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Background: The endoplasmic reticulum (ER) regulates critical processes, including lipid synthesis, which can be affected by transmembrane proteins localized in the ER membrane. One of them, the transmembrane protein 147 (TMEM147) has been recently implicated for its role in hepatocellular carcinoma (HCC) tumorigenesis, though the mechanisms remain unclear. Here, we investigated the role of TMEM147 in HCC and its underlying mechanisms. Methods: Expression of TMEM147 was examined in human HCC and adjacent non-tumorous tissues using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effects of TMEM147 on HCC progression were investigated both in vitro and in vivo. Proteins interacting with TMEM147 were identified using RNAseq analysis, immunoprecipitation, and mass spectrometry analyses. Lipidomic analysis and enzyme-linked immunosorbent assay (ELISA) were employed to identify and analyze cholesterol and 27-hydroxycholesterol (27HC) contents. Comprehensive experimental methods were used to demonstrate ferroptosis in HCC cells. The fatty acid content of macrophages affected by TMEM147 was quantified using ELISA. Macrophage phenotypes were determined using various assays, such as immunofluorescence assay and flow cytometry analysis. Results: TMEM147 mRNA and protein levels were upregulated in HCC, and increased TMEM147 expression was associated with poor survival. TMEM147 promoted tumor cell proliferation and metastasis in vitro and in vivo. It was found to interact with the key sterol reductase DHCR7, which affected cellular cholesterol homeostasis and increased extracellular 27HC levels in HCC. TMEM147 promoted the expression of DHCR7 by enhancing the activity of the transcription factor, STAT2. 27HC upregulated the expression of glutathione peroxidase 4 in HCC, leading to ferroptosis resistance and promotion of HCC proliferation. HCC cell-derived 27HC activated PPARγ signaling and enhanced lipid metabolism in macrophages, thereby activating M2 polarization, and then promoted the invasion and migration of HCC. Conclusions: Our results indicated that TMEM147 confers ferroptosis resistance and M2 macrophage polarization, which are mainly dependent on the upregulation of cellular cholesterol homeostasis and 27HC secretion, leading to cancer growth and metastasis. Our findings suggest that the TMEM147/STAT2/DHCR7/27HC axis in the tumor microenvironment may serve as a promising therapeutic target for HCC.

PDF file - 142K, Supplementary Table S1. Alteration of protein expression in sorafenib-resistant cells compared with parental cells in the presence or absence of sorafenib.

PDF file - 327K, Supplementary Fig. S4.Akt inhibition or depletion synergizes with sorafenib to inhibit cell growth by regulating autophagic and apoptotic pathways in sorafenib-resistant HCC cells.

PDF file - 224K, Supplementary Fig. S2.Sorafenib activates the Akt pathway independent of PP2A in hepatocellular carcinoma cells.

PDF file - 109K, Supplementary Fig. S 1. Sorafenib-resistant cells responded poorly to sorafenib treatment in vivo.Parental Huh7 and Huh7-SR tumors were established in mice, which received different treatments for 15 days and then euthanize as described in Fig. 1, and Materials and Methods. The size (mm3) of tumors was recorded. "**" (P

Sorafenib is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), but the acquired resistance to sorafenib results in limited benefits. Activation of Akt is thought to be responsible for mediating the acquired resistance to sorafenib. The present study aims to examine the underlying mechanism and seek potential strategies to reverse this resistance. Two sorafenib-resistant HCC cell lines, which had been established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition and apoptosis in vitro and in vivo. Sustained exposure to sorafenib activated Akt via the feedback loop of mTOR but independent of protein phosphatase 2A in HCC cells. Autophagy participated in the resistance to sorafenib as inhibition of autophagy reduced the sensitivity of sorafenib-resistant HCC cells to sorafenib, whereas activation of autophagy by rapamycin had the opposite effect. However, rapamycin did not show a synergistic effect with sorafenib to inhibit cell proliferation, while it also activated Akt via a feedback mechanism in sorafenib-resistant HCC cells. Inhibition of Akt reversed the acquired resistance to sorafenib by switching autophagy from a cytoprotective role to a death-promoting mechanism in the sorafenib-resistant HCC cells. Akt inhibition by GDC0068 synergized with sorafenib to suppress the growth of sorafenib-resistant HCC tumors that possessed the sorafenib-resistant feature in vivo. The results have provided evidence for clinical investigation of GDC0068, a novel ATP-competitive pan-Akt inhibitor, as the second-line treatment after the failure of sorafenib-medicated molecular targeted therapy for advanced HCC. Mol Cancer Ther; 13(6); 1589–98. ©2014 AACR.

