Combined identification of three lncRNAs in serum as effective diagnostic and prognostic biomarkers for hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) is a common, highly invasive malignant tumor associated with a high mortality rate. This study aimed to identify the effective diagnostic and prognostic biomarkers for HBV-related HCC. With HBV-related HCC RNA-sequencing data of The Cancer Genome Atlas (TCGA) database, 159 differentially expressed long noncoding RNAs (lncRNAs) between HBV-related HCC and para-carcinoma normal samples were identified, and 12 lncRNAs were eventually assessed for deeper research. Classification analysis developed a three-lncRNA signature of AC005332.5, ELF3-AS1 and LINC00665, which was demonstrated to be the most discriminatory with an AUC (Area under the curve) value of 0.913 (95% CI: 0.8610-0.9665) and verified in validation patients. The expression levels of AC005332.5, ELF3-AS1 and LINC00665 were significantly changed with different tumor stages or grades. Survival analysis revealed that AC005332.5, ELF3-AS1 and LINC00665 were highly associated with the prognosis of overall survival. Additionally, the lncRNA signature yielded statistical significance to predict clinical outcomes independently from other clinical variables in validation patients, as suggested in the multivariate Cox hazards analysis. Conclusively, a three-lncRNA signature of AC005332.5, ELF3-AS1 and LINC00665 may serve as an excellent diagnostic biomarker for HBV-related HCC and potential prognostic significance for HBV-related HCC sufferers.

Fabrication of avidin-stabilized gold nanoclusters with dual emissions and their application in biosensing.

Protein-stabilized gold nanoclusters (Prot-Au NCs) have been widely used in biosensing and cell imaging owing to their excellent optical properties and low biotoxicity. However, several Prot-Au NCs reported in the literature do not retain the biological role of the protein, which greatly limits their ability to directly detect biomarkers. This study demonstrated for the first time the successful synthesis of dual-function avidin-stabilized gold nanoclusters (Av-Au NCs) using a one-pot method. The resulting Av-Au NCs exhibited intense blue and red emissions under 374 nm excitation. Furthermore, the Av-Au NCs retained the native functionality of avidin to bind to biotin. When DNA strands modified with biotin at both ends (i.e., linker chains) were mixed with Av-Au NCs, large polymers were formed, indicating that Av-Au NCs could achieve fluorescence signal amplification by interacting with biotin. Taking advantage of the aforementioned properties, we constructed a novel enzyme-free fluorescent biosensor based on the Av-Au NCs-biotin system to detect DNA. The designed fluorescent biosensor could detect target DNA down to 0.043 nM, with a wide line range from 0.2 nM to 20 µM. Thus, these dual-functional Av-Au NCs were shown to be an excellent fluorescent material for biosensing.

Long noncoding RNA CCAT2 promotes hepatocellular carcinoma proliferation and metastasis through up-regulation of NDRG1.

BACKGROUND: Emerging studies demonstrate that long noncoding RNAs (lncRNAs) play crucial roles in hepatocarcinogenesis through various mechanisms. LncRNA CCAT2 was a newly discovered lncRNA and amplified in several cancers. However, the mechanisms involved in function of CCAT2 in hepatocellular carcinoma (HCC) remain to be explored. METHODS: CCAT2 expressions in HCC tissues and cell lines were measured by RT-qPCR. MTS assay, colony formation assay, wound-healing assay and transwell assay were used to explore the biological functions of CCAT2 on HCC cells proliferation and metastasis. Experiments in vivo were carried out to confirm these effects. The underlying mechanisms were analyzed by western blot and dual-luciferase reporter assay. RESULTS: In this study, we found that CCAT2 were significantly elevated in HCC tissues and cell lines, and it promoted HCC cells proliferation and metastasis both in vitro and in vivo. Additionally, we identified that NDRG1 was a downstream target of CCAT2. Meanwhile, depletion of CCAT2 inhibited cellular proliferation and metastasis behaviors induced by NDRG1- overexpression. Analysis of mechanism underlying these effects revealed that CCAT2 increased the expression of NDRG1 by enhancing its promoter activity. Furthermore, the active region between CCAT2 and NDRG1 promoter was confirmed by dual-luciferase reporter assay. CONCLUSIONS: All these observations demonstrate that CCAT2 acts as an oncogene by up-regulating NDRG1, which may have the potential to be used as a promising prognostic biomarker and therapeutic target for HCC.

