Publications by authors named "Wenyu Lin"

73 Publications

Fatty Acids Activate the Transcriptional Coactivator YAP1 to Promote Liver Fibrosis via p38 Mitogen-Activated Protein Kinase.

Cell Mol Gastroenterol Hepatol 2021 Jun 9. Epub 2021 Jun 9.

Liver Center, Massachusetts General Hospital, Boston, Massachusetts. Electronic address:

Background & Aims: Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)-related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program.

Methods: We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes.

Results: YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells.

Conclusions: These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.
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http://dx.doi.org/10.1016/j.jcmgh.2021.06.003DOI Listing
June 2021

The risk of hepatitis C virus recurrence in hepatitis C virus-infected patients treated with direct-acting antivirals after achieving a sustained virological response: A comprehensive analysis.

Liver Int 2021 May 29. Epub 2021 May 29.

Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Background & Aims: The risk for hepatitis C virus (HCV) recurrence persists after HCV eradication with direct-acting antivirals (DAAs), particularly in patients with ongoing high-risk behaviours. Our aim was to assess the risk of HCV recurrence (late relapse and/or reinfection) post-sustained virological response (SVR).

Methods: We searched the literature for studies reporting HCV recurrence rates post-SVR in PubMed, Web of Science and the Cochrane Library. Identified publications were divided into groups based on patient risk for HCV reinfection: low-risk HCV mono-infection, high-risk HCV mono-infection and a human immunodeficiency virus (HIV)/HCV coinfection. The HCV recurrence rate for each study was calculated by using events divided by the person-years of follow-up (PYFU). HCV recurrence was defined as confirmed, detectable HCV RNA post-SVR.

Results: In the 16 studies of low-risk patients, the pooled recurrence rate was 0.89/1000 PYFU (95% confidence interval [CI], 0.16-2.03). For the 19 studies of high-risk patients, the pooled recurrence rate was 29.37/1000 PYFU (95% CI, 15.54-46.91). For the eight studies of HIV/HCV-coinfected patients, the pooled recurrence rate was 23.25/1000 PYFU (95% CI, 4.24-53.39). The higher pooled estimates of recurrence in the high-risk and HIV/HCV-coinfected populations were predominantly driven by an increase in reinfection rather than late relapse.

Conclusions: The HCV recurrence risk after achieving SVR with all-oral DAAs therapy is low, and the risk of HCV recurrence in high-risk and HIV/HCV-coinfected populations was driven by an increase in reinfection rather than late relapse.
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http://dx.doi.org/10.1111/liv.14976DOI Listing
May 2021

Differentially expressed immune response genes in COVID-19 patients based on disease severity.

Aging (Albany NY) 2021 03 29;13(7):9265-9276. Epub 2021 Mar 29.

Fuyang Infectious Disease Clinical College of Anhui Medical University, Fuyang 236015, Anhui Province, P.R. of China.

Background: Dysregulated immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are thought to underlie the progression of coronavirus disease 2019 (COVID-19). We sought to further characterize host antiviral and cytokine gene expression in COVID-19 patients based on illness severity.

Methods: In this case-control study, we retrospectively analyzed 46 recovered COVID-19 patients and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We assessed changes in antiviral gene expression between healthy controls and patients with mild/moderate (MM) and severe/critical (SC) disease.

Results: We found that type I interferon signaling (IFNA2, TLR8, IFNA1, IFNAR1, TLR9, IRF7, ISG15, APOBEC3G, and MX1) and genes encoding proinflammatory cytokines (IL12B, IL15, IL6, IL12A and IL1B) and chemokines (CXCL9, CXCL11 and CXCL10) were upregulated in patients with MM and SC disease. Moreover, we found that IFNA1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), and Fas-associated protein with death domain (FADD) were significantly downregulated ( < 0.05) in the SC group compared to the MM group. We also observed that microRNA (miR)-155 and miR-130a levels were markedly higher in the MM group compared to the SC group.

Conclusion: COVID-19 is associated with the activation of host antiviral genes. Induction of the IFN system appears to be particularly important in controlling SARS-CoV-2 infection, as decreased expression of IFNA1, APOBEC3G and FADD genes in SC patients, relative to MM patients, may be associated with disease progression.
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http://dx.doi.org/10.18632/aging.202877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064215PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth, metastasis and sorafenib resistance through STAT3 activation.

J Cell Mol Med 2021 Feb 7;25(3):1568-1582. Epub 2021 Jan 7.

Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.