PDF file - 225K, Supplementary Fig. S5.Interactions of Akt, autophagic and apoptotic pathways in sorafenib-resistant HCC cells.(A) Immunoblots of lysates of HepG2-SR and Huh7-SR cells following incubation with sorafenib (10 μM), GDC0068 (5 μM) or in combination for 48 h.

PDF file - 166K, Supplementary Fig. S3. Sorafenib-resistant cells express different levels of p-Akt and autophagic proteins in vivo. Lysates of tumors harvested from (Fig. S1) at the end of experiments were subjected to immunoblotting.

Targeting energy metabolism holds the potential to effectively treat a variety of malignant diseases, and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) is a key regulator of energy metabolism. However, PGC1α's role in cancer, especially in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we reported that PGC1α was significantly downregulated in HCC cell lines and specimens. Moreover, reduced expression of PGC1α in tumor cells was correlated with poor prognosis. PGC1α overexpression substantially inhibited cell proliferation and induced apoptosis in vitro and in vivo. On the contrary, the knockdown of PGC1α produced the opposite effect. The mechanism was at least partially due to the upregulation of mitochondrial pyruvate carrier 1 (MPC1) caused by PGC1α, which promoted mitochondrial biogenesis by binding to nuclear respiratory factor 1 (NRF1). Consequently, the production of cellular reactive oxygen species (ROS) caused by mitochondrial oxidation was elevated above a critical threshold for survival. Furthermore, we found that PGC1α could enhance the antitumor activity of sorafenib and doxorubicin in HCC through ROS accumulation-mediated cell death. These results indicate that PGC1α/NRF1-MPC1 axis is involved in HCC progression and could be a promising target for HCC treatment.

Background & aims Pyogenic liver abscesses (PLA) are space‐occupying lesions in the liver that produce high morbidity and mortality. The clinical characteristics and prognosis of abscesses is different depending on the bacterial culture results and require different strategies for management. The aim of this study was to investigate the clinical characteristics and prognostic factors of patients with PLA. Methods Clinical features, laboratory tests and etiology of PLA between 2006 to 2011 and 2012 to 2017 in a single hospital were retrospectively reviewed. The incidence and mortality of PLA caused by Escherichia coli and Klebsiella pneumoniae were compared and the risk factors for multiple organ dysfunction (MODS) and endophthalmitis were evaluated. Results Among the 1,572 PLA patients, the proportion with PLA increased from 333 (21.2%) in 2006‐2011 to 1,239 (78.8%) in 2012‐2017 without any investigation and treatment procedure differences. K pneumoniae was the main isolate in analysed pus cultures (85.6%). The mortality rate of patients with K pneumoniae infection was lower in the latter period (6.7% vs 0.7%, P = .035). Multivariate analyses revealed that age, fever, MODS and length of hospital stay were factors affecting poor prognosis (death + unhealed/uncured) in PLA patients after treatment and that cardiovascular disease, pleural effusion and pulmonary infection were risk factors for MODS, while diabetes mellitus was the only risk factor for endophthalmitis. Most patients (95.5%) with PLA recovered after abscess drainage/puncture and antibiotic therapy. Conclusions Pleural effusion, fever, MODS and length of hospital stays were factors useful in predicting PLA outcomes.

Pancreatic cancer is a malignancy of the digestive system characterized by poor prognosis. A number of prognostic messenger RNA (mRNA) signatures have been identified by using the high-throughput expression profiles. MicroRNAs (miRNA) play a critical role in regulating multiple cellular functions. However, no such integrated analysis of miRNAs and mRNAs for studying the prognostic mechanisms of pancreatic cancer has been reported. In this study, we first identified prognostic mRNAs and miRNAs based on The Cancer Genome Atlas datasets, and then performed an enrichment analysis to explore the underlying biological mechanisms involved in pancreatic cancer prognosis at the mRNA level. Furthermore, we performed an integrated analysis of mRNAs and miRNAs to identify prognostic subpathways, which were closely associated with pancreatic cancer genes and tumor hallmarks and involved in hypoxia, oxidative phosphyorylation and xenobiotic metabolisms. Meanwhile, we performed a random walk algorithm based on global network, prognostic mRNAs and miRNAs, and identified top risk mRNAs as the prognostic signature. Finally, an independent testing set was used to confirm the predictive power of the top mRNA signature, and most of these genes involved were known oncogenes. In conclusion, we performed a series of integrated analyses by comprehensively exploring pancreatic cancer prognosis and systematically optimized the prognostic signature for clinical use.