LINC00628 suppresses migration and invasion of hepatocellular carcinoma by its conserved region interacting with the promoter of VEGFA.

Accumulated evidence revealed that numerous long noncoding RNAs (lncRNAs) have been found to be involved in the development and progression of hepatocellular carcinoma (HCC). LINC00628, a member of lncRNAs, has been reported to act as a tumor suppressor in gastric cancer and breast cancer. However, its potential role in HCC still remains unknown. Herein, we characterized the function of LINC00628 in HCC. Our investigation has revealed that LINC00628 were dramatically decreased in HCC tissues and cells, and inhibited the migration and invasion of HCC cells in vitro and in vivo. Moreover, LINC00628 exerted its tumor suppressive function by repressing the vascular endothelial growth factor A (VEGFA) promoter activity. A highly conserved region element in LINC00628 was identified by a cross-species comparative analysis, which is required for LINC00628 exerted its function. Dual-luciferase reporter assay showed that the conserved sequence mediated the interaction with a specific region of VEGFA promoter, resulting in a decrease of VEGFA expression. In conclusion, our results demonstrated that LINC00628 could function as a tumor suppressor in HCC via its conserved sequence elements interacting with a particular region of VEGFA promoter, suggesting that LINC00628 may serve as a novel promising target for diagnosis and therapy in HCC.

Rap1b enhances the invasion and migration of hepatocellular carcinoma cells by up-regulating Twist 1.

Rap1b was found be dysregulated in several types of cancers. Previously, we have demonstrated that Rap1b affects proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells. However, the definite function of Rap1b in HCC remains unknown. Here, we reported that Rap1b was significantly up-regulated in HCC tissues compared with the non-tumoral liver tissues. Overexpression of Rap1b promoted tumor growth and migration in vitro and tumor formation in vivo. Oppositely, inhibition of Rap1b suppressed the proliferation and migration of HCC cells. Mechanism study revealed that Rap1b could up-regulate Twist 1 expression by enhancing its promoter activity. We concluded that Rap1b increased Twist 1 expression by targeting its promoter activity to induce proliferation and migration of HCC cells.

Long-Noncoding RNA Colorectal Neoplasia Differentially Expressed Gene as a Potential Target to Upregulate the Expression of IRX5 by miR-136-5P to Promote Oncogenic Properties in Hepatocellular Carcinoma.

BACKGROUND/AIMS: The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene was first found to be activated in colorectal neoplasia. Now, it also has been found to be upregulated in many other solid tumors. Whether CRNDE affects tumorigenesis remains unknown. METHODS: We conducted bioinformatics, real-time polymerase chain reaction (PCR), Western blot analysis, cell proliferation assay, colony formation assay, wound healing assay, cell migration and invasion assays, RNA immunoprecipitation, and reporter vector construction and luciferase assays. RESULTS: CRNDE was upregulated in hepatocellular carcinoma (HCC). The overexpression of CRNDE promoted HCC cellular proliferation, migration, and invasion in intro and in vivo, and acted as an oncogene in HCC progression. Furthermore, CRNDE impaired miR-136-5P expression in a RISC manner, and a reciprocal repression feedback loop was possible between CRNDE and miR-136-5P. We found that the neighboring mRNA of CRNDE was IRX5, and IRX5 increased the tumorigenicity of HCC cells. IRX5 was a potential downstream target gene of miR-136-5P. MiR-136 regulated IRX5 by interacting with its 3'UTR. In addition, miR-136-5P was involved in the CRNDE-regulated expression of IRX5. CONCLUSION: CRNDE acted as a tumor oncogene by exhibiting oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5P-IRX5 regulatory network in HCC. CRNDE may be considered to be a potential target for HCC therapies based on its ability to upregulate IRX5, and it deserves further investigation.