The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.
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http://dx.doi.org/10.1111/jcmm.16256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875922PMC
February 2021

Virus detection using nanoparticles and deep neural network-enabled smartphone system.

Sci Adv 2020 Dec 16;6(51). Epub 2020 Dec 16.

Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02139, USA.

Emerging and reemerging infections present an ever-increasing challenge to global health. Here, we report a nanoparticle-enabled smartphone (NES) system for rapid and sensitive virus detection. The virus is captured on a microchip and labeled with specifically designed platinum nanoprobes to induce gas bubble formation in the presence of hydrogen peroxide. The formed bubbles are controlled to make distinct visual patterns, allowing simple and sensitive virus detection using a convolutional neural network (CNN)-enabled smartphone system and without using any optical hardware smartphone attachment. We evaluated the developed CNN-NES for testing viruses such as hepatitis B virus (HBV), HCV, and Zika virus (ZIKV). The CNN-NES was tested with 134 ZIKV- and HBV-spiked and ZIKV- and HCV-infected patient plasma/serum samples. The sensitivity of the system in qualitatively detecting viral-infected samples with a clinically relevant virus concentration threshold of 250 copies/ml was 98.97% with a confidence interval of 94.39 to 99.97%.
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http://dx.doi.org/10.1126/sciadv.abd5354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744080PMC
December 2020

SLC7A11/xCT in cancer: biological functions and therapeutic implications.

Am J Cancer Res 2020 1;10(10):3106-3126. Epub 2020 Oct 1.

Laboratory of Cancer Biology, Key Lab of Biotherapy in Zhejiang, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine Hangzhou 310016, Zhejiang, China.

Amino acid transporters mediate substrates across cellular membranes and their fine-tuned regulations are critical to cellular metabolism, growth, and death. As the functional component of system Xc-, which imports extracellular cystine with intracellular glutamate release at a ratio of 1:1, SLC7A11 has diverse functional roles in regulating many pathophysiological processes such as cellular redox homeostasis, ferroptosis, and drug resistance in cancer. Notably, accumulated evidence demonstrated that SLC7A11 is overexpressed in many types of cancers and is associated with patients' poor prognosis. As a result, SLC7A11 becomes a new potential target for cancer therapy. In this review, we first briefly introduce the structure and function of SLC7A11, then discuss its pathological role in cancer. We next summarize current available data of how SLC7A11 is subjected to fine regulations at multiple levels. We further describe the potential inhibitors of the SLC7A11 and their roles in human cancer cells. Finally, we propose novel insights for future perspectives on the modulation of SLC7A11, as well as possible targeted strategies for SLC7A11-based anti-cancer therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642655PMC
October 2020

The Role of Obstetric Factors, miRNA-30d and miRNA-181a in Postpartum Women with Pelvic Organ Prolapse.

Risk Manag Healthc Policy 2020 28;13:2309-2316. Epub 2020 Oct 28.

Department of Gynecology, Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University, Fuzhou, People's Republic of China.

Background: The diagnosis of postpartum pelvic organ prolapse (POP) relies on symptoms combined with pelvic organ prolapse-quantification (POP-Q) and lacks serological indicators. The objective of this study was to assess serum elastin, type I collagen, miRNA-30d, and miRNA-181a in the early postpartum period to identify hematologic predictors of POP.

Material And Methods: The study included 1013 42- to 60-day-postpartum women who had delivered at Quanzhou Women's and Children's Hospital from October 1, 2016, to October 31, 2017. This study was performed in accordance with the Declaration of Helsinki. The pregnancy and childbirth characteristics and pelvic floor function were evaluated. Forty cases with and without POP were matched, and serum elastin and type I collagen were determined by enzyme-linked immunosorbent assay (ELISA). Reverse-transcription polymerase chain reaction (RT-PCR) was used to detect miRNA-30d and miRNA-181a in 15 pairs.

Results: Of the 1013 women recruited, 699 (69.00%) were diagnosed with POP. The mean age was 29.00 years old, and the mean body mass index (BMI) was 22.6 kg/m. In the univariate analysis, age ≥35 years (OR, 1.449; 95% CI, 0.965, 2.298), postpartum BMI ≥ 24 (OR, 4.402; 95% CI, 2.657, 6.148), neonatal weight ≥4 kg (OR, 4.832; 95% CI, 1.373, 17.290) and vaginal delivery (OR, 2.751; 95% CI, 1.855, 4.081) were risk factors for postpartum POP. There were no significant differences in the concentrations of serum elastin and type I collagen between the groups (P=0.52; P=0.26). There were significant differences in the concentrations of miRNA-30d and miRNA-181a between the groups (P=0.004; P=0.003).

