by enhancing Smad2/3 nuclear translocation.