Serum Of Hepatocellular Carcinoma Patients Research Articles

Clinical significance of serum miR-487b in HBV-related hepatocellular carcinoma and its potential mechanism

Background Hepatocellular carcinoma (HCC) is a malignant tumour with high mortality. In recent years, microRNA (miRNA) has been recognized in the diagnosis and prognosis of cancer. miR-487b has been found to play a role in a variety of cancers. The purpose of this study is to explore the role of miR-487b in the diagnosis and prognosis of hepatitis B virus (HBV)-related HCC, and its influence on the biological behaviour of HCC cells. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression level of miR-487b in the serum of HCC patients, HBV patients, and healthy people. The ROC curve was used to evaluate the role of miR-487b in the diagnosis of HCC. The prognostic significance of miR-487b in HCC was analyzed by the Kaplan-Meier survival curve and Cox regression model. The effects of miR-487b on cell proliferation, migration, and invasion were explored through MTT assay and transwell assays. Results The expression level of miR-487b in the serum of HBV-related HCC patients was significantly higher than that of CHB patients and normal healthy people. The expression level of miR-487b can distinguish HCC patients from CHB patients or normal healthy people. High expression of miR-487b was associated with poor prognosis, which could be used as an independent prognostic factor for HCC. The upregulation of miR-487b promoted cell proliferation, migration, and invasion. Conclusion miR-487b can be used as a biomarker for the diagnosis and prognosis of HCC.

Adipose most abundant 2 protein is a predictive marker for cisplatin sensitivity in cancers

Cisplatin (CDDP) is one of the chemotherapeutic drugs being used to treat various cancers. Although effective in many cases, as high doses of CDDP cause cytotoxic effects that may worsen patients’ condition, therefore, a marker of sensitivity to CDDP is necessary to enhance the safety and efficiency of CDDP administration. This study focused on adipose most abundant 2 (APM2) to examine its potential as a marker of CDDP sensitivity. The relationship of APM2 expression with the mechanisms of CDDP resistance was examined in vitro and in vivo using hepatocellular carcinoma (HCC) cells, tissues and serum of HCC patients (n = 71) treated initially with intrahepatic arterial infusion of CDDP followed by surgical resection. The predictability of serum APM2 for CDDP sensitivity was assessed in additional 54 HCC patients and 14 gastric cancer (GC) patients. APM2 expression in CDDP-resistant HCC was significantly higher both in serum and the tissue. Bioinformatic analyses and histological analyses demonstrated upregulation of ERCC6L (DNA excision repair protein ERCC6-like) by APM2, which accounts for the degree of APM2 expression. The serum APM2 level and chemosensitivity for CDDP were assessed and cut-off value of serum APM2 for predicting the sensitivity to CDDP was determined to be 18.7 µg/mL. The value was assessed in HCC (n = 54) and GC (n = 14) patients for its predictability of CDDP sensitivity, resulted in predictive value of 77.3% and 100%, respectively. Our study demonstrated that APM2 expression is related to CDDP sensitivity and serum APM2 can be an effective biomarker of HCC and GC for determining the sensitivity to CDDP.Trial registration: This study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN000028487).

Serum exosomal long noncoding RNAs lnc-FAM72D-3 and lnc-EPC1-4 as diagnostic biomarkers for hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs), such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. However, lncRNA expression in the serum of HCC patients is still unclear. To identify candidate lncRNAs for HCC diagnosis, we purified exosomal total RNA from the serum of healthy volunteers (controls) and hepatitis, cirrhosis, and HCC patients. To assess the function of lncRNAs, small interfering RNAs and overexpression vectors were designed and cell viability and cell apoptosis assays conducted. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients. qPCR assays showed that lnc-FAM72D-3, lnc-GPR89B-15, lncZEB2-19, and lnc-EPC1-4 are differentially expressed in HCC. Furthermore, the expression level of lnc-EPC1-4 correlated with age. While the expression levels of lnc-GPR89B-15 and lnc-EPC1-4 correlated with serum alpha-fetoprotein level. lnc-FAM72D-3 knockdown decreased cell viability and promoted cell apoptosis, indicating that lnc-FAM72D-3 functions as an oncogene in HCC. In contrast, lnc-EPC1-4 overexpression inhibited cell proliferation and induced cell apoptosis, indicating that it functions as a tumor suppressor gene. Collectively, these findings show that lnc-FAM72D-3 and lnc-EPC1-4 play a novel role that might contribute to hepatocarcinogenesis and identify potential candidate biomarkers for HCC diagnosis.

