Quantitative Characterization of Protein N-Linked Core-Fucosylation by an Efficient Glycan Truncation Strategy.

Dysregulation of protein core-fucosylation plays a pivotal role in the onset, progression, and immunosuppression of cancer. However, analyzing core-fucosylation, especially the accurate determination of the core-fucosylation (CF) site occupancy ratio, remains challenging. To address these problems, we developed a truncation strategy that efficiently converts intact glycopeptides with hundreds of different glycans into two truncated forms, i.e., a monosaccharide HexNAc and a disaccharide HexNAc+core-fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of the CF occupancy ratio with high reproducibility. Notably, three times CF sites were identified using this strategy compared to conventional methods based on intact glycopeptides. Application of this platform to characterize protein core-fucosylation in two breast cancer cell lines, i.e., MDA-MB-231 and MCF7, yields a total of 1615 unique glycosites and about 900 CF sites from one single LC-MS/MS analysis. Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes and provides insights into developing new therapeutic strategies to aid precision medicine. Given the robust performance of this platform, it would have broad application in discovering novel biomarkers based on the CF glycosylation pattern, investigating cancer mechanisms, as well as detecting new intervention targets.

Multi-omics pan-cancer study of SPTBN2 and its value as a potential therapeutic target in pancreatic cancer.

SPTBN2 is a protein-coding gene that is closely related to the development of malignant tumors. However, its prognostic value and biological function in pan-cancer, especially pancreatic cancer (PAAD), have not been reported. In the present study, a novel exploration of the value and potential mechanism of SPTBN2 in PAAD was conducted using multi-omics in the background of pan-cancer. Via various database analysis, up-regulated expression of SPTBN2 was detected in most of the tumor tissues examined. Overexpression of SPTBN2 in PAAD and kidney renal clear cell cancer patients potentially affected overall survival, disease-specific survival, and progression-free interval. In PAAD, SPTBN2 can be used as an independent factor affecting prognosis. Mutations and amplification of SPTBN2 were detected, with abnormal methylation of SPTBN2 affecting its expression and the survival outcome of PAAD patients. Immunoassay results demonstrate that SPTBN2 was a potential biomarker for predicting therapeutic response in PAAD, and may influence the immunotherapy efficacy of PAAD by regulating levels of CD8 + T cells and neutrophil infiltration. Results from an enrichment analysis indicated that SPTBN2 may regulate the development of PAAD via immune pathways. Thus, SPTBN2 is a potential prognostic biomarker and immunotherapy target based on its crucial role in the development of PAAD.

[Corrigendum] Metformin reverses multidrug resistance in human hepatocellular carcinoma Bel­7402/5­fluorouracil cells.

Subsequently to the publication of the above paper, the authors have reviewed its content and the primary data, and have realized that the western blots selected to show the ß­actin experiments featured in Fig. 4A and Fig. 3C were the same blot, albeit with a different exposure time. The control blots correctly presented for Fig. 3C were inadvertently copied into Fig. 4A owing to an error made during the figure compilation process. The revised version of Fig. 4, containing the correct ß­actin blots for Fig. 4A, is shown below. Note that this error did not significantly affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors thank the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 10: 2891­2897, 2014; DOI: 10.3892/mmr.2014.2614].

Case Report: An Undefined Liver Lesion in a Young Man With Severe Aplastic Anemia: A Teachable Moment.

In this work, we reported a young man complaining of asthenia and intermittent fever for 10 days, and an ultrasound showed an undefined lesion on his liver. Facing the patient's situation with severe agranulocytosis, anemia, and thrombocytopenia, we passed through a tough diagnostic process for choosing an appropriate treatment for him with an ambiguous result of pathological biopsy. The undefined liver lesion was successfully solved by withdrawing the androgen for observation, without lobectomy. The lesion gradually diminished over 2 years of follow-up.

Emodin Protects Against Acute Pancreatitis-Associated Lung Injury by Inhibiting NLPR3 Inflammasome Activation via Nrf2/HO-1 Signaling.