Tumor cells primarily utilize aerobic glycolysis for energy production, a phenomenon known as the Warburg effect, but the involvement of Warburg effect in liver cancer cell metastasis is not well understood. In present study, our results indicate a positive correlation between glucose metabolism level and metastatic potential of hepatocellular carcinoma (HCC). We also observed that a long noncoding RNA-SOX2OT (lncRNA-SOX2OT) can not only increase the metastatic potential of HCC but also promote a pyruvate kinase M2 (PKM2)-mediated activation of glucose metabolism. Inhibition of PKM2 in HCC cells greatly compromises lncRNA-SOX2OT in promoting Warburg effect and metastasis. Furthermore, miR-122-5p was found being a direct target of lncRNA-SOX2OT in regulating PKM2 expression. Thus, our findings reveal that lncRNA-SOX2OT, a regulator of PKM2, could predispose HCC patients to metastases and may serve as a candidate for metastatic prediction and therapies in HCC patients.

Purpose Itaconate is well known for its strong anti-inflammatory and antioxidant effect, but little is known about the potential role of long non-coding RNAs (lncRNAs) in the underlying mechanisms of hepatic ischemia-reperfusion (IR) injury. The aim of our study is to identify lncRNAs related to IR injury and itaconate-mediated protection and to demonstrate the mechanism by which itaconate acts in liver IR injury from the new perspective of lncRNAs. Methods 4-Octyl itaconate (OI), a membrane-permeable derivative of itaconate, was used as a substitute for itaconate in our study. By using a mouse model of hepatic IR injury, serum and liver samples were collected to measure indexes of liver injury. Then, the liver samples of the mice were subjected to RNA sequencing (RNA-seq) and subsequent bioinformatics analysis. Results Itaconate attenuated liver IR injury. A total of 138 lncRNAs and 156 messenger RNAs (mRNAs) were markedly differentially expressed in the IR-damaged liver tissues pretreated with OI compared with the matched liver tissues treated with vehicle. Functional analysis indicated that lncRNAs may indirectly participate in the effects of itaconate. Furthermore, 41 mRNAs were examined for the protein–protein interaction (PPI) network analysis, and a key gene cluster was defined. Then, combined the coexpression analysis and the cis and trans regulatory function prediction of lncRNAs, some “candidate” lncRNA-mRNA pairs which might relate to itaconate-mediated liver protection were identified, while the relationship requires future validation. Conclusion Our study revealed that itaconate could protect the liver against IR injury and that lncRNAs might play a role in this process. Our study provides a novel way to investigate the mechanism by which itaconate affects hepatic IR injury and exerts its anti-inflammatory and antioxidative stress effects., Graphical Abstract

Peroxisome proliferator-activated receptors (PPARs) α and γ have been shown to be protective in hepatic ischemia/reperfusion (I/R) injury. However, the precise role of PPARγ coactivator-1α (PGC-1α), which can coactivate both of these receptors, in hepatic I/R injury, remains largely unknown. This study was designed to test our hypothesis that PGC-1α is protective during hepatic I/R injury in vitro and in vivo. Our results show that endogenous PGC-1α is basally expressed in normal livers and is moderately increased by I/R. Ectopic PGC-1α protects against hepatic I/R and hepatocyte anoxia/reoxygenation (A/R) injuries, whereas knockdown of endogenous PGC-1α aggravates such injuries, as evidenced by assessment of the levels of serum aminotransferases and inflammatory cytokines, necrosis, apoptosis, cell viability, and histological examination. The EMSA assay shows that the activation of PPARα and PPARγ is increased or decreased by the overexpression or knockdown of PGC-1α, respectively, during hepatic I/R and hepatocyte A/R injuries. In addition, the administration of specific antagonists of either PPARα (MK886) or PPARγ (GW9662) can effectively decrease the protective effect of PGC-1α against hepatic I/R and hepatocyte A/R injuries. We also demonstrate an important regulatory role of PGC-1α in reactive oxygen species (ROS) metabolism during hepatic I/R, which is correlated with the induction of ROS-detoxifying enzymes and is also dependent on the activations of PPARα and PPARγ. These data demonstrate that PGC-1α protects against hepatic I/R injury, mainly by regulating the activation of PPARα and PPARγ. Thus, PGC-1α may be a promising therapeutic target for the protection of the liver against I/R injury.