Comprehensive Analysis of circRNA, lncRNA, miRNA and mRNA Expression Profiles and Their Competing Endogenous RNA Networks in Hepatitis B Virus-Related Hepatocellular Carcinoma.

Pursuing knowledge about circular RNA (circRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression profiles and their competing endogenous RNA (ceRNA) networks in hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) was the focus of this research. Expression patterns of circRNAs, lncRNAs, miRNAs, and mRNAs were searched for in relation to HBV-related HCC using whole-transcriptome sequencing. The expression levels of chosen circRNA, lncRNA, miRNA, and mRNA were analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The potential connections and roles of ceRNA were deduced via bioinformatics research. The sum of 284 circRNAs, 2,927 lncRNAs, 693 miRNAs, and 5566 mRNAs were discovered to be expressed at considerably different levels in HBV-related HCC tissue and adjacent normal tissue. And the most significantly up- and down-regulated circRNAs, lncRNAs, miRNAs, and mRNAs were verified in HBV-related HCC by qRT-PCR. The circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks of HBV-related HCC were established, and the ceRNA regulatory networks revealed the gene expression mechanisms controlled by ncRNAs. Collectively, we revealed the contribution of various circRNA, lncRNA, miRNA, and mRNA expression profiles and identified their ceRNA regulatory networks in HBV-related HCC, providing a theoretical basis for further exploration.

Horseradish peroxidase-catalyzed polyacrylamide gels: monitoring their polymerization with BSA-stabilized gold nanoclusters and their functional validation in electrophoresis.

Polyacrylamide gel (PAG) is extensively used as a matrix for biomolecular analysis and fractionation. However, the traditional polymerization catalyst system N,N,N',N'-tetramethylethylenediamine (TEMED)/ammonium persulphate (APS) of PAG presents non-negligible toxicity. Herein, we utilized the green and efficient bio-enzyme horseradish peroxidase (HRP) to catalyze the gel polymerization of polyacrylamide. At the same time, the efficacy of this gel system in separating nucleic acids and proteins was confirmed by applying the gel system in electrophoresis. This study aims to explore a higher biosafety polyacrylamide gel polymerization catalytic system which can be applied to electrophoresis technology. Furthermore, in order to differentiate between the bio-enzymatic catalytic system and the traditional toxic catalytic system during polymerization, aggregation-induced luminescence (AIE) of bovine serum albumin-stabilized gold nanoclusters (BSA-Au NCs) was used to monitor the polymerization reaction of the system. The results indicated that the fluorescence intensity of the polymeric system containing BSA-Au NCs increased with the polymerization of the monomers. Subsequently, we assessed whether certain components of nucleic acid electrophoresis and protein electrophoresis such as sodiumdodecylsulfate (SDS) and TBE buffer (Tris-boric acid, EDTA, pH 8.3) would affect the polymerization of the polyacrylamide gels catalyzed by the biological enzymes. The experimental conditions were also optimized to explore the optimal concentration of the ternary system of HRP, H2O2 and ACAC. Our results suggested that the bioenzyme-catalyzed system could be a feasible alternative to the TEMED/APS-catalyzed system, which also could provide new insights into the methods of monitoring the polymerization system.

Increased Platelet Distribution Width Predicts 3-Year Recurrence in Patients with Hepatocellular Carcinoma After Surgical Resection.