AIM: Lung injury is a common complication of acute pancreatitis (AP), which leads to the development of acute respiratory distress syndrome and causes high mortality. In the present study, we investigated the therapeutic effect of emodin on AP-induced lung injury and explored the molecular mechanisms involved. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly divided into AP (n=24) and normal (n=6) groups. Rats in the AP group received a retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct and then randomly assigned to untreated, emodin, combined emodin and ML385, and dexamethasone (DEX) groups. Pancreatic and pulmonary injury was assessed using H&E staining. In in vitro study, rat alveolar epithelial cell line L2 cells were exposed to lipopolysaccharide and treated with emodin. Nrf2 siRNA pool was applied for the knockdown of Nrf2. The contents of the pro-inflammatory cytokines in the bronchoalveolar lavage fluid and lung were determined using enzyme-linked immunosorbent assay. The expressions of related mRNAs and proteins in the lung or L2 cells were detected using real-time polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence. KEY FINDINGS: Emodin administration alleviated pancreatic and pulmonary injury of rats with AP. Emodin administration suppressed the production of proinflammatory cytokines, downregulated NLRP3, ASC and caspase-1 expressions and inhibited NF-κB nuclear accumulation in the lung. In addition, Emodin increased Nrf2 nuclear translocation and upregulated HO-1 expression. Moreover, the anti-inflammatory effect of emodin was blocked by Nrf2 inhibitor ML385. CONCLUSION: Emodin effectively protects rats against AP-associated lung injury by inhibiting NLRP3 inflammasome activation via Nrf2/HO-1 signaling.

CBX2 Regulates Proliferation and Apoptosis via the Phosphorylation of YAP in Hepatocellular Carcinoma.

Chromobox 2 (CBX2), a chromobox family protein, is a crucial component of the polycomb group complex: polycomb repressive complex 1 (PRC1). Research on CBX2 as an oncogene has been published in recent years. However, the connection between CBX2 and hepatocellular carcinoma (HCC) has not been studied. In this article, based on the results of immunohistochemical (IHC) staining of HCC and adjacent liver tissue microarrays, we found that high CBX2 expression is associated with poor prognosis in HCC patients. The results of a CCK8 assay, a clonogenic survival assay and a nude mouse tumorigenicity assay showed that knockdown of CBX2 inhibited the proliferation of HCC cells. According to the results of Annexin V-FITC/propidium iodide (PI) staining-based fluorescence activated cell sorting (FACS) analysis, knockdown of CBX2 increased HCC cell apoptosis. Furthermore, the RNA-seq results revealed that knockdown of CBX2 inhibited the expression of WTIP, which is an inhibitor of the Hippo pathway. We used western blotting to validate the mechanism and discovered that knockdown of CBX2 increased the phosphorylation of YAP, which explains why knockdown of CBX2 inhibits proliferation and increases apoptosis in HCC cells. In conclusion, CBX2 could be a potential target for HCC anticancer treatment.

Cloning of pcB and pcA Gene from Gracilariopsis lemaneiformis and Expression of a Fluorescent Phycocyanin in Heterologous Host.

In order to study the assembly mechanism of phycocyanin in red algae, the apo-phycocyanin genes (pcB and pcA) were cloned from Gracilariopsis lemaneiformis. The full length of phycocyanin ß-subunit (pcB) contained 519 nucleotides encoding a protein of 172 amino acids, and the full length of phycocyanin α-subunit(pcA) contained 489 nucleotides encoding a protein of 162 amino acids. Expression vector pACYCDuet-pcB-pcA was constructed and transformed into E. coli BL21 with pET-ho-pcyA (containing ho and pcyA gene to synthesize phycocyanobilin). The recombinant strain showed fluorescence activity, indicating the expression of optically active phycocyanin in E. coli. To further investigate the possible binding sites between phycocyanobilin and apo-phycocyanin, Cys-82 and Cys-153 of the ß subunit and the Cys-84 of the α subunit were respectively mutated, and four mutants were obtained. All mutant strains had lower fluorescence intensity than the non-mutant strains, which indicated that these mutation sites could be the active binding sites between apo-phycocyanin and phycocyanobilin (PCB). This research provides a supplement for the comprehensive understanding of the assembly mechanism of optically active phycocyanin in red algae.

Proteomics analysis of site-specific glycoforms by a virtual multistage mass spectrometry method.