A Novel lncRNA NONHSAT053785 Acts as an Independent Risk Factor for Intrahepatic Metastasis of Hepatocellular Carcinoma.

PurposeLong noncoding RNAs (lncRNAs) in body fluids have been considered as promising novel biomarkers for tumor-related diseases. The present study aimed to investigate the expression level of lncRNA NONHSAT053785 in serum and its correlation with clinical characteristics of hepatocellular carcinoma (HCC) patients.MethodsThe droplet digital PCR (ddPCR) was used to measure the serum levels of NONHSAT053785 in 112 HCC patients, 96 chronic hepatitis B (CHB) patients, and 99 healthy controls (HC). The correlation between NONHSAT053785 and clinical characteristics was analyzed by chi-square test and Spearman correlation test. The risk factors of intrahepatic metastasis (IM) were detected by univariate and multivariate analyses. Furthermore, the diagnostic value of NONHSAT053785 in HCC and its predictive ability in IM were evaluated by the receiver operating characteristic (ROC) curves.ResultsThe level of NONHSAT053785 was significantly increased in the serum of HCC patients and was higher in HCC patients with IM as compared to those without. Additionally, the expression level of NONHSAT053785 was significantly related to IM, Child–Pugh classification, and peripheral blood indicators such as liver metabolic enzymes and positively correlated to IM, Barcelona Clinic Liver Cancer (BCLC) staging, and some peripheral blood indicators. Furthermore, the serum NONHSAT053785 was indicated as an independent predictor for IM in the elderly, non-smoking, drinking, and tumor size ≥5 cm subjects. The area under the ROC curve (AUC) was 0.801 (P <0.0001) for diagnosis of HCC and 0.678 (P =0.0015) for predicting IM.ConclusionThe increase in serum NONHSAT053785 levels was related to an increased risk of IM, and hence, may serve as a novel biomarker for the diagnosis of HCC and the prediction of IM.

Serum STIP1, a Novel Indicator for Microvascular Invasion, Predicts Outcomes and Treatment Response in Hepatocellular Carcinoma.

Background: Previous studies reported that stress-induced phosphoprotein 1 (STIP1) can be secreted by hepatocellular carcinoma (HCC) cells and is increased in the serum of HCC patients. However, the therapy-monitoring and prognostic value of serum STIP1 in HCC remains unclear. Here, we aimed to systemically explore the prognostic significance of serum STIP1 in HCC.Methods: A total of 340 HCC patients were recruited to this study; 161 underwent curative resection and 179 underwent transcatheter arterial chemoembolization (TACE). Serum STIP1 was detected by enzyme-linked immunosorbent assay (ELISA). Optimal cutoff values for serum STIP1 in resection and TACE groups were determined by receiver operating characteristic (ROC) analysis. Prognostic value was assessed by Kaplan-Meier, log-rank, and Cox regression analyses. Predictive values of STIP1 for objective response (OR) to TACE and MVI were evaluated by ROC curves and logistic regression.Results: Serum STIP1 was significantly increased in HCC patients when compared with chronic hepatitis B patients or health donors (both P < 0.05). Optimal cutoff values for STIP1 in resection and TACE groups were 83.43 and 112.06 ng/ml, respectively. High pretreatment STIP1 was identified as an independent prognosticator. Dynamic changes in high STIP1 status were significantly associated with long-term prognosis, regardless of treatment approaches. Moreover, post-TACE STIP1 was identified as an independent predictor for OR, with a higher area under ROC curve (AUC-ROC) than other clinicopathological features. Specifically, pretreatment STIP1 was significantly increased in patients with microvascular invasion (MVI), and was confirmed as a novel, powerful predictor for MVI.Conclusions: Serum STIP1 is a promising biomarker for outcome evaluation, therapeutic response assessment, and MVI prediction in HCC. Integration serum STIP1 detection into HCC management might facilitate early clinical decision making to improve the prognosis of HCC.