Background: Dysregulation of RNA binding protein (RBP) expression has been reported in various malignant tumors, and it is related to the occurrence and development of cancer. However, the role of RBPs in colon cancer remains unclear. Methods: We downloaded the RNA sequencing data of colon cancer from The Cancer Genome Atlas (TCGA) database, and determined the differently expressed RBPs between normal and cancer tissues. Then, through a series of bioinformatics analysis, we systematically studied the expression and prognostic value of these RBPs.Result: A total of 490 different expression differently expressed RBPs were identified, including 323 up-regulated and 167 down regulated RBPs. Five RBPs (PNLDC1, NSUN6, NOL3, PPARGC1A, LRRFIP2) were identified as prognosis related genes for the construction of prognostic model. Further analysis showed that the overall survival rate (OS) of patients in the high-risk subgroup was worse than that in the low-risk subgroup based on this model. The area under the characteristic curve of time-dependent receiver was 0.691 in TCGA and 0.624 in GEO, which confirmed the prognostic model to be a good one. We also established a nominal map based on the internal validation in 5 RBPs mRNAs and TCGA sequeues, showing a good ability to differentiate colon cancer.Conclusions: We screened RBPs expression differences between colon cancer and adjacent non tumor colon tissues using the TCGA database to identify potential gene biomarkers.Besides,a very effective prediction model was constructed and tested based on the differential expression of RBPs using the TCGA and Gene Expression Omnibus (GEO) database.We also Validated of the relationship between the expression of five RBPs and prognosis

Three-dimensional (3D) visualization involves feature extraction and 3D reconstruction of CT images using a computer processing technology. It is a tool for displaying, describing, and interpreting 3D anatomy and morphological features of organs, thus providing intuitive, stereoscopic, and accurate methods for clinical decision-making. It has played an increasingly significant role in the diagnosis and management of liver diseases. Over the last decade, it has been proven safe and effective to use 3D simulation software for pre-hepatectomy assessment, virtual hepatectomy, and measurement of liver volumes in blood flow areas of the portal vein; meanwhile, the use of 3D models in combination with hydrodynamic analysis has become a novel non-invasive method for diagnosis and detection of portal hypertension. We herein describe the progress of research on 3D visualization, its workflow, current situation, challenges, opportunities, and its capacity to improve clinical decision-making, emphasizing its utility for patients with liver diseases. Current advances in modern imaging technologies have promised a further increase in diagnostic efficacy of liver diseases. For example, complex internal anatomy of the liver and detailed morphological features of liver lesions can be reflected from CT-based 3D models. A meta-analysis reported that the application of 3D visualization technology in the diagnosis and management of primary hepatocellular carcinoma has significant or extremely significant differences over the control group in terms of intraoperative blood loss, postoperative complications, recovery of postoperative liver function, operation time, hospitalization time, and tumor recurrence on short-term follow-up. However, the acquisition of high-quality CT images and the use of these images for 3D visualization processing lack a unified standard, quality control system, and homogeneity, which might hinder the evaluation of application efficacy in different clinical centers, causing enormous inconvenience to clinical practice and scientific research. Therefore, rigorous operating guidelines and quality control systems need to be established for 3D visualization of liver to develop it to become a mature technology. Herein, we provide recommendations for the research on diagnosis and management of 3D visualization in liver diseases to meet this urgent need in this research field.

Circular RNAs (circRNAs) are a class of non-coding RNAs with a loop structure; however, their functions remain largely unknown. Growing evidence suggests that circRNAs play a pivotal role in the progression of malignant diseases. However, the expression profiles and function of circRNAs in hepatocellular carcinoma (HCC) remain unclear. We investigated the expression of microtubule-associated serine/threonine kinase 1 (MAST1) circRNA (circMAST1) in HCC and healthy tissues using bioinformatics, quantitative real-time PCR (qRT-PCR), and fluorescence in situ hybridization. Luciferase reporter assays were performed to assess the interaction between circMAST1 and miR-1299. Proliferation assays, colony formation assays, flow cytometry, transwell assays, and western blotting were also performed. A mouse xenograft model was also used to determine the effect of circMAST1 on HCC growth in vivo. CircMAST1 was upregulated in HCC tissues and cell lines; silencing via small interfering RNA inhibited migration, invasion, and proliferation of HCC cell lines in vitro as well as tumor growth in vivo. Furthermore, the expression of circMAST1 was positively correlated with catenin delta-1 (CTNND1) and negatively correlated with microRNA (miR)-1299 in HCC clinical samples. Importantly, circMAST1 sponged miR-1299 to stabilize the expression of CTNND1 and promoted tumorigenic features in HCC cell lines. We found that circMAST1 may serve as a novel biomarker for HCC. Moreover, circMAST1 elicits HCC progression by sponging miRNA-1299 and stabilizing CTNND1. Our data provide potential options for therapeutic targets in patients with HCC.