Determination of site-specific glycoforms is the key to reveal the micro-heterogeneity of protein glycosylation at proteome level. Herein, we presented an integrated virtual multistage MS strategy to identify intact glycopeptides, which allowed the determination of site-specific glycoforms. In this strategy, the enzymatically de-glycosylated peptides and intact glycopeptides were mixed and analyzed in the same LC-MS/MS run. The acquired MS2 spectra of intact glycopeptides allowed determination of the glycans, and the MS2 spectra of the de-glycosylated peptides enabled the identification of peptide backbone sequences. Compared with the conventional multistage strategy, the peptide backbones could be directly identified by the MS2 of the de-glycopeptides with higher sensitivity. This strategy was first validated by analyzing the glycosites and site-specific glycoforms of mouse liver tissues. Then, it was applied to differential analysis of the glycoproteomes of hepatocellular carcinoma (HCC) and adjacent liver tissues. Compared with the identification scheme using only MS2 spectra of intact glycopeptides or glycosites, this approach enabled quantitative analysis on two levels, i.e. glycosites and site-specific glycoforms, simultaneously. Thus, it could be a powerful tool to characterize the subtle differences in the macro- and micro-heterogeneity of protein glycosylation for different samples.

Cloning of nitrite reductase gene from Haematococcus pluvialis and transcription and enzymatic activity analysis at different nitrate and phosphorus concentration.

Haematococcus pluvialis is an economic microalga to produce astaxathin. To study the nitrogen metabolic process of H. pluvialis, the transcription level and enzyme content of nitrite reductase at different nitrate and phosphorus concentrations were studied. In this research, nitrite reductase gene (nir) was first cloned from H. pluvialis, which consists of 5592 nucleotides and includes 12 introns. The cDNA ORF is 1776 bp, encoding a 592 amino acid protein with two conserved domains. Phylogenetic analysis showed that the nir gene in H. pluvialis had the highest affinity with other freshwater green algae. Nitrogen and phosphorus play an important role in the growth of H. pluvialis. The single factor experiments of nitrogen on growth conditions showed that the group with 0.2 g/L NaNO3 had a relative high biomass. The single factor experiments of phosphorus on growth conditions showed that the group with 0.06 g/L K2HPO4 had a relative high biomass. The transcription level and enzymatic activity of nitrite reductase were detected at different nitrate and phosphorus concentrations. In the absence of nitrogen and phosphorus in the medium, nitrite reductase activity is the highest. This research provides theoretical guidance for optimization of culture medium for H. pluvialis and also provides an experimental basis for understanding of nitrogen metabolism pathway in H. pluvialis.

MiR-3196, a p53-responsive microRNA, functions as a tumor suppressor in hepatocellular carcinoma by targeting FOXP4.

Increasing evidences demonstrate that miRNAs play an important role in development and progression of hepatocellular carcinoma (HCC). Recent studies indicate that miR-3196 regulates tumorigenesis in breast and lung cancer. However, its role and regulatory mechanism remains unknown in hepatocellular carcinoma. Here, we found that miR-3196 was downregulated in HCC tissues and decreased miR-3196 was correlated with tumor size (P=0.0297) and TNM stage (P=0.034). Forced miR-3196 suppressed HCC cell growth and chemoresistance in vivo and in vitro. Further mechanistic studies revealed that the tumor suppressor p53 transcriptionally upregulated miR-3196 expression by binding to its promoter region in HCC cells. Additional, we also found that FOXP4 was a downstream target of miR-3196 and increased miR-3196 inhibited FOXP4 expression which led to HCC growth suppression and cell apoptosis increase. Collectively, our data shed a new role of miR-3196 in HCC and indicates that p53-dependent, miR-3196-medicated FOXP4 pathway inhibits the tumorigenesis of HCC.

Cloning of the pks3 gene of Aurantiochytrium limacinum and functional study of the 3-ketoacyl-ACP reductase and dehydratase enzyme domains.