Clinical relevance of increased serum preneoplastic antigen in hepatitis C-related hepatocellular carcinoma.

BACKGROUNDThe prognosis of hepatocellular carcinoma (HCC) patients remains poor despite advances in treatment modalities and diagnosis. It is important to identify useful markers for the early detection of HCC in patients. Preneoplastic antigen (PNA), originally reported in a rat carcinogenesis model, is increased in the tissues and serum of HCC patients.AIMTo determine the diagnostic value of PNA for discriminating HCC and to characterize PNA-positive HCC.METHODSPatients with hepatitis C virus (HCV)-related hepatic disorders were prospectively enrolled in this study, which included patients with hepatitis, with cirrhosis, and with HCC. A novel enzyme-linked immunosorbent assay was developed to measure serum PNA concentrations in patients.RESULTSSerum PNA concentrations were measured in 89 controls and 141 patients with HCV infections (50 hepatitis, 44 cirrhosis, and 47 HCC). Compared with control and non-HCC patients, PNA was increased in HCC. On receiver operating characteristic curve analysis, the sensitivity of PNA was similar to the HCC markers des-γ-carboxy-prothrombin (DCP) and α-fetoprotein (AFP), but the specificity of PNA was lower. There was no correlation between PNA and AFP and a significant but weak correlation between PNA and DCP in HCC patients. Importantly, the correlations with biochemical markers were completely different for PNA, AFP, and DCP; glutamyl transpeptidase was highly correlated with PNA, but not with AFP or DCP, and was significantly higher in PNA-high patients than in PNA-low patients with HCV-related HCC.CONCLUSIONPNA may have the potential to diagnose a novel type of HCC in which glutamyl transpeptidase is positively expressed but AFP or DCP is weakly or negatively expressed.

Rapid determination of trace elements in serum of hepatocellular carcinoma patients by inductively coupled plasma tandem mass spectrometry

The occurrence and development of hepatocellular carcinoma (HCC) is related to the level of trace elements. The mechanism of changes in trace element concentration and the identification of HCC risks associated with them are not clear. Therefore, an accurate and reliable approach for quantification of the level of trace elements in the serum of liver cancer patients is of great significance. In this study, we proposed a novel strategy for accurate determination of trace elements such as Mn, Fe, Ni, Cu, Zn, and Se in the serum of HCC patients based on inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). In the MS/MS mode, N2O was used as the reaction gas, and an octopole reaction system (ORS) was employed to provide interference-free conditions and a sufficiently low limit of detection (LOD) for the determination of analytical elements. The internal standard elements with similar masses and similar mass spectrometry behavior were selected to correct the matrix effect. The LODs of Mn, Fe, Ni, Cu, Zn, and Se were as low as 0.013, 0.25, 0.010, 0.020, 0.17, and 0.008 μg L−1, respectively. Bovine serum (SRM 1598a) was adopted as the standard reference material to evaluate the accuracy of the method, and the developed method was successfully applied to determine the content of Mn, Fe, Cu, Zn, Ni, Se in serum from HCC patients. The obtained results were in agreement with that of sector field ICP-MS (SF-ICP-MS). The contents of Fe and Cu in the serum of HCC patients were higher compared to that of healthy controls, while the contents of Zn and Se were lower than that of healthy controls. Furthermore, no significant differences were observed in the contents of Mn and Ni in the serum of HCC patients and healthy controls.