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

Background Patients with hepatocellular carcinoma (HCC) suffer from a high fatality rate, likely due to increased incidence of tumor relapse and metastasis. Understanding the molecular mechanisms that contribute to HCC development and progression is vital for the discovery of new treatment targets. This study aims to explore the expression profiles and functions of immunoglobulin superfamily member 3 (IGSF3) in HCC. Methods We evaluated IGSF3 levels in HCC and normal tissues using bioinformatics, western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemistry. We also conducted proliferation assays, colony formation assays, flow cytometry, cell migration assay, cell invasion assay, qRT-PCR, and western blotting in HCC cell lines. Immunofluorescence and western blotting further used to study the IGSF3 pathway. A mouse xenograft model was utilized to examine the influence of IGSF3 on HCC growth in vivo. Results IGSF3 levels were higher in HCC tissues and cell lines. Silencing of IGSF3 via lentiviral vector system (LV) inhibited migration, invasion, and growth of HCC cell lines in vitro as well as tumor growth in vivo. Overexpression of IGSF3 promoted result in vitro. Importantly, we found that IGSF3 activates the NF-κB pathway to promote tumorigenic features in HCC cell lines. Conclusions We found that IGSF3 can be used as a novel biomarker for HCC detection. Moreover, IGSF3 elicits HCC progression by activating the NF-κB pathway. As such, our data provides potential options for therapeutic targets in patients with HCC.

Background Neuropilin-1 (NRP-1) activates signaling pathways as multifunctional co-receptors in cancer cells. However, its role and how it is regulated by miRNAs in cholangiocarcinoma (CCA) have not yet been investigated. Methods The expression of NRP-1, miR-320 and key molecules involved in cell proliferation, migration and related signaling pathways were detected by immunohistochemistry, immunoblotting and qRT-PCR. Stable transfectants depleted of NRP-1 were generated. The regulatory effect of miR-320 on NRP-1 was evaluated by luciferase reporter assays. Cell proliferation, cell cycle distribution and migration were examined. Xenograft tumor models were established to assess tumorigenesis, tumor growth and lung metastasis. Results CCA tissues expressed higher levels of NRP-1 than adjacent normal biliary tissues, and its expression negatively correlated with miR-320. NRP-1 depletion inhibited cell proliferation and induced cell cycle arrest in the G1/S phase by upregulating p27, and downregulating cyclin E and cyclin-dependent kinase 2; and reduced cell migration by inhibiting the phosphorylation of focal adhesion kinase. NRP-1 depletion suppressed tumorigenesis, tumor growth and lung metastasis by inhibiting cell proliferation and tumor angiogenesis in experimental animals. Depletion of NRP-1 inhibited the activation of VEGF/VEGFR2, EGF/EGFR and HGF/c-Met pathways stimulated by respective ligands. MiR-320 negatively regulated the expression of NRP-1 by binding to the 3’-UTR of NRP-1 promoter, and miR-320 mimics inhibited cell proliferation and migration, and the growth of established tumors in animals by downregulating NRP-1. Conclusions The present results indicate that NRP-1 is negatively regulated by miR-320, and both of them may be potentially therapeutic targets for CCA. This article is protected by copyright. All rights reserved.

Sorafenib displays a limited efficacy for advanced hepatocellular carcinoma (HCC). Some patients with HCC initially respond to sorafenib, but eventually succumb to the disease, indicating that the acquired resistance to sorafenib reduces its beneficial effects. No alternative drugs are available after the failure of sorafenib therapy. Therefore, investigation of the mechanisms underlying the acquired resistance and development of second-line treatments for sorafenib-resistant HCC are urgently required. In this study, sorafenib-resistant HCC cells generated from sorafenib-sensitive human HCC cells were shown to overproduce hepatocyte growth factor (HGF) and overexpress c-Met kinase and its phosphorylated form, leading to the activation of Akt and ERK (extracellular signaling-regulated kinase) pathways. Use of specific c-Met inhibitors enhanced the effects of sorafenib by inhibiting the growth of sorafenib-resistant HCC cells. Akt inhibitors, a class of second-line therapeutic drugs under investigation for treating HCC in clinical trials, enhanced the effects of sorafenib, but also activated the c-Met pathway in sorafenib-resistant cells. Dual inhibition of Akt and c-Met by their respective inhibitors, MK2206 and capmatinib, additively or synergistically suppressed sorafenib-resistant HCC cells in vitro and sorafenib-resistant HCC xenografts in mice. The anticancer activities of MK2206 mainly rely on its ability to induce cell apoptosis and autophagic death, while capmatinib treatment leads to cell cycle arrest at phase G1. These results provide strong evidence for further investigation on the clinical utility of dual inhibition of Akt and c-Met, particularly MK2206 and capmatinib, as a second-line therapy for advanced HCC that has acquired resistance to sorafenib.