Aurantiochytrium limacinum has received attention because of its abundance of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). DHA is synthesized through the polyketide synthase (PKS) pathway in A. limacinum. The related enzymes of the PKS pathway are mainly expressed by three gene clusters, called pks1, pks2 and pks3. In this study, the full-length pks3 gene was obtained by polymerase chain reaction amplification and Genome Walking technology. Based on a domain analysis of the deduced amino acid sequence of the pks3 gene, 3-ketoacyl-ACP reductase (KR) and dehydratase (DH) enzyme domains were identified. Herein, A. limacinum OUC168 was engineered by gene knock-in of KR and DH using the 18S rDNA sequence as the homologous recombination site. Total fatty acid contents and the degree of unsaturation of total fatty acids increased after the kr or dh gene was knocked in. The cloning and functional study of the pks3 gene of A. limacinum establishes a foundation for revealing the DHA synthetic pathway. Gene knock-in of the enzyme domain associated with PKS synthesis has the potential to provide effective recombinant strains with higher DHA content for industrial applications.

One type of duplex appendix: horseshoe appendix.

A horseshoe appendix is a subtype of duplex appendix, in which the appendix is shaped like a circle that may lead to an internal hernia and result in intestinal necrosis. This subtype is extremely rare, with only 13 cases reported worldwide to date, and easily triggers a series of medico-legal consequences due to the neglect of another infection base of the appendix. We describe a 22-year-old man who presented with a 3-day history of fever and was diagnosed with pneumonia. After receiving antibiotics for 3 days in the Department of Pneumology, he was found to have a periappendiceal abscess. He underwent appendectomy after 3 days of conservative treatment failure in the Department of General Surgery. During the operation, we found that he had a horseshoe appendix with the two bases forming a circle, each communicating with the cecum. We provide a review of 13 cases presented in the literature, with a discussion of the clinical features, diagnosis, and surgical approach of the horseshoe appendix to make the general surgeon get a clear concept of this type of appendicitis.

Fluorescence imaging of hepatocellular carcinoma with a specific probe of COX-2.

Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer. Although the standard treatment method based on surgery has generally extended life, it still causes the second and sixth most prevalent cancer-related death in men and women, respectively. The recurrence of cancer caused by unclear resection margins and any remaining undiscovered metastatic nodules should take a large proportion of responsibility for the poor prognosis after resective surgery. Therefore, a practical and effective method that can be used during hepatectomy to specifically identify HCC is a potentially significant area deserving attention. Tests involving fluorescence have been used in many biological systems. In this study, we use a probe that can combine with cyclooxygenase-2 (COX-2) and subsequently emit fluorescence to identify HCC cells and heteroplastic tumors in a mouse model. The results show that this specific probe can clearly differentiate HCC, with differences that could be observed with the naked eye in human samples. The biotechnology of knocking down COX-2 and its inhibitor were used on human HCC cell line SMMC7721, and the outcomes confirmed the above results. The toxic effect also showed that the probe had no harmful effect on normal liver cells. Taken together, our study demonstrates that a COX-2-specific fluorescence probe may be a new and effective method to identify HCC, especially during surgery.

Long Non-Coding RNA Linc-USP16 Functions As a Tumour Suppressor in Hepatocellular Carcinoma by Regulating PTEN Expression.

BACKGROUND/AIMS: Recent evidence has indicated the crucial regulatory roles of long non-coding RNAs (lncRNAs) in tumour biology. In hepatocellular carcinoma (HCC), aberrant expression of lncRNAs plays an essential role in HCC tumourigenesis. However, the potential roles and regulatory mechanisms of the novel human lncRNA, Linc-USP16, in HCC are unclear. METHODS: To investigate the function of Linc-USP16 in HCC, we first analysed the expression levels of Linc-USP16 in HCC patient tissues and cell lines via q-RT-PCR and established overexpressed or knockdown HCC cell lines. RESULTS: Here, we found that Linc-USP16 expression was significantly down-regulated in HCC patient tissues and cell lines. Further functional experiments suggested that Linc-USP16 could directly increase PTEN expression by acting as a competing endogenous RNA (ceRNA) for miR-21 and miR-590-5p. These interactions led to repression of AKT pathway and inhibition of HCC cell proliferation and migration. CONCLUSION: Thus, our data showed that Linc-USP16, as a tumour suppressor, plays an important role in HCC pathogenesis and provides a new therapeutic strategy for HCC treatment.