The prognostic value of neuromedin U in patients with hepatocellular carcinoma

BackgroundNeuromedin U (NMU) is a neuropeptide belonging to the neuromedin family. Recently, significant associations between NMU and several cancers have been reported. However, no studies have examined the association between NMU and hepatocellular carcinoma (HCC). The purpose of this study was to examine the role of NMU in HCC.MethodsAn enzyme-linked immunosorbent assay was used to measure the level of NMU protein in the sera of patients with hepatic hemangioma and HCC. NMU and cytokine mRNA expression was assessed in HCC samples via RT-qPCR. A tissue microarray consisting of 228 HCC peri- and intra-tumor tissues was used to detect NMU expression via immunohistochemical analysis. The association between NMU expression and overall survival (OS) and disease-free survival (DFS) was analyzed by Kaplan-Meier curves, the log-rank test, and Cox proportional hazard model.ResultsThe level of NMU protein was increased in the sera of HCC patients (p = 0.006). NMU was expressed in intercellular space, rather than in hepatocytes or HCC cells. The prognosis of HCC patients with high NMU expression in peri-tumor tissue was significantly poorer than that of patients with low NMU expression (OS: p = 0.002, DFS: p = 0.033). Peri-tumor NMU expression was also a significant independent prognostic factor for OS (hazard ratio: 1.541, 95% confidence interval: 1.092–2.175, p = 0.014). The level of NMU expression was positively associated with M2 macrophage percentage and the levels of type-2 inflammatory cytokines in HCC tissue.ConclusionsNMU may serve as a novel prognostic biomarker for HCC patients, although further validation is needed in the future. The activation of M2 macrophages and a type-2 inflammatory response may involve in the role of NMU in patients with HCC.

Serum microRNA let-7a-1/let-7d/let-7f and miRNA 143/145 Gene Expression Profiles as Potential Biomarkers in HCV Induced Hepatocellular Carcinoma.

Background:Egypt has the highest prevalence of hepatitis C virus (HCV) worldwide. Which make liver cirrhosis and hepatocellular carcinoma (HCC) major health concerns in Egypt. Circulating microRNAs (miRNAs) have been investigated as biomarkers for malignancies. We investigated miRNA gene expression of Lethal-7 (let-7) cluster: let7-a-1, let-7d-1, let-7f-1 and miRNA (miR)143/145 cluster in sera of HCC patients and chronic HCV patients. Methods:The study included 40 post HCV-Hepatocellular carcinoma patients, 40 chronic HCV patients divided into 2 subgroups, 20 cirrhotic patients and 20 non-cirrhotic patients, and 40 apparently healthy subjects as a control group. Gene expression analysis for studied miRNAs was done using quantitative SYBR Green reverse-transcription Real-Time polymerase chain reaction (PCR). Results:We found that Let-7a-1 gene expression was significantly downregulated in the serum of HCV-HCC patients than in HCV non HCC cirrhotic group and was significantly upregulated in the serum of liver cirrhosis patients than HCV non-cirrhotic group. miR-143 and miR-145 expressions were significantly downregulated in the serum of HCC patients than in control group and miR-143 was significantly downregulated in the serum of non-cirrhotic HCV patients than in control group. Conclusion:The downregulation of serum let-7-a1, miR-143, and miR-145 gene expression may exhibit significant influence on the development of HCC in chronic HCV Egyptian patients and can be used as biomarkers for HCC diagnosis.

MicroRNA-30e and MicroRNA-223 Expression for Early Diagnosis of Hepatocellular Carcinoma Associated with Hepatitis C Virus

Background. The goals of this study were to elucidate the use of the expression of microRNA-30e (miR-30e) and microRNA-223 (miR-223) as diagnostic biomarkers for early diagnosis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection. Methods. The study included three groups, the first group included thirty patients with HCC associated with HCV, the second group included thirty patients with cirrhosis with HCV and the third group included thirty healthy control subjects. Blood samples were obtained for determination of serum expression of miR-30e and miR-223 by real time polymerase chain reaction. Results. There was significant decrease of miR-30e of expression in patients with HCC (0.16 ± 0.1) compared to both patients with cirrhosis (0.4 ± 0.2, P&lt;0.01) and healthy control subjects (1.2 ± 0.4, P&lt;0.001). There was also significant reduction of miR-223 expression levels in patients with HCC (0.2 ± 0.1) compared to patients with cirrhosis (0.5 ± 0.2, P&lt;0.01) and healthy control subjects (1.0 ± 0.1, P&lt;0.001). There was also significant decrease of miR-30e expression in late stage versus early stage of HCC (0.1 ± 0.0 vs. 0.22 ± 0.1, P&lt;0.001), while there was no significant difference of alpha-fetoprotein (AFP) between patients with late and early HCC. Also, values of miR-223 had significantly reduced levels in late HCC compared to its expression values in early HCC (0.1± 0.0 vs. 0.23 ± 0.2, P&lt;001). Conclusion. There was significant reduction of expression of miR-30e and miR-223 in serum of HCC patients compared to either patients with cirrhosis or healthy subjects. These results show that the combined use of both biomarkers had better sensitivity in the diagnosis of HCC compared to AFP.