Additional file 1: Table S1. Descriptive summary of datasets used in the study. Table S2. Genes of the 00982_1 subpathway. Table S3. Predictive power of published gene signatures. Figure S1. Dataset Search strategy in GEO database. Figure S2. Flow diagram of dataset selection strategies. Figure S3. Accumulative predictive abilities of signatures. Figure S4. Genes in the path:00982_1 subpathway. Figure S5. Collision classification for GSE57495 and GSE79668 datasets. Figure S6. Prognostic capacity of path:00982_1 signature for classical subtype. by Moffitt classification. Figure S7. Association of path:00982_1 subpathway with other pathways.

Introduction. The purpose of this study was to investigate the difference in surgical outcomes between symptomatic and asymptomatic patients with primary hyperparathyroidism (PHPT) and between patients with high serum calcium and those with normal blood calcium, as well as to explore the epidemiological trend of PHPT in northern China. Methods. Clinicopathologic data of 197 patients (50 men and 147 women) with PHPT who underwent surgery at the First Affiliated Hospital of Harbin Medical University from 2008 to 2017 were analyzed. Changes in clinicopathology were compared among different subgroups of patients. Patients were categorized into subgroups based on serum calcium levels, whether or not they presented with symptoms, and admission time. Results. Of the total patients, 82.23% had hypercalcemic primary hyperparathyroidism (HCPHPT), 17.77% had normocalcemic primary hyperparathyroidism (NCPHPT), 45.18% had symptomatic primary hyperparathyroidism (SPHPT), and 54.82% had asymptomatic primary hyperparathyroidism (ASPHPT). Seventy-seven cases of PHPT involved thyroid nodules, with 22 confirmed as papillary thyroid carcinoma, and 29 confirmed as nodular goiter. There was no significant difference in the success rate of surgery, postoperative recurrence rate, and the symptoms of temporary hypocalcemia between the HCPHPT and NCPHPT groups, and between the SPHPT and ASPHPT groups. The incidence of PHPT has increased threefold since 2013. Conclusions. Symptoms and serum calcium levels did not affect the results of surgical treatment for PHPT. The incidence of PHPT in northern China is increasing. Moreover, PHPT manifestation has shifted from the symptomatic to the asymptomatic form. Thyroid surgery should be performed in PHPT patients with thyroid nodules.

Vanadium and its compounds exhibit concentration- and time-dependent anticancer effects on various types of tumor; however, the effects of sodium orthovanadate (SOV) on anaplastic thyroid carcinoma (ATC) have not yet been reported. In the present study, the anticancer effects of SOV on ATC were evaluated. In vitro experiments, including cell viability assays, plate colony formation assays, cell cycle analysis and apoptosis analysis were used to study the role of SOV in ATC. Using in vivo experiments, the effects of SOV on the growth and apoptosis of an ATC-xenograft tumor were studied by comparing the SOV-treatment with the control group. The results revealed that treatment of the human ATC cell line 8505C with SOV inhibited cell viability, induced G2/M phase cell cycle arrest, stimulated apoptosis and reduced mitochondrial membrane potential in a concentration-dependent manner. These findings were confirmed in vivo in a nude mouse ATC xenograft model. In conclusion, the present study demonstrated that SOV inhibited human ATC by regulating proliferation, cell cycle progression and apoptosis, thus suggesting that SOV may be considered a novel option for the treatment of ATC.