Deep sequencing and comprehensive expression analysis identifies several molecules potentially related to human poorly differentiated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) that is graded histologically as poorly differentiated has a high recurrence, metastasis and poor prognosis. We sought to determine the regulatory mechanisms of HCC tumorigenesis and to identify molecules closely related to poorly differentiated HCC. High-throughput sequencing was used to construct microRNA (miRNA) and mRNA expression profiles for poorly differentiated HCC tissues and adjacent tissues. Network analysis was carried out to study miRNA-target interactions. Integrating the miRNA and mRNA data of HCC with four tumor grades from The Cancer Genome Atlas (TCGA) portal enabled the identification of potential closely related molecules for early diagnosis of poorly differentiated HCC. Electronic validation of RNA-seq data and survival analysis was also performed. In total, 1051 differentially expressed genes and 165 differentially expressed miRNAs were identified between HCC tumor and paired non-tumorous tissue. Based on 3718 miRNA-target interactions, we established an miRNA-target interaction network; the target genes were mainly involved in bile acid biosynthesis and bile secretion. Integrating expression data of HCC from TCGA indicated that two proteins, TM4SF1 and ANXA2, are convincing indicators for initial diagnosis of poorly differentiated HCC. According to the survival analysis, three proteins, ANXA2, C8orf33 and IGF2BP3, were identified as being associated with the survival time of HCC patients. Moreover, we suggest that hsa-miR-1180 may be an effective biomarker for poorly differentiated HCC. Three molecules, TM4SF1, ANXA2 and C8orf33, are potential biomarkers for distinguishing poorly differentiated from well-differentiated HCC.

MicroRNA-205-5p regulates the chemotherapeutic resistance of hepatocellular carcinoma cells by targeting PTEN/JNK/ANXA3 pathway.

Hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive system, and patients with advanced HCC have a poor outlook, partly due to resistance to chemotherapeutic drugs. Previous studies have implicated microRNAs in the regulation of chemoresistance, and we have previously shown that microRNA (miR)-205-5p is down-regulated in multiple hepatoma cell lines. Here, we investigate whether miR-205-5p is involved in chemotherapeutic resistance in HCC. Expression of miR-205-5p was measured by real-time quantitative reverse transcription PCR and cell viability was determined using a CCK-8 cell viability assay. Expression of proteins in the PTEN/JNK/ANXA3 pathway were assessed via Western blotting. We found that miR-205-5p expression was down-regulated in all HCC cell lines investigated. In addition, miR-205-5p expression was upregulated by 5-fluorouracil (5-Fu) treatment in Bel-7402 (Bel) cells. Interestingly, miR-205-5p expression was increased in multidrug-resistant Bel-7402/5-Fu (Bel/Fu) cells, compared with Bel cells. We next demonstrated that sensitivity to 5-Fu was increased in Bel/Fu cells after treatment with a miR-205-5p inhibitor. Similarly, increased resistance to 5-Fu was observed in Bel cells after transfection with a miR-205-5p mimic. We injected nude mice with Bel/5-Fu cells to promote tumor growth, and found that co-treatment with a miR-205-5p antagomir and 5-Fu slowed tumor growth more than either treatment alone. Finally, we found that these effects were all associated with changes in the PTEN/JNK/ANXA3 pathway. In conclusion, inhibition of miR-205-5p may reverse chemotherapeutic resistance to 5-Fu, and this may occur via the PTEN/JNK/ANXA3 pathway.

CT-721, a Potent Bcr-Abl Inhibitor, Exhibits Excellent In Vitro and In Vivo Efficacy in the Treatment of Chronic Myeloid Leukemia.

Kinase inhibitors that target Bcr-Abl are highly effective in the treatment of chronic myeloid leukemia (CML). However, these inhibitors are often invalidated due to the drug resistance. Therefore, the discovery and development of novel Bcr-Abl inhibitors is required to overwhelm the drug resistance in the treatment of CML resistant to the currently used first-line Bcr-Abl inhibitors. Herein we have described a newly developed Bcr-Abl inhibitor CT-721, which displayed potent inhibitory effects on wild-type and T315I mutant Bcr-Abl. It functioned as a typically ATP-competitive inhibitor, superior to other existing Bcr-Abl inhibitors. CT-721 also demonstrated time-dependent inhibition of Bcr-Abl activation and the resultant downstream signaling transduction pathways in Bcr-Abl positive cells. Furthermore, CT-721 induced cell apoptosis and cell cycle arrest, and efficaciously inhibited tumor growth in Bcr-Abl-expressed K562 and KU812 xenograft models in a mechanism-based manner. Further PK/PD studies revealed a positive in vivo correlation between the compound concentration and inhibition of Bcr-Abl activity. Taken together, CT-721 is a potent and time-dependent Bcr-Abl kinase inhibitor, and has shown strong in vitro and in vivo anti-CML activities with a favorable pharmacokinetic profile, differentiating it from other Bcr-Abl kinase inhibitors already approved and current in development for the treatment of CML.