Conclusion: miRNA-30d and miRNA-181a tended to be increased in women with POP and could be potential clinical predictors.
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http://dx.doi.org/10.2147/RMHP.S268235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604264PMC
October 2020

Cost-effectiveness and accuracy of cervical cancer screening with a high-risk HPV genotyping assay vs a nongenotyping assay in China: an observational cohort study.

Cancer Cell Int 2020 28;20:421. Epub 2020 Aug 28.

Department of Gynecology, Laboratory of Gynecologic Oncology, Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University, 18 Daoshan Road, Fuzhou, 350001 Fujian People's Republic of China.

Background: New screening techniques may affect the optimal approaches for the prevention of cervical cancer. We evaluated the cost-effectiveness and accuracy of alternative screening strategies to provide evidence for cervical cancer screening guidelines in China.

Methods: In total, 32,306 women were enrolled. The current screening with Cervista high-risk human papillomavirus (HR-HPV) nongenotyping and cytology cotesting (Cervista cotesting) was compared with PCR-reverse dot blot HR-HPV genotyping and cytology cotesting (PCR-RDB cotesting). All eligible participants were divided into Arm 1, in which both HR-HPV assays were performed, and Arms 2 and 3, in which the PCR-RDB HPV or Cervista HR-HPV assay, respectively, was performed. Outcome indicators included the cases, sensitivity, negative predictive value (NPV), colposcopy referral rate and cost of identifying cervical intraepithelial neoplasia of grade 2/3 or worse (CIN2+/CIN3+).

Results: Among the eligible participants, 18.4% were PCR-RDB HR-HPV-positive, while 16.9% were Cervista HR-HPV-positive, which reflects good agreement (k = 0.73). PCR-RDB cotesting identified more CIN3+ cases than Cervista cotesting in the first round of screening in Arm 1 (37 vs 32) and Arms 2/3 (252 vs 165). The sensitivity and NPV of PCR-RDB cotesting for identifying CIN3+ in Arm 1 (sensitivity: 94.9% vs 86.5%; NPV: 99.9% vs 99.7%) and Arms 2/3 (sensitivity: 95.1% vs 80.9%; NPV: 99.9% vs 99.6%) were higher than those of Cervista cotesting, but the cost was similar.

Conclusions: The PCR-RDB HR-HPV genotyping and Cervista HR-HPV assay results were consistent. PCR-RDB cotesting possesses optimal cost-effectiveness for cervical cancer screening in China, which has the highest number of cases globally but low screening coverage.
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http://dx.doi.org/10.1186/s12935-020-01512-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453699PMC
August 2020

Pyruvate Kinase M2 Tetramerization Protects against Hepatic Stellate Cell Activation and Liver Fibrosis.

Am J Pathol 2020 11 15;190(11):2267-2281. Epub 2020 Aug 15.

Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China; Pathophysiology, School of Medicine, Jinan University, Guangzhou, China. Electronic address:

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The in vitro and in vivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs in vitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.
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http://dx.doi.org/10.1016/j.ajpath.2020.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786052PMC
November 2020

COVID-19 induced liver function abnormality associates with age.

Aging (Albany NY) 2020 07 28;12(14):13895-13904. Epub 2020 Jul 28.

Department of Respiratory, The Second People's Hospital of Fuyang, Fuyang 236015, Anhui Province, P.R. China.

Background: Coronavirus disease 2019 (COVID-19) is a novel infectious disease that may cause fever, dry cough, fatigue and shortness of breath. The impact of COVID-19 on liver function is not well described.

Results: We found that the overall frequency of LFT abnormality was 17.6%. Frequency of LFT abnormality was significantly greater in patients with severe/critical (SC) COVID-19 compared to those with mild/moderate (MM) COVID-19 (32.4% vs 11.6%, p=0.011). Among patients with LFT abnormality, the median age was significantly higher in the SC group compared to the MM group (52 vs 39 years, p=0.021).

Conclusion: COVID-19 is frequently associated with mild liver function abnormality, particularly in individuals with severe/critical COVID-19 who were older. Liver function should be monitored carefully during infection, with judicious use of hepatotoxic agents where possible and avoidance of prolonged hypotension to minimize liver injury in older patients.