Increasing evidence supports a role for N-myc downstream-regulated gene 2 (NDRG2) deregulation in tumorigenesis. We investigated the roles and mechanisms of NDRG2 in human cholangiocarcinoma (CCA) progression. In the present study, expression of NDRG2, microRNA (miR)-181c and leukemia inhibitory factor (LIF) in human CCA and adjacent nontumor tissues were examined. The effects of NDRG2 on CCA tumor growth and metastasis were determined both in vivo and in vitro. The role of the NDRG2/LIF/miR-181c signaling pathway in cholangiocarcinogenesis and metastasis were investigated both in vivo and in vitro. The results showed that human CCA tissues exhibited decreased levels of NDRG2 and increased levels of miR-181c and LIF compared with nontumor tissues. NDRG2 could inhibit CCA cell proliferation, chemoresistance, and metastasis both in vitro and in vivo. We found that NDRG2 is a target gene of miR-181c, and the down-regulation of NDRG2 was attributed to miR-181c overexpression in CCA. Furthermore, miR-181c can be activated by LIF treatment, whereas NDRG2 could inhibit LIF transcription through disrupting the binding between Smad, small mothers against decapentaplegic complex and LIF promoter. Down-regulation of NDRG2 and overexpression of miR-181c or LIF are significantly associated with a poorer overall survival (OS) in CCA patients. Finally, we found that a combination of NDRG2, miR-181c, and LIF expression is a strong predictor of prognosis in CCA patients. Conclusion: These results establish the counteraction between NDRG2 and LIF/miR-181c as a key mechanism that regulates cholangiocarcinogenesis and metastasis. Our results elucidated a novel pathway in NDRG2-mediated inhibition of cholangiocarcinogenesis and metastasis and suggest new therapeutic targets, including NDRG2, LIF, miR-181c, and transforming growth factor beta, in CCA prevention and treatment. (Hepatology 2016;64:1606-1622)

// Shuyao Tang 1 , Gang Tan 2 , Xian Jiang 1 , Peng Han 1 , Bo Zhai 2 , Xuesong Dong 1 , Haiquan Qiao 1 , Hongchi Jiang 1 , Xueying Sun 1 1 The Hepatosplenic Surgery Center, Department of General Surgery, the First Affiliated Hospital of Harbin Medical University, Harbin, China 2 Department of General Surgery, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, China Correspondence to: Xueying Sun, email: kevsun88@hotmail.com , k.sun@auckland.ac.nz Keywords: sorafenib, hepatocellular carcinoma, drug resistance, LncRNA, miRNA Received: June 13, 2016 Accepted: September 21, 2016 Published: September 28, 2016 ABSTRACT Sorafenib resistance remains a major obstacle for the effective treatment of hepatocellular carcinoma (HCC), and a number of miRNAs contribute to this resistance. However, the regulatory networks of miRNAs are very complex, thus inhibiting a single miRNA may sequentially activate other compensatory pathways. In the present study, we generated an artificial long non-coding RNA (AlncRNA), which simultaneously targets multiple miRNAs including miR-21, miR-153, miR-216a, miR-217, miR-494 and miR-10a-5p. These miRNAs have been shown to be upregulated in sorafenib-resistant cells and participate in the mechanisms underlying sorafenib resistance. The AlncRNA contains tandem sequences of 6 copies of the complementary binding sequences to the target miRNAs and is expressed by an adenoviral vector (Ad5-AlncRNA). Infection of Ad5-AlncRNA into sorafenib-resistant HCC cells blocked the function of miRNAs, and sequentially inhibited the downregulation of PTEN and activation of AKT. Ad5-AlncRNA significantly inhibited proliferation and induced apoptosis of sorafenib-resistant cells and enhanced the effects of sorafenib in vitro and in animal models. Inhibition of autophagy decreased the sensitivity of sorafenib-resistant cells to Ad5-AlncRNA, while its induction had the opposite effect. These results indicate that targeting multiple miRNAs by the artificial lncRNA could be a potential promising strategy for overcoming sorafenib resistance in the treatment of HCC.