Notch1 regulates the JNK signaling pathway and increases apoptosis in hepatocellular carcinoma.

Notch1-induced pathways are involved in cell growth, apoptosis, motility, and invasion in many cancers. In the present study, the expression of Notch1 and NICD1 was detected in hepatocellular carcinoma (HCC) tissues using in-vitro assays. And then, we explored cell biology and signaling pathways using Notch1 siRNA or plasmids. Here, the expression of Notch1 and NICD1 was significantly decreased in HCC tissues. In-vitro, Notch1 plasmids inhibited cell proliferation, migration and invasion, but enhanced apoptosis of HepG2 and Hep3B cells. Conversely, si-Notch1 enhanced cell proliferation, migration and invasion, but inhibited apoptosis of HepG2 and Hep3B cells. Mechanically, Notch1 decreased the expression of cyclin D1, MMP-9 and Bcl-2, but increased the expression of p-JNK, Bax and cleaved caspase 3 in HepG2 and Hep3B cells. Besides, si-JNK or JNK inhibitor SP600125 affected the activation of Notch1 signaling pathway, and prevents cell apoptosis. In conclusion, Notch1 regulates the JNK signaling pathway and increases apoptosis in HCC. Because patients with HCC have a poor prognosis, Notch1 pathway may provide a novel treatment strategy.

miR-186 affects the proliferation, invasion and migration of human gastric cancer by inhibition of Twist1.

Recent evidence shows that miRNAs are dysregulated in a variety of cancers including gastric cancer (GC), and emerging as key oncogenes or tumor suppressors. In this study, qRT-PCR was used to analyze the expression of miR-186 in GC tissues and adjacent non-cancerous tissues, and then more in-vitro experiments were used to investigate the role of miR-186 in GC cells. Here, we identified miR-186 was generally down-regulated in GC tissues; however, Twist1 was generally up-regulated in GC tissues. Moreover, miR-186 and Twist1 were associated with larger tumor size and advanced clinical stage of GC. In-vitro experiments demonstrated that ectopic overexpression of miR-186 inhibited GC cell proliferation, invasion and migration; however, inhibited expression of miR-186 enhanced cell proliferation, invasion and migration. Furthermore, the luciferase reporter assay demonstrated Twist1 as a direct target of miR-186. Finally, over-expression of Twist1 abrogated inhibitory impact of miR-186 on cell proliferation, invasion and migration. In conclusion, miR-186 affects the proliferation, invasion and migration of human gastric cancer by inhibition of Twist1, and could be a tumor suppressor in GC development. Thus, miR-186 may be served as a candidate prognostic biomarker and target for new therapies in human gastric cancer.

Metformin mediates resensitivity to 5-fluorouracil in hepatocellular carcinoma via the suppression of YAP.

Metformin plays an anti-proliferative role in tumor cells in many types of cancer. However, the correlation between metformin and sensitivity to chemotherapeutic agents in hepatocellular carcinoma (HCC) and the relevant mechanism are unclear. The present study showed that HCC patients with type 2 diabetes mellitus benefited from metformin administration, with a longer overall survival. Metformin resensitized Bel-7402/5-fluorouracil (Bel/Fu) cells to 5-fluorouracil (5-Fu) in vitro and in vivo, and the combination of metformin and 5-Fu inhibited cell proliferation, promoted cell apoptosis and induced G0/G1 cell cycle arrest in the Bel/Fu cells. Moreover, metformin repressed YAP by both decreasing the total protein expression and accelerating the phosphorylation of YAP. The inhibition of YAP subsequently promoted the expression of PTEN, and suppressed the Akt pathway. Therefore, the expression of P-gp and MRP1 was downregulated. Taken together, our findings suggested that metformin may increase sensitivity to chemotherapeutic agents by suppressing YAP in hepatocellular carcinoma.