Methods: The No. 2 People's Hospital of Fuyang City in China has admitted a total of 159 patients with confirmed COVID-19 since the outbreak from January 2020 to March 2020. We analyzed the incidence of liver function test (LFT) abnormality in these patients with confirmed COVID-19 infection.
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http://dx.doi.org/10.18632/aging.103720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425469PMC
July 2020

Dexmedetomidine promotes the progression of hepatocellular carcinoma through hepatic stellate cell activation.

Exp Mol Med 2020 07 6;52(7):1062-1074. Epub 2020 Jul 6.

Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, 510315, Guangzhou, China.

Dexmedetomidine (DEX) is an anesthetic that is widely used in the clinic, and it has been reported to exhibit paradoxical effects in the progression of multiple solid tumors. In this study, we sought to explore the mechanism by which DEX regulates hepatocellular carcinoma (HCC) progression underlying liver fibrosis. We determined the effects of DEX on tumor progression in an orthotopic HCC mouse model of fibrotic liver. A coculture system and a subcutaneous xenograft model involving coimplantation of mouse hepatoma cells (H22) and primary activated hepatic stellate cells (aHSCs) were used to study the effects of DEX on HCC progression. We found that in the preclinical mouse model of liver fibrosis, DEX treatment significantly shortened median survival time and promoted tumor growth, intrahepatic metastasis and pulmonary metastasis. The DEX receptor (ADRA2A) was mainly expressed in aHSCs but was barely detected in HCC cells. DEX dramatically reinforced HCC malignant behaviors in the presence of aHSCs in both the coculture system and the coimplantation mouse model, but DEX alone exerted no significant effects on the malignancy of HCC. Mechanistically, DEX induced IL-6 secretion from aHSCs and promoted HCC progression via STAT3 activation. Our findings provide evidence that the clinical application of DEX may cause undesirable side effects in HCC patients with liver fibrosis.
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http://dx.doi.org/10.1038/s12276-020-0461-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080602PMC
July 2020

Evaluation of PCR-Reverse Dot Blot Human Papillomavirus Genotyping Test in Predicting Residual/Recurrent CIN 2+ in Posttreatment Patients in China.

Cancer Manag Res 2020 1;12:2369-2379. Epub 2020 Apr 1.

Laboratory of Gynecologic Oncology, Fujian Provincial Maternity and Children's Health Hospital, Affiliated Hospital of Fujian Medical University, Fuzhou, People's Republic of China.

Objective: To assess the clinical value of the PCR-reverse dot blot human papillomavirus genotyping test during follow-up of patients with CIN grade 2 or worse (CIN 2+).

Methods: Four hundred patients with CIN 2+ receiving treatment from January 2008 to January 2017 were included in our study. Postoperative follow-up procedures comprised HPV examination and cervical cytology every 3-6 months for the first 2 years and then followed up every 6-12 months. A pathology examination was performed when there was a positive funding for HPV 16/18 or an abnormal ThinPrep cytology test (TCT) with or without positive for HR-HPV according to the American Society for Coloscopy and Cervical Pathology (ASCCP) guidelines.

Results: The median follow-up period was 27.10±12.47 months (ranging from 3 to 50 months). During follow-up, 12.00% (48/400) of the women developed residual/recurrent disease. The highest risk in CIN 2+ and CIN 3+ residual/recurrence was HPV-16/-18 (hazard ratio (HR)=12.898, 95% CI= 6.849-24.289; HR= 20.726, 95% CI= 9.64-44.562, respectively). Among the different follow-up methods, type-specific (TP) HR-HPV persistent infection showed the highest cumulative incidence risk (CIR) (84.62%, 95% CI=73.29-95.94) and HR (5.38, 95% CI= 2.596-11.149) during the 4-year follow-up period. At the CIN 2+ and CIN 3+ endpoints, TP-HPV testing had relatively high sensitivity (84.62%, 95% CI=73.29-95.94 and 89.28%, 95% CI= 77.83-100.00, respectively) and specificity (78.07%, 95% CI= 72.70-83.44 and 75.73%, 95% CI= 70.30-81.17, respectively). However, at the CIN 2+/CIN 3+ endpoint, TCT follow-up had a sensitivity of 60.42%/62.16% (95% CI=46.58-72.25/46.54-77.79) and specificity of 90.18%/88.72% (95% CI=86.95-93.41/85.35-92.10).

Conclusion: TP HR-HPV follow-up can provide a reliable and sensitive clinical reference for CIN 2+ postoperative patients.
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http://dx.doi.org/10.2147/CMAR.S237490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132552PMC
April 2020

2', 5'-Oligoadenylate Synthetase 2 (OAS2) Inhibits Zika Virus Replication through Activation of Type Ι IFN Signaling Pathway.

