Yu-zhao Wang

A Long Noncoding RNA Activated by TGF-β Promotes the Invasion-Metastasis Cascade in Hepatocellular Carcinoma

The role of TGF-β-induced epithelial-mesenchymal transition (EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs in TGF-β signaling is still unknown. In this study, we observed that the lncRNA-activated by TGF-β (lncRNA-ATB) was upregulated in hepatocellular carcinoma (HCC) metastases and associated with poor prognosis. lncRNA-ATB upregulated ZEB1 and ZEB2 by competitively binding the miR-200 family and then induced EMT and invasion. In addition, lncRNA-ATB promoted organ colonization of disseminated tumor cells by binding IL-11 mRNA, autocrine induction of IL-11, and triggering STAT3 signaling. Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, these findings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCC patients to metastases and may serve as a potential target for antimetastatic therapies.

Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans

In recent years, long noncoding RNAs (lncRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of lncRNAs to hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) remain largely unknown. Differentially expressed lncRNAs between HBV‐related HCC and paired peritumoral tissues were identified by microarray and validated using quantitative real‐time polymerase chain reaction. Liver samples from patients with HBV‐related HCC were analyzed for levels of a specific differentially expressed lncRNA High Expression In HCC (termed lncRNA‐HEIH); data were compared with survival data using the Kaplan‐Meier method and compared between groups by the log‐rank test. The effects of lncRNA‐HEIH were assessed by silencing and overexpressing the lncRNA in vitro and in vivo. The expression level of lncRNA‐HEIH in HBV‐related HCC is significantly associated with recurrence and is an independent prognostic factor for survival. We also found that lncRNA‐HEIH plays a key role in G0/G1 arrest, and further demonstrated that lncRNA‐HEIH was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of EZH2 target genes. Conclusions: Together, these results indicate that lncRNA‐HEIH is an oncogenic lncRNA that promotes tumor progression and leads us to propose that lncRNAs may serve as key regulatory hubs in HCC progression. (HEPATOLOGY 2011

Repression of the miR‐17‐92 cluster by p53 has an important function in hypoxia‐induced apoptosis

We here report that miR‐17‐92 cluster is a novel target for p53‐mediated transcriptional repression under hypoxia. We found the expression levels of miR‐17‐92 cluster were reduced in hypoxia‐treated cells containing wild‐type p53, but were unchanged in hypoxia‐treated p53‐deficient cells. The repression of miR‐17‐92 cluster under hypoxia is independent of c‐Myc. Luciferase reporter assays mapped the region responding to p53‐mediated repression to a p53‐binding site in the proximal region of the miR‐17‐92 promoter. Chromatin immunoprecipitation (ChIP), Re‐ChIP and gel retardation assays revealed that the binding sites for p53‐ and the TATA‐binding protein (TBP) overlap within the miR‐17‐92 promoter; these proteins were found to compete for binding. Finally, we show that pri‐miR‐17‐92 expression correlated well with p53 status in colorectal carcinomas. Over‐express miR‐17‐92 cluster markedly inhibits hypoxia‐induced apoptosis, whereas blocked miR‐17‐5p and miR‐20a sensitize the cells to hypoxia‐induced apoptosis. These data indicated that p53‐mediated repression of miR‐17‐92 expression likely has an important function in hypoxia‐induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

MicroRNA‐23a Antisense Enhances 5‐Fluorouracil Chemosensitivity Through APAF‐1/Caspase‐9 Apoptotic Pathway in Colorectal Cancer Cells

Current literature provided information that alteration in microRNA expression impacted sensitivity or resistance of certain tumor types to anticancer treatment, including the possible intracellular pathways. The microRNA‐23a (miR‐23a)‐regulated apoptosis in response to the 5‐fluorouracil (5‐FU)‐induced mitochondria‐mediated apoptotic pathway was determined in this study. The miR‐23a expression in 5‐FU‐treated and untreated colon cancer cells and tissues was assessed using real‐time PCR analysis. To determine the function of miR‐23a in the regulation of 5‐FU‐induced apoptosis, cell‐proliferation, cytotoxicity, and apoptosis analyses were performed. Dual luciferase reporter assay was used to identify the apoptosis‐related target gene for miR‐23a. The activity of caspases‐3, ‐7, and ‐9 were also assessed in miR‐23a antisense and 5‐FU treated tumor cells. A xenograft tumor model was established to evaluate the biological relevance of altered miR‐23a expression to the 5‐FU‐based chemotherapy in vivo. We found that the expression of miR‐23a was increased and the level of apoptosis‐activating factor‐1 (APAF‐1) was decreased in 5‐FU‐treated colon cancer cells compared to untreated cells. The activation of the caspases‐3 and 7 was increased in miR‐23a antisense and 5‐FU‐treated colon cancer cells compared to negative control. APAF‐1, as a target gene of miR‐23a, was identified and miR‐23a antisense‐induced increase in the activation of caspase‐9 was observed. The overexpression of miR‐23a antisense up‐regulated the 5‐FU induced apoptosis in colon cancer cells. However, the miR‐23a knockdown did not increase the antitumor effect of 5‐FU in xenograft model of colon cancer. This study shows that miR‐23a antisense enhanced 5‐FU‐induced apoptosis in colorectal cancer cells through the APAF‐1/caspase‐9 apoptotic pathway. J. Cell. Biochem. 115: 772–784, 2014.

Characterization of the genotype and integration patterns of hepatitis B virus in early‐ and late‐onset hepatocellular carcinoma

Early‐onset hepatocellular carcinoma (HCC) accounts for 15%‐20% of total HCC cases in Asia, and the incidence is increasing. The low frequency of cirrhosis and poor prognosis of early‐onset HCC suggests that its mechanisms may differ from late‐onset HCC. Although hepatitis B virus (HBV) infection is epidemiologically associated with HCC, the role of HBV in early‐onset HCC remains poorly understood. Here, we report a comparative study of HBV subgenotypes and integration in early‐ (≤30) and late‐onset (≥70) HBV‐associated HCC using a novel high‐throughput viral integration detection method. We report that HBV B2 is predominantly present in early‐onset HCC. HBV integration is a common phenomenon, both in early‐ and late‐onset HCC, which favors integrating into human repeat regions. Moreover, we found a breakpoint in 8q24 located between c‐Myc and plasmocytoma variant translocation 1 (PVT1), which was detected in 12.4% (14 of 113) of early‐onset HCCs, but only 1.4% (2 of 145) in late‐onset HCCs. HBV integrating this site results in c‐MYC, PVT1, and microRNA‐1204 overexpression in tumors, thereby potentially contributing to the development of early‐onset HCC. Conclusion: HBV genotype and integration patterns may be distinct in early‐onset HCC. Our results may shed light on HCC risk factors in young HBV carriers. Further studies are needed to elucidate at which time in tumor development this integration event occurs and whether it plays an important, causative role in HCC development or progression. (Hepatology 2015;61:1821‐1831)

A novel, cheap and effective fusion expression system for the production of recombinant proteins

To develop faster, less expensive methods for expression and purification of proteins, the annexin B1-intein fusion expression system was constructed. The interest proteins fused to the annexin B1-intein tag were purified in a single-step method based on the Ca2+-binding activity of annexin B1, and the annexin B1-intein fusion tag was removed based on the self-cleaving activity of the intein. Moreover, we found that in some cases, fusion to annexin B1 can promote the solubility of heterologous proteins. The production of soluble and highly active of interleukin-2 and low-molecular single-chain urokinase in our results proved that the system was a novel, cheap and effective fusion expression system for the production of valuable soluble recombinant proteins in Escherichia coli.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.