Background Whether continuous intraoperative nerve monitoring (C-IONM) can further reduce the incidence of recurrent laryngeal nerve injury compared with intermittent intraoperative nerve monitoring (I-IONM) in high-risk thyroid surgery is still controversial. This observational study aimed to evaluate the incidence of vocal cord paralysis (VCP) in high-risk thyroid surgeries performed with I-IONM and C-IONM. Materials and methods High-risk thyroid surgical patients operated with I-IONM or C-IONM by the same group of surgeons in the thyroid surgery department of our institution between January 2014 and February 2018 were analyzed. Differences in the incidence rates of temporary and permanent VCP between the two groups were compared. A P-value Results A total of 344 patients who underwent high-risk thyroid surgery (550 nerves at risk [NARs]) were observed, with 238 patients (374 NARs) operated with I-IONM and 106 patients (173 NARs) operated with C-IONM. The incidence of temporary and permanent VCP was 1.9% (7/374) and 0.8% (3/374) in the I-IONM group and 1.2% (2/173) and 0% (0/173) in the C-IONM group, respectively, showing no statistical difference (P = 0.726 and P = 0.555). The incidence rate of impending recurrent laryngeal nerve injuries successfully prevented in the C-IONM group was 5.2% (9/173). Conclusions Both I-IONM and C-IONM are equally safe and effective in high-risk thyroid surgery. C-IONM can help predict impending recurrent laryngeal nerve injury in real time and has a good warning feature, thereby minimizing critical maneuvers in high-risk thyroid surgery.

Background The poor viability of transplanted mesenchymal stem cells (MSCs) hampers their therapeutic efficacy for ischemic heart disease. Micro RNA s are involved in regulation of MSC survival and function. The present study was designed to investigate the molecular effects of mi R ‐15a/15b on MSC survival, focusing on the role of vascular endothelial growth factor receptor 2. Methods and Results We first harvested donor luc(Luciferase)‐ MSC s (5×10 5 ) isolated from the luciferase transgenic mice with FVB background. Luc‐ MSC s were transfected with miR‐15a/15b mimics or inhibitors and cultured under oxygen glucose deprivation condition for 12 hours to mimics the harsh microenvironment in infarcted heart; they were subjected to MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide?Thiazolyl Blue Tetrazolium Bromide) assay, bioluminescence imaging, quantitative reverse transcription–polymerase chain reaction, transferase‐mediated deoxyuridine triphosphate–digoxigenin nick‐end labeling assay, and flow cytometry. Furthermore, the levels of vascular endothelial growth factor receptor 2, protein kinase B, p(Phosphorylate)‐protein kinase B, Bcl‐2, Bax, and caspase‐3 proteins were available by Western blotting assay. In vivo, acute myocardial infarction was induced in 24 mice by coronary ligation, with subsequent receipt of Luc‐ MSC s, Luc‐ MSC s+miR‐15a/15b inhibitors, or PBS treatment. The therapeutic procedure and treatment effects were tracked and assessed using bioluminescence imaging and echocardiographic measurement. Next, ex vivo imaging and immunohistochemistry were conducted to verify the distribution of MSC s. We demonstrated that miR‐15a/15b targeted vascular endothelial growth factor receptor 2 to modulate MSC survival, possibly via phosphatidylinositol 3‐kinase/protein kinase B signaling pathway, which was proved by bioluminescence imaging, immunohistochemistry analysis, and echocardiographic measurement. Conclusions Luc‐ MSC s could be followed dynamically in vitro and in vivo by bioluminescence imaging, and the role of mi R ‐15a/b could be inferred from the loss of signals from luc‐ MSC s. This finding may have practical clinical implications in mi R ‐15a/15b–modified MSC transplantation in treating myocardial infarction.

OBJECTIVE The low pre- and intraoperative diagnostic rates in follicular thyroid carcinoma (FTC) often lead to inadequate surgical resection and necessitate further completion surgery. Therefore, the preoperative prediction of FTC in thyroid nodules is essential. DESIGN AND PATIENT: Patients were categorized into two data sets: the modelling data set, which included 3649 patients admitted to our centre between January 2014 and December 2016, and the validation data set, which included 1253 patients admitted between January and December 2017. Patient data from the FTC and non-FTC groups were initially included in a modelling data set to establish a preoperative prediction model. This model was subsequently employed in a validation data set for external validation of the predictive value. The positivity rate for FTC predicted by the model was compared with that of the intraoperative frozen sections. RESULTS The preoperative serum thyroglobulin level, nodule diameter, calcification status, solidity and blood supply were selected as predictors for the model. The regression equation was as follows: Y = 0.010 × (thyroglobulin level) + 0.556 × (nodule diameter) + 0.675 × (calcification status) + 2.355 × (nodule component) + 1.072*(blood flow) - 9.787. The model positively predicted FTC at values of Y ≥ -4.11. The accuracy, sensitivity, specificity, positive likelihood ratio and negative likelihood ratio of the prediction model were 89.2%, 90.2%, 87.7%, 39.2 and 0.11, respectively. External validation of the model demonstrated acceptable results. The positive prediction rate of the model was 90.7% (78/86), which was significantly higher than that of the intraoperative frozen sections (10.5% [9/86]; P