Viruses 2020 04 8;12(4). Epub 2020 Apr 8.

Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China.

Background: 2', 5'-oligoadenylate synthetase 2 (OAS2) has been known as an antiviral interferon-stimulated gene (ISG). However, the role of OAS2 on Zika virus (ZIKV) replication is still unknown. In this study, we sought to explore the effect of OAS2 on ZIKV replication and its underlying mechanism.

Methods: We performed RNA-Seq in A549 cells with or without ZIKV infection. OAS2 or RIG-I was overexpressed by plasmid transfection or knocked down by siRNA in A549 cells. Expression levels of mRNA and protein of selected genes were detected by RT-qPCR and Western Blot, respectively. Interferon stimulated response element (ISRE) activity was examined by dual luciferase assay.

Results: We found that ZIKV infection induced OAS2 expression through a RIG-I-dependent pathway. OAS2 overexpression inhibited ZIKV replication, while OAS2 knockdown increased ZIKV replication. We observed that OAS2 inhibited ZIKV replication through enhanced IFNβ expression, leading to the activation of the Jak/STAT signaling pathway.

Conclusion: ZIKV infection induced OAS2 expression, which in turn exerted its anti-ZIKV activities through the IFN-activated Jak/STAT signaling pathway.
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http://dx.doi.org/10.3390/v12040418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232345PMC
April 2020

HR-HPV viral load quality detection provide more accurate prediction for residual lesions after treatment: a prospective cohort study in patients with high-grade squamous lesions or worse.

Med Oncol 2020 Mar 30;37(5):37. Epub 2020 Mar 30.

Laboratory of Gynecologic Oncology, Fujian Provincial Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University, 18 Daoshan Road, Fuzhou, Fujian, 350001, People's Republic of China.

The relationship between high-risk-human-papillomavirus (HR-HPV) viral loads and residual/recurrence lesion is uncertain. This study aimed to evaluate the clinical value of HR-HPV viral loads to predict the residual/recurrence lesions among women with high-grade squamous lesions or worse (≥ HSIL) after surgery. Finally, 301 women who underwent primary screening of cervical cancer using polymerase-chain-reaction-(PCR)-reverse-dot-blot-(RDB) human papillomavirus (HPV) genotyping and cytology assays were enrolled. They received surgery and took HR-HPV viral loads with a BioPerfectus Multiplex Real-Time PCR assay. Colposcopy biopsies were performed in patients with HPV-16/18(+) and/or TCT ≥ ASCUS with HR-HPV(+). The risk of HR-HPV viral loads and potentials factors for residual/recurrence lesions were analyzed and the optimal cut-off values of HR-HPV viral loads were calculated. The significant differences were found in residual/recurrence lesions among patients with different ages, margin status, cytology and HR-HPV at 6 months (all P < 0.05). Interestingly, HPV viral loads were observed significant differences in the group of residual lesions, not recurrence group. Furthermore, except for HPV-31/33, the viral loads of HP-16/52/58 were significant differences in residual lesions. The cut-off level of HR-HPV viral loads was 5.22 copies/10,000 cells, providing viable triage for the risk of residual lesions. Compared with different follow-up methods, the HR-HPV viral loads ≥ 5.22copies/10,000 cells (HR 3.39, 95% CI 1.57-7.35) had a higher risk for developing residual lesions. HR-HPV viral loads can be a reliable predictor of residual lesions. Furthermore, women with viral loads ≥ 5.22 copies/10,000 cells may have higher risk for residual disease and should be give a more aggressive treatment and follow-up strategy.
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http://dx.doi.org/10.1007/s12032-020-01363-zDOI Listing
March 2020

LncRNAs regulate metabolism in cancer.

Int J Biol Sci 2020 10;16(7):1194-1206. Epub 2020 Feb 10.

Laboratory of Cancer Biology, Key Lab of Biotherapy in Zhejiang, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, Zhejiang, China.

Metabolic reprogramming is a hallmark of cancer. Mammalian genome is characterized by pervasive transcription, generating abundant non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are freshly discovered functional ncRNAs exerting extensive regulatory impact through diverse mechanisms. Emerging studies have revealed widespread roles of lncRNAs in the regulation of various cellular activities, including metabolic pathways. In this review, we summarize the latest advances regarding the regulatory roles of lncRNAs in cancer metabolism, particularly their roles in mitochondrial function, glucose, glutamine, and lipid metabolism. Moreover, we discuss the clinical application and challenges of targeting lncRNAs in cancer metabolism. Understanding the complex and special behavior of lncRNAs will allow a better depiction of cancer metabolic networks and permit the development of lncRNA-based clinical therapies by targeting cancer metabolism.
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http://dx.doi.org/10.7150/ijbs.40769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053319PMC
February 2020

[F]-Alfatide PET imaging of integrin αvβ3 for the non-invasive quantification of liver fibrosis.