Identification of Protein Expression Changes in Hepatocellular Carcinoma through iTRAQ.

Background Hepatocellular carcinoma (HCC) is a malignant tumor associated with a poor prognosis. Serum biomarkers of HCC have the potential to improve the diagnosis, provide a means to monitor the tumors, and predict their malignancy. Proteins that are expressed differentially between HCC patients and normal controls have the potential to be biomarkers. Method Serum samples from 10 confirmed HCC patients and 10 controls were collected. The differentially expressed proteins in the serum were identified using an isobaric tags for relative and absolute quantitation- (iTRAQ-) based method. Potential serum biomarkers were validated by ELISA in another 20 HCC patients and 20 controls. Their expression data in HCC were extracted from The Cancer Genome Atlas (TCGA) dataset. Results A total of 260 proteins were measured in the serum of HCC patients and compared to those in sex- and age-matched normal controls. Forty-one proteins displayed significant changes, with 26 being downregulated and 15 being upregulated. Upregulated proteins included alpha-1-antitrypsin (A1AT) and peroxiredoxin 2 (PRDX2), and downregulated proteins included paraoxonase 1 (PON1) and C-reactive protein (CRP). We then used ELISA to measure serum levels of A1AT, PRDX2, PON1, and CRP in another 20 patients with HCC and found that only PON1 levels were consistent with the iTRAQ result. In TCGA dataset, PON1 expression was downregulated in HCC tissues (P < 0.001) and low expression of PON1 was associated with poor survival in HCC patients (P < 0.001) and low expression of PON1 was associated with poor survival in HCC patients (Conclusions PON1 could act as a biomarker for HCC to assist in the diagnosis of HCC.

Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 108/cm2. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

Tumor-Derived Soluble MICA Obstructs the NKG2D Pathway to Restrain NK Cytotoxicity.

The natural killer group 2D (NKG2D) receptor and its ligands play important roles in immune surveillance. In this study, we observed that the average serum soluble MICA (sMICA) concentration of 174 hepatocellular carcinoma (HCC) patients was significantly higher than that in 80 healthy subjects (602.17 ± 338.15 vs. 72.26 ± 87.88 pg/ml, t = 3.107, P=0.002). The levels of serum sMICA in 44 HCC patients with initial levels above 400 pg/ml declined significantly after surgical removal of the liver cancer tissue (P<0.001). Moreover, the mean survival time of HCC patients who had sMICA above 400 pg/ml was significantly shorter than that HCC patients with lower sMICA levels (P<0.001). Using the reporter cell line (NKG2D-2B4) in which activation of the NKG2D receptor pathway results in GFP expression based on the stimulation of immobilized rMICA, we showed that the number of GFP-expressing cells decreased sharply in presence of sMICA. Upon adding sMICA, the release of cytokines IFN-γ, TNF-α, and IL-8 by NK cell line (NKL) under stimulation of immobilized rMICA was blocked. Using MICA-expressing cells as the target cells, we observed that about 80% of target cells were killed by NKL at E:T of 10:1, but in presence of sMICAhigh serum of HCC patients, the dead target cells were reduced to 30.8%. Compared in presence of sMICAlow serum from HCC patients, there were 63.7% of target cells dead (p=0.043). Thus, our data suggested that sMICA obstructs the activation of NKG2D pathway to protect tumor cells from NK cell-mediated cytotoxicity.