Background: Platelet distribution width (PDW) is a marker of platelet anisocytosis that increases with platelet activation. The clinical implications of PDW in HCC are not well-defined. This study aimed to determine whether PDW could predict recurrence in patients with HCC after resection. Methods: Between January and December 2008, 471 patients with HCC were recruited retrospectively. The clinicopathological characteristics of patients with HCC were analyzed based on the relationship between the two PDW groups. Kaplan-Meier curves and multivariate Cox regression analyses were used to evaluate the relationship between PDW and disease-free survival (DFS). A novel nomogram was developed based on the identified independent risk factors. Its accuracy was evaluated using a calibration curve and concordance index. The predictive value was evaluated using a receiver operating characteristic (ROC) curve. Results: PDW was significantly associated with direct bilirubin, total bilirubin, urea, and prothrombin time. Patients with PDW ≥ 17.1 were a significantly shorter DFS than those with PDW < 17.1 (17.98% vs 49.83%, p< 0.001). Multivariate analysis determined that alpha-fetoprotein (AFP), carcinoembryonic antigen, microvascular invasion (MVI), tumor size, and tumor number were the independent variables associated with DFS. Patients with PDW ≥ 17.1 had a hazard ratio of 1.381 (95% confidence interval: 1.069-1.783, p = 0.014) for DFS. AFP, PDW, MVI, tumor size, and tumor number were identified as preoperative independent risk factors for DFS and used to establish the nomogram. Calibration curve analysis revealed that the standard curve fitted well with the predicted curve. ROC curve analysis demonstrated the high efficiency of the nomogram. Conclusion: Increased PDW may predict recurrence-free survival in patients with HCC. Our nomogram model also performed well in predicting patient prognoses.

Long noncoding RNA MIR31HG inhibits hepatocellular carcinoma proliferation and metastasis by sponging microRNA-575 to modulate ST7L expression.

BACKGROUND: Emerging evidences have indicated that long noncoding RNAs (lncRNAs) play essential roles in the development and progression of cancers. Dysregulation of lncRNA MIR31HG has recently been reported in several types of cancers, and researches on the function of MIR31HG in cancers suggested that MIR31HG could act as either oncogene or tumor suppressor. But the functional involvement of MIR31HG has not been studied in hepatocellular carcinoma (HCC). METHODS: In this study, MTS assays, colony formation assay, Wound-healing assay, Transwell assy, and tumor xenografts experiments were used to identify biological effects of MIR31HG on HCC cells HCC proliferation and metastasis in vitro and in vivo. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to show the interactions of MIR31HG and miR-575. The bioinformatics methods were completed to find the target genes of miR-575. And Dual-luciferase reporter assay and Western blot analysis were further used to confirm the target gene of miR-575. RESULTS: We found that overexpression of MIR31HG obviously suppressed HCC proliferation and metastasis in vitro and in vivo, whereas knockdown of MIR31HG had the opposite effects. Besides, overexpression of MIR31HG significantly decreased the expression of microRNA-575 (miR-575), which plays an oncogenic role in HCC. Moreover, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay revealed that MIR31HG exerted tumor-suppressive functions by binding directly to miR-575, and there was a reciprocal inhibition between MIR31HG and miR-575 in the same RNA-induced silencing complex (RISC). Furthermore, overexpression of MIR31HG enhanced the expression of suppression of tumorigenicity 7 like (ST7L), which was identified as a downstream target gene of miR-575. Thus, MIR31HG positively regulated ST7L expression through sponging miR-575, and acted as tumor suppressor in HCC. CONCLUSIONS: Overall, our study illuminates the role of MIR31HG as a miRNA sponge in HCC, and sheds new light on lncRNA-directed diagnostics and therapeutics in HCC.

LINC00052/miR-101-3p axis inhibits cell proliferation and metastasis by targeting SOX9 in hepatocellular carcinoma.