J Hepatol 2020 07 5;73(1):161-169. Epub 2020 Mar 5.

Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Massachusetts General Hospital, Boston, USA. Electronic address:

Background & Aims: The vitronectin receptor integrin αvβ3 drives fibrogenic activation of hepatic stellate cells (HSCs). Molecular imaging targeting the integrin αvβ3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvβ3 on activated HSCs (aHSCs) in the injured liver. In this study, we sought to compare differences in the uptake of [F]-Alfatide between normal and injured liver to evaluate its utility for assessment of hepatic fibrogenesis.

Methods: PET with [F]-Alfatide, non-enhanced CT, histopathology, immunofluorescence staining, immunoblotting and gene analysis were performed to evaluate and quantify hepatic integrin αvβ3 levels and liver fibrosis progression in mouse models of fibrosis (carbon tetrachloride [CCl] and bile duct ligation [BDL]). The liver AUC divided by the blood AUC over 30 min was used as an integrin αvβ3-PET index to quantify fibrosis progression. Ex vivo analysis of frozen liver tissue from patients with fibrosis and cirrhosis verified the animal findings.

Results: Fibrotic mouse livers showed enhanced [F]-Alfatide uptake and retention compared to control livers. The radiotracer was demonstrated to bind specifically with integrin αvβ3, which is mainly expressed on aHSCs. Autoradiography and histopathology confirmed the PET imaging results. Further, the mRNA and protein level of integrin αvβ3 and its signaling complex were higher in CCl and BDL models than controls. The results obtained from analyses on human fibrotic liver sections supported the animal findings.

Conclusions: Imaging hepatic integrin αvβ3 with PET and [F]-Alfatide offers a potential non-invasive method for monitoring the progression of liver fibrosis.

Lay Summary: Integrin αvβ3 expression on activated hepatic stellate cells (aHSCs) is associated with HSC proliferation during hepatic fibrogenesis. Herein, we show that a radioactive tracer, [F]-Alfatide, binds to integrin αvβ3 with high affinity and specificity. [F]-Alfatide could thus be used as a non-invasive imaging biomarker to track hepatic fibrosis progression.
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http://dx.doi.org/10.1016/j.jhep.2020.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363052PMC
July 2020

Microrna-130a Downregulates HCV Replication through an atg5-Dependent Autophagy Pathway.

Cells 2019 04 10;8(4). Epub 2019 Apr 10.

Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.
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http://dx.doi.org/10.3390/cells8040338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523735PMC
April 2019

A Long Noncoding RNA Regulates Hepatitis C Virus Infection Through Interferon Alpha-Inducible Protein 6.

Hepatology 2019 03 13;69(3):1004-1019. Epub 2019 Feb 13.

Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.
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http://dx.doi.org/10.1002/hep.30266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393205PMC
March 2019

Tyrosine kinase SYK is a potential therapeutic target for liver fibrosis.

Hepatology 2018 09 21;68(3):1125-1139. Epub 2018 May 21.

Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.

Spleen tyrosine kinase (SYK) plays a critical role in immune cell signaling pathways and has been reported as a biomarker for human hepatocellular carcinoma (HCC). We sought to investigate the mechanism by which SYK promotes liver fibrosis and to evaluate SYK as a therapeutic target for liver fibrosis. We evaluated the cellular localization of SYK and the association between SYK expression and liver fibrogenesis in normal, hepatitis B virus (HBV)-infected, hepatitis C virus (HCV)-infected and non-alcoholic steatohepatitis (NASH) liver tissue (n=36, 127, 22 and 30, respectively). A polymerase chain reaction (PCR) array was used to detect the changes in transcription factor (TF) expression in hepatic stellate cells (HSCs) with SYK knockdown. The effects of SYK antagonism on liver fibrogenesis were studied in LX-2 cells, TWNT-4 cells, primary human HSCs, and three progressive fibrosis/cirrhosis animal models, including a CCL mouse model, and diethylnitrosamine (DEN) and bile duct ligation (BDL) rat models. We found that SYK protein in HSCs and hepatocytes correlated positively with liver fibrosis stage in human liver tissue. HBV or HCV infection significantly increased SYK and cytokine expression in hepatocytes. Increasing cytokine production further induced SYK expression and fibrosis-related gene transcription in HSCs. Up-regulated SYK in HSCs promoted HSC activation by increasing the expression of specific TFs related to activation of HSCs. SYK antagonism effectively suppressed liver fibrosis via inhibition of HSC activation, and decreased obstructive jaundice and reduced HCC development in animal models. Conclusion: SYK promotes liver fibrosis via activation of HSCs and is an attractive potential therapeutic target for liver fibrosis and prevention of HCC development. (Hepatology 2018).
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http://dx.doi.org/10.1002/hep.29881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138581PMC
September 2018