LINC00978 promotes the progression of hepatocellular carcinoma by regulating EZH2-mediated silencing of p21 and E-cadherin expression

Long non-coding RNAs (lncRNAs) have been suggested as important regulators of cancer development and progression in hepatocellular carcinoma (HCC). Nevertheless, the clinical value and biological roles of LINC00978 in HCC remain unclear. In this study, we detected the expression of LINC00978 in tumor tissues and serum of HCC patients, examined the roles of LINC00978 in HCC progression and elucidated the underlying molecular mechanisms. We found that LINC00978 expression was upregulated in tumor tissues and serum of HCC patients. Higher serum levels of LINC00978 could distinguish HCC patients from hepatitis and liver cirrhosis patients and healthy controls. LINC00978 knockdown inhibited HCC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. Overexpression of LINC00978 led to the opposite effects. LINC00978 knockdown also inhibited HCC growth and metastasis in mouse tumor models. Mechanistically, LINC00978 bound to EZH2 and mediated its accumulation at the promoter region of p21 and E-cadherin genes, leading to the trimethylation of H27K3 and the inhibition of p21 and E-cadherin expression. Moreover, the simultaneous depletion of p21 and E-cadherin expression reversed the inhibitory effects of LINC00978 knockdown on HCC cell proliferation, migration, and invasion. Taken together, these findings suggest that LINC00978 promotes HCC progression by inhibiting p21 and E-cadherin expression via EZH2-mediated epigenetic silencing. LINC00978 may represent a novel biomarker for HCC diagnosis, prognosis, and therapy.

Hepatocellular carcinoma serum derived exosomal HIF-1α induces phosphoinositide-3 kinase/protein kinase B signaling to induce the angiogenesis and proliferation of HCC.

BackgroundTumor exosomes are nanovesicles mostly secreted by tumor cells that play roles of paracrine signaling during tumor progression, including tumor-stromal interactions, activation of proliferative pathways and inducing immunosuppression. The hypoxia-inducible factor (HIF) family serve role of the principal molecular mediators of hypoxia in physiological and pathophysiological course. HIF-1α excreted from tumor and stromal cells serves an important role in tumor angiogenesis, especially in hepatocellular carcinoma (HCC). However, exosomal HIF-1α from HCC serum has not been studied extensively. The present study aimed to determine role of HIF-1α derived from HCC serum exosomes in the development of HCC.MethodsWe extracted exosomes from the serum of 48 patients suffering HCC and 24 healthy individuals. The exosomes were examined by transmission electron microscopy and nanoparticle tracking analysis. The concentrations of exosomal HIF-1α were measured by ELISA. In vitro experiments were performed to testify the exosomal role in human umbilical endothelial cells (HUVECs) and Huh-7 cells by tube formation, cell viability and western blotting analyses. Nude mouse xenograft Huh-7 tumors were generated by injecting HCC patient serum exosomes, healthy individuals’ serum exosomes and PBS into nude mice. Tumors in vivo were isolated, weighed and subjected to immunohistochemical assays.ResultsHCC patients exhibited a greater capacity to convert HIF-1α in serum exosomes than normal individuals (P=0.0007), and high serum exosomal HIF-1α levels were correlated with Barcelona Clinic Liver Cancer (BCLC) staging (P=0.031) and the phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway. The results of flow cytometry analysis demonstrated that the HUVECs cocultured with HCC exosomes turned down the percentage of HUVECs in the G1 phase but turned up the percentage of HUVECs in the S phase (P<0.05). HUVECs and Huh-7 cells cultured with HCC serum exosomes enhanced the proliferation of HUVECs and Huh-7 cells, and the tube formation of HUVECs, while exosomes from healthy individuals and the controls did not. The results demonstrated that serum exosomes from patients with HCC appeared to accelerate tumor growth compared to the control group (P<0.05) and immunohistochemical analysis revealed that the nude mice injected with exosomes from HCC patient serum exhibited higher microvessel density (MVD) (P<0.05).ConclusionsHCC serum derived exosomes could induce angiogenesis and the proliferation of HCC potentially via the HIF-1α-PI3K/AKT signaling pathway, it making the prevention and treatment of HCC angiogenesis and proliferation available.