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in a variety of diseases, including many tumors, such as hepatocellular carcinoma (HCC). However, the function and mechanisms responsible for these molecules in HCC are not thoroughly understood. In our previous study, we found that LINC00052 was acted as a tumor suppressor in HCC. In this study, we performed transcription microarray analysis to investigate the target gene of LINC00052, and found that knockdown of LINC00052 significantly increased the expression of SRY-related HMG-box gene 9 (SOX9), which plays an oncogenic role in HCC. Moreover, luciferase reporter assay revealed that LINC00052 promoted miR-101-3p expression by enhancing its promoter activity. In addition, online database analysis tools and luciferase assays showed that miR-101-3p could target SOX9. Quantitative real-time polymerase chain reaction (qRT-PCR) demonstrated that miR-101-3p was downregulated in HCC tissues and HCC cell lines. And we found a positive relationship between LINC00052 and miR-101-3p, and a negative relationship between miR-101-3p and SOX9 in HCC tissues. Besides, miR-101-3p was involved in LINC00052 inhibits HCC cells proliferation and metastasis. At the molecular level, LINC00052 downgulated SOX9 to inhibit HCC cells proliferation and metastasis by interacting with miR-101-3p. It might be a potential application for HCC therapy.

LINC00052 upregulates EPB41L3 to inhibit migration and invasion of hepatocellular carcinoma by binding miR-452-5p.

Numerous studies have demonstrated that a class of long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma (HCC) and they are closely related with tumorigenesis. Our previous studies indicated that LINC00052 was a downregulated lncRNA in HCC and acted as a tumor suppressor gene. Using transcription microarray analysis, we found that knockdown of LINC00052 resulted in EPB41L3 downregulation. However, the function of EPB41L3 and the mechanism of LINC00052 downregulating EPB41L3 in HCC remain unclear. In this study, we found that overexpression of LINC00052 could upregulate the EPB41L3 expression and it might serve as a tumor suppressor gene in HCC. Database analysis showed that miR-452-5P could target LINC00052. The binding regions between LINC00052 and miR-452-5P were confirmed by luciferase assays. Moreover, LINC00052 inhibited cell malignant behavior by increasing miR-452-5P expression, suggesting that LINC00052 was negatively regulated by miR-452-5P. In addition, overexpression of miR-452-5P resulted in a decrease of EPB41L3 expression, suggesting that EPB41L3 was as a target of miR-452-5P. In conclusion, these results demonstrated that a novel pathway was mediated by LINC00052 in HCC.

Differential expression of microRNAs in plasma of patients with prediabetes and newly diagnosed type 2 diabetes.

AIMS: MicroRNAs (miRNAs) are present in plasma and have emerged as critical regulators of gene expression at posttranscriptional level, and thus are involved in various human diseases, including diabetes. The objective of this study was to screen and validate differentially expressed plasma miRNAs in prediabetes and newly diagnosed type 2 diabetes (T2D). METHODS: In this study, we screened differentially expressed plasma miRNAs in prediabetes and newly diagnosed T2D by miRNA microarray analysis, and validated the expression of candidate miRNAs using quantitative reverse transcription polymerase chain reaction assays. Furthermore, we performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses to disclose functional enrichment of genes predicted to be regulated by the differentially expressed miRNAs. RESULTS: Notably, our results revealed that hsa-miR-1249, hsa-miR-320b, and hsa-miR-572 (P < 0.05) were differentially expressed among the three groups, which yielded an area under the receiver operator characteristics curve (AUC) of 0.784 [95 % confidence interval (CI) 0.685-0.883], 0.946 (95 % CI 0.906-0.985), and 0.843 (95 % CI 0.766-0.920) discriminating T2D patients from NGT control groups, respectively, while the AUC was 0.887 (95 % CI 0.818-0.957), 0.635 (95 % CI 0.525-0.744), and 0.69 (95 % CI 0.580-0.793) discriminating prediabetes patients from NGT control groups, respectively. In addition, GO and KEGG pathway analyses showed that genes predicted to be regulated by differentially expressed miRNAs were significantly enriched in several related biological processes and pathways, including the development of multicellular organisms, signal transduction, cell differentiation, apoptosis, cell metabolism, ion transport regulation, and other biological functions. CONCLUSIONS: Taken together, our results showed differentially expressed miRNAs in T2D and prediabetes. Plasma hsa-miR-1249, hsa-miR-320b, and hsa-miR-572 may serve as novel biomarkers for diagnosis and potential targets for the treatment for prediabetes and T2D.