Intermittent hypoxia is a proinflammatory stimulus resulting in IL-6 expression and M1 macrophage polarization.

Hepatol Commun 2017 06 18;1(4):326-337. Epub 2017 May 18.

Massachusetts General Hospital Gastrointestinal Unit Boston MA.

The biological factors that promote inflammation or nonalcoholic steatohepatitis (NASH) in the setting of nonalcoholic fatty liver disease remain incompletely understood. Clinical studies have demonstrated an association between obstructive sleep apnea (OSA) and both inflammation and fibrosis in NASH, but the mechanism has not been identified. In this study, we use modeling to examine the impact of intermittent hypoxia on the liver. Hepatocyte, stellate cell, and macrophage cell lines were exposed to intermittent or sustained hypoxia. Candidate genes associated with inflammation, fibrosis, and lipogenesis were analyzed. Circulating cytokines were assessed in human serum of patients with nonalcoholic fatty liver disease. Intermittent hypoxia results in significant induction of interleukin (IL)-6 expression in both hepatocytes and macrophages. The increase in IL-6 expression was independent of hypoxia inducible factor 1 induction but appeared to be in part related to antioxidant response element and nuclear factor kappa B activation. Mature microRNA 365 (miR-365) has been demonstrated to regulate IL-6 expression, and we found that miR-365 expression was decreased in the setting of intermittent hypoxia. Furthermore, macrophage cell lines showed polarization to an M1 but not M2 phenotype. Finally, we found a trend toward higher circulating levels of IL-6 in patients with OSA and NASH. : Intermittent hypoxia acts as a potent proinflammatory stimulus, resulting in IL-6 induction and M1 macrophage polarization. Increased IL-6 expression may be due to both induction of antioxidant response element and nuclear factor kappa B as well as inhibition of miR-365 expression. Higher levels of IL-6 were observed in human samples of patients with OSA and NASH. These findings provide biological insight into mechanisms by which obstructive sleep apnea potentiates inflammation and fibrosis in patients with fatty liver disease. ( 2017;1:326-337).
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http://dx.doi.org/10.1002/hep4.1045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721395PMC
June 2017

MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway.

J Virol 2018 04 14;92(7). Epub 2018 Mar 14.

Liver Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. We identified that miR-130a regulates the target gene and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.
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http://dx.doi.org/10.1128/JVI.02009-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972888PMC
April 2018

Overexpression of c-Met in bone marrow mesenchymal stem cells improves their effectiveness in homing and repair of acute liver failure.

Stem Cell Res Ther 2017 07 5;8(1):162. Epub 2017 Jul 5.

Department of Infectious Disease, the First Affiliated Hospital with Nanjing Medical University, Nanjing, China.

Background: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has emerged as a novel therapy for acute liver failure (ALF). However, the homing efficiency of BMSCs to the injured liver sites appears to be poor. In this study, we aimed to determine if overexpression of c-Met in BMSCs could promote the homing ability of BMSCs to rat livers affected by ALF.

Methods: Overexpression of c-Met in BMSCs (c-Met-BMSCs) was attained by transfection of naive BMSCs with the lenti-c-Met-GFP. The impact of transplanted c-Met-BMSCs on both homing and repair of ALF was evaluated and compared with lenti-GFP empty vector transfected BMSCs (control BMSCs).

Results: After cells were transfected with the lenti-c-Met-GFP vector, the BMSCs displayed very high expression of c-Met protein as demonstrated by Western blot. In addition, in vitro transwell migration assays showed that the migration ability of c-Met-BMSCs was significantly increased in comparison with that of control BMSCs (P < 0.05), and was dependent on hepatocyte growth factor (HGF). Furthermore, rats with ALF that received transplanted c-Met-BMSCs showed significantly improved homing ability to the injured liver; this was accompanied by elevated survival rates and liver function in the ALF rats. Parallel pathological examination further confirmed that transplantation of c-Met-BMSCs ameliorated liver injury with reduced hepatic activity index (HAI) scores, and that the effects of c-Met-BMSCs were more profound than those of control BMSCs.