Evaluation of serum long noncoding RNA NEAT and MiR-129-5p in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent form of cancer. Various long non coding RNA (lncRNAs) and micro RNA have been confirmed to have a role in the progression of HCC. Our aim was to investigate for the first time the expression profile of serum level of LNC NEAT (nuclear enrich abundant transcript) and MiR-129-5p in HCC patients and their relations with patient's clinical and biochemical investigations rather than previous studies on tissue cell lines. Our study includes 72 subjects divided into 36 as control subjects and 36 patients with HCC. Complete physical and laboratory investigations were done on all subjects. RNAs were extracted from sera of all subjects. RNAs were reversed transcribed into cDNAs using Qiagen, Valenica, CA. Quantitative PCR (qPCR) was performed using Rotor gene Q System (Qiagen). Relative NEAT1 expression level was significantly increased in serum of HCC patients 4.7 (1.31-6.82) (p < .0001). Meanwhile MiR-129-5p relative expression level was significantly decreased in serum of HCC patients 0.17 (0.14-20) (p < .0001). Also there was negative significant correlation between the expression level of LNC NEAT and MiR-129-5p in HCC group (p < .0001). ROC curve analysis revealed that LNC NEAT; AUC = 0.981, p < .0001, cutoff value (1.02), sensitivity 100%, specificity 88.9%. MiR-129-5p; AUC = 0.997, p < .0001, cutoff value (0.43), sensitivity 100%, specificity 97.2%. Serum LNC NEAT and MiR-129-5p could be used as potential biomarkers for HCC cancer diagnosis and prognosis.

A comparative characterization of the circulating miRNome in whole blood and serum of HCC patients

miRNAs are considered promising non-invasive biomarkers. Serum represents the major source of biomarkers, being readily accessible for many analytical tests. Recently, whole blood has drawn increasing interest in biomarker studies due to the presence of cancer-interacting cells and circulating cancer cells. Although Hepatocellular Carcinoma (HCC) is the seventh most frequent cancer worldwide, fragmented information exists regarding the miRNome characterization in blood and serum. We profiled the circulatory miRNome of paired serum and blood samples from 20 HCC patients, identifying 274 miRNA expressed in serum and 670 in blood, most of them still uncharacterized. 157 miRNA significantly differ between the two biofluids with 28 exclusively expressed in serum. Six miRNA clusters significantly characterize the two compartments, with the cluster containing miR-4484, miR-1281, miR-3178, miR-3613-3p, miR-4532, miR-4668-5p, miR-1825, miR-4487, miR-455-3p, miR-940 having the highest average expression in serum compared to blood. The ontological analysis revealed a role of these miRNAs in cancer progression, vascular invasion and cancer immune surveillance thought the regulation of DUSP1, PD-L1 and MUC1. Taken together, these results provide the most comprehensive contribution to date towards a complete miRNome profile of blood and serum for HCC patients. We show a consistent portion of circulatory miRNAs being still unknown.

MicroRNA-1277 Inhibits Proliferation and Migration of Hepatocellular Carcinoma HepG2 Cells by Targeting and Suppressing BMP4 Expression and Reflects the Significant Indicative Role in Hepatocellular Carcinoma Pathology and Diagnosis After Magnetic Resonance Imaging Assessment.