Conclusions: Overexpression of c-Met promotes the homing of BMSCs to injured hepatic sites in a rat model of ALF, thereby improving the efficacy of BMSC therapy for ALF repair.
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http://dx.doi.org/10.1186/s13287-017-0614-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499016PMC
July 2017

HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus.

Front Microbiol 2017 20;8:240. Epub 2017 Feb 20.

Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital Boston, MA, USA.

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.
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http://dx.doi.org/10.3389/fmicb.2017.00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316548PMC
February 2017

Apolipoprotein B100 is required for hepatitis C infectivity and Mipomersen inhibits hepatitis C.

World J Gastroenterol 2016 Dec;22(45):9954-9965

Esperance AK Schaefer, James Meixiong, Christina Mark, Daniel L Motola, Dahlene Fusco, Andrew Yang, Cynthia Brisac, Shadi Salloum, Wenyu Lin, Lee F Peng, Raymond T Chung, Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA 02114, United States.

Aim: To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection.

Methods: In this study, we utilize a gene editing tool, transcription activator-like effector nucleases (TALENs), to generate human hepatoma cells with a stable genetic deletion of to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDA-approved antisense inhibitor of apoB100, on HCV using cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method.

Results: We found that apoB100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALEN-mediated gene deletion of apoB (), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apoB did not play a role in HCV entry or replication. However, the virus produced by cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus.

Conclusion: ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.
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http://dx.doi.org/10.3748/wjg.v22.i45.9954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5143762PMC
December 2016

IQGAP2 is a novel interferon-alpha antiviral effector gene acting non-conventionally through the NF-κB pathway.

J Hepatol 2016 11 9;65(5):972-979. Epub 2016 Jul 9.

Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Background & Aims: Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response.

Methods: We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection.

Results: We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties.

Conclusions: Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation.

Lay Summary: In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver.
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http://dx.doi.org/10.1016/j.jhep.2016.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656012PMC
November 2016

Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

Hepatology 2016 12 5;64(6):1951-1968. Epub 2016 Oct 5.

Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells.

Conclusion: HIV accentuates an HCV-driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFβ1 up-regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951-1968).
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http://dx.doi.org/10.1002/hep.28766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116014PMC
December 2016

HCV induces transforming growth factor β1 through activation of endoplasmic reticulum stress and the unfolded protein response.

Sci Rep 2016 Mar 1;6:22487. Epub 2016 Mar 1.

Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB, and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.
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http://dx.doi.org/10.1038/srep22487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772380PMC
March 2016

EFTUD2 on innate immunity.

Oncotarget 2015 Oct;6(32):32313-4

Department of Medicine, Liver Center and Gastrointestinal Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.18632/oncotarget.5863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741694PMC
October 2015

Mapping the Diversity of Follicular Helper T Cells in Human Blood and Tonsils Using High-Dimensional Mass Cytometry Analysis.

Cell Rep 2015 Jun 11;11(11):1822-33. Epub 2015 Jun 11.

Agency for Science, Technology and Research (A(∗)STAR), Singapore Immunology Network (SIgN), Singapore 138648, Singapore. Electronic address:

Single-cell analysis technologies such as mass cytometry allow for measurements of cellular heterogeneity with unprecedented dimensionality. Here, we applied dimensionality reduction and automated clustering methods on human T helper (T(H)) cells derived from peripheral blood and tonsils, which showed differential cell composition and extensive T(H) cell heterogeneity. Notably, this analysis revealed numerous subtypes of follicular helper T (T(FH)) cells that followed a continuum spanning both blood and tonsils. Furthermore, we identified tonsillar CXCR5(lo)PD-1(lo)CCR7(lo) T(FH) cells expressing interferon-γ (IFN-γ), interleukin-17 (IL-17), or Foxp3, indicating that T(FH) cells exhibit diverse functional capacities within extrafollicular stages. Regression analysis demonstrated that CXCR5(lo)PD-1(-) and CXCR5(lo)PD-1(lo) cells accumulate during childhood in secondary lymphoid organs, supporting previous findings that these subsets represent memory T(FH) cells. This study provides an in-depth comparison of human blood and tonsillar T(FH) cells and outlines a general approach for subset discovery and hypothesizing of cellular progressions.
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http://dx.doi.org/10.1016/j.celrep.2015.05.022DOI Listing
June 2015
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