Our study aimed to investigate the roles and possible regulatory mechanism of miR-1277 in the development of hepatocellular carcinoma (HCC). HCC patients were identified from patients who were diagnosed with focal liver lesions using magnetic resonance imaging (MRI). The expression levels of miR-1277 in the serum of HCC patients and HepG2 cells were measured. Then miR-1277 mimic, miR-1277 inhibitor, or scramble RNA was transfected into HepG2 cells. The effects of miR-1277 overexpression and suppression on HepG2 cell proliferation, migration, and invasion were then investigated. Additionally, the expression levels of epithelial–mesenchymal transition (EMT)-related markers, including E-cadherin, β-catenin, and vimentin, were detected. Target prediction and luciferase reporter assay were performed to explore the potential target of miR-1277. miR-1277 was significantly downregulated in the serum of HCC patients and HepG2 cells. Suppression of miR-1277 promoted HepG2 cell proliferation, migration, and invasion, whereas overexpression of miR-1277 had opposite effects. In addition, after miR-1277 was suppressed, the expressions of E-cadherin and β-catenin were significantly increased, while the expressions of vimentin were markedly decreased. Bone morphogenetic protein 4 (BMP4) was identified as the direct target of miR-1277. Knockdown of BMP4 reversed the effects of miR-1277 suppression on HepG2 cell migration and invasion, as well as the expressions of E-cadherin, β-catenin, and vimentin. Our results indicate that downregulation of miR-1277 may promote the migration and invasion of HepG2 cells by targeting BMP4 to induce EMT. Combination of MRI and miR-1277 level will facilitate the diagnosis and treatment of HCC.

Oncogenic Wnt3a: A Candidate Specific Marker and Novel Molecular Target for Hepatocellular Carcinoma.

Background and aim: It is of the utmost importance for the specific diagnosis and effective therapy of hepatocellular carcinoma (HCC). Abnormality of oncogenic Wingless-type MMTV integration site family member 3a (Wnt3a) has been associated with progression of HCC. In this study, we aimed to evaluate Wnt3a as a novel biomarker and target for HCC.Methods: Circulating Wnt3a levels were quantitatively detected in a cohort of chronic liver diseases by an enzyme-linked immune-absorbent assay (ELISA). Hepatic Wnt3a expression in HCC and para-cancerous tissues was analyzed by immunohistochemistry. Prognostic value of Wnt3a for HCC was discovered in the cohort from the Cancer Genome Atlas (TCGA). Dynamic alterations of Wnt3a levels were detected in the hepatocarcinogenesis model induced by 2-acetylaminofluorene. Effects of Wnt3a on biological behaviors were evaluated in vitro and in vivo based on Crispr/Cas9.Results: Up-regulated Wnt3a levels were observed in serum of HCC patients with high specificity and sensitivity for HCC diagnosis. Combination of Wnt3a and AFP could improve sensitivity to 93.9% in serological detection. In addition, Wnt3a expression in HCC tissues was significantly higher than that in para-cancerous tissues. The cohort of TCGA demonstrated that high Wnt3a expression led to a poor survival of HCC patients, especially in cases at advanced stages. Furthermore, the hepatocarcinogenesis model showed that Wnt3a dynamically increased in the development of HCC. Functionally, silencing Wnt3a by Crispr/Cas9 suppressed the proliferation, colony formation, induced cell cycle arrest of HCC cells by de-activating Wnt/β-catenin pathway in vitro, and inhibited xenograft tumor growth in vivo.Conclusions: Oncogenic Wnt3a could be considered as a candidate biomarker and novel target for HCC.

Alterations of glutamine and glutamate levels in patients and rats with hepatocellular carcinoma

The alteration of glutamine (Gln) and glutamate (Glu) levels in hepatocellular carcinoma (HCC) has yet to be thoroughly investigated. In this study, 28 HCC patients and 33 healthy people were enrolled. HCC in rats were induced by diethylnitrosamine. Human and rat sera and the extract of rat liver tissues were derivatized with phenylisothiocyanate (PITC) and then separated by an ACQUITY BEH C18 column with 0.1% formic acid and methanol as mobile phase. The UPLC-UV method showed high specificity. Gln and Glu concentrations ranging within 15.6–500 µg/mL showed good linear relationship with the peak area. The average recoveries were 85%–115%. Compared with those in the healthy group, Gln levels in the sera of HCC patients and HCC rats significantly (p < 0.05) decreased, whereas Glu levels increased. In HCC rats, Gln levels in the serum decreased by 36.3%, whereas Gln levels in the liver increased by 33.4%, with no statistical difference in the Gln/Glu ratio. Our results indicated that changes in Gln transporter activity may be closely related to the occurrence and development of HCC, which contributes to the HCC prevention and therapy and thus warrant further study.