👤 Yanting Lan

Nasopharyngeal carcinoma (NPC) is one of the most common cancers. To investigate the gene mutation profile of NPC patients, we performed whole exome sequencing (WES) in tumor cells, peripheral blood cells, and circulating tumor cells (CTCs) of primitive and metastatic NPC patients, and explored its clinical significance. Primitive tumor cells, white blood cells, and CTCs of patients were collected and hybridized with probes targeting whole exons. Mutational signatures, signaling pathways, and cancer associated genes from CTCs cells of two primitive and two metastatic patients were analyzed using gene ontology (GO) method. The mutational landscape of four primitive tumors showed that there were more These changes are strongly relevant to their clinical characteristics and therapeutic strategy. Show less

GBM (glioblastoma multiforme) is the most common and aggressive brain tumor with no curative options available. Therefore, it is imperative to develop novel potent therapeutic drugs for GBM treatment. Here, we show that regorafenib, an oral multi-kinase inhibitor, exhibits superior therapeutic efficacy over temozolomide, the first-line chemotherapeutic agent for GBM treatment both Show less

Prostate cancer is the most common malignancy in men in developed countries. Overexpression of enhancer of zeste homolog 2 (EZH2), the major histone H3 lysine 27 methyltransferase, has been connected to prostate cancer malignancy. However, its downstream genes and pathways have not been well established. Here, we show tumor suppressor Hepatocyte Nuclear Factor 1β (HNF1B) as a direct downstream target of EZH2. EZH2 binds HNF1B locus and suppresses HNF1B expression in prostate cancer cell lines, which is further supported by the reverse correlation between EZH2 and HNF1B expression in clinical samples. Consistently, restored HNF1B expression significantly suppresses EZH2-mediated overgrowth and EMT processes, including migration and invasion of prostate cancer cell lines. Mechanistically, we find that HNF1B primarily binds the promoters of thousands of target genes, and differentially regulates the expression of 876 genes. We also identify RBBP7/RbAP46 as a HNF1B interacting protein which is required for HNF1B-mediated repression of SLUG expression and EMT process. Importantly, we find that higher HNF1B expression strongly predicts better prognosis of prostate cancer, alone or together with lower EZH2 expression. Taken together, we have established a previously underappreciated axis of EZH2-HNF1B-SLUG in prostate cancer, and also provide evidence supporting HNF1B as a potential prognosis marker for metastatic prostate cancer. Show less

Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation. Show less

In our preliminary screening, expression of miR-338-5p was found to be higher in primary colorectal cancer (CRC) with metastasis. The autophagy related gene- phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3) appeared to be targeted by miR-338-5p. Here, we provide solid evidence in support of PIK3C3 involved in miR-338-5p related metastasis of CRC in vitro and in vivo. The potential clinical relevance of miR-338-5p and its target gene was analysed on benign colorectal polyps and primary CRCs by QPCR. Mouse spleen xenograft experiment was performed to examine the importance of miR-338-5p for metastasis. PIK3C3 was one of target genes of miR-338-5p. In primary CRCs, expression of miR-338-5p is positively related to tumour staging, distant metastasis and poor patient survival. Patients with higher ratios of miR-338-5p/PIK3C3 also had significantly poor overall survival, supporting their significance in the progression of CRC. Over-expression of miR-338-5p promotes CRC metastasis to the liver and lung in vivo, in which PIK3C3 was down-regulated in the metastatic tumours. In contrast, overexpression of PIK3C3 in miR-338-5p stable cells inhibited the growth of metastatic tumours. Both migration and invasion of CRC in vitro induced by miR-338-5p are mediated by suppression of PIK3C3. Using forward and reverse approaches, autophagy was proved to involve in CRC migration and invasion induced by miR-338-5p. MiR-338-5p induces migration, invasion and metastasis of CRC in part through PIK3C3-related autophagy pathway. The miR-338-5p/PIK3C3 ratio may become a prognostic biomarker for CRC patients. FUND: NCKU Hospital, Taiwan, Ministry of Science and Technology, Taiwan. Show less

Melanocortin 4 receptor: (MC4R) and Myostatin (MSTN) are two important growth trait-related genes in animals. In this study, we showed that two SNPs, MC4R-719A>G and MSTN-519C>T, found in the promoters of the MC4R and MSTN genes, respectively, are both associated with growth traits in Spinibarbus hollandi. Furthermore, we observed that there were significant associations between the expression levels of the MC4R and MSTN genes and these two growth trait-related SNPs. The expression level of MC4R gene in brain was lower in GG genotype fish with extremely high growth performance than that in AA genotype fish with extremely low growth performance. Expression level of the MSTN gene in muscle was lower in TT genotype fish with extremely high growth performance than that in CC and CT genotype fish with lower growth performance. The results indicated that these SNPs located in the promoters of MC4R and MSTN are associated with growth-related traits through modification of gene expression levels. The MSTN and MC4R SNPs may have useful application in effective marker-assisted selection aimed to increase output in S. hollandi. Show less

The efficiency of in vitro embryo production remains low compared with that observed in vivo. Recent studies have independently shown that cyclic adenosine monophosphate (cAMP) modulation prior to in vitro maturation (IVM) supplementation improves oocyte developmental competence. In this context, special cAMP modulators have been applied during IVM as promising alternatives to improve this biotechnology. Accordingly, this study was conducted to evaluate the effects of treatment with cilostazol, a PDE3 inhibitor, during pre-IVM culture on oocyte meiotic maturation in yak. Immature yak cumulus-oocyte complexes (COCs) were treated in vitro without (control) or with 5μM cilostazol for 0, 2, or 4h prior to IVM. Results showed that the presence of cilostazol in pre-IVM medium significantly increased the percentages of oocytes at metaphase II stage compared with that in the control groups (P<0.05). Moreover, pre-IVM with cilostazol significantly enhanced intraoocyte cAMP and glutathione (GSH) levels at the pre-IVM or IVM phase relative to the no pre-IVM groups (P<0.05). After in vitro fertilization (IVF) and parthenogenetic activation (PA), the developmental competences of oocytes and embryo quality were improved significantly after pre-IVM with cilostazol compared with the control groups (P<0.05), given that the cleavage and blastocyst formation rates and the total number of blastocyst cells were increased. The presence of cilostazol also increased the levels of mRNA expression for adenylate cyclase 3 (ADCY3) and protein kinase 1 (PKA1), as well as decreased the abundance of phosphodiesterase 3A (PDE3A) in COCs and IVF blastocysts, compared with their control counterparts (P<0.05). The results demonstrated that the meiotic progression of immature yak oocytes could be reversibly affected by cAMP modulators. By contrast, treatment with cilostazol during pre-IVM positively affected the developmental competence of yak oocytes, probably by improving intraoocyte cAMP and GSH levels and regulating mRNA expression patterns. We concluded that appropriate treatment with cilostazol during pre-IVM would be beneficial for oocyte maturation in vitro. Show less

The oncogenic herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is known to encode four viral interferon regulatory factors (vIRF1 to -4) to subvert the host antiviral immune response, but their detailed DNA-binding profiles as transcription factors in the host remain uncharacterized. Here, we first performed genome-wide vIRF2-binding site mapping in the human genome using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). vIRF2 was capable of binding to the promoter regions of 100 putative target genes. Importantly, we confirmed that vIRF2 can specifically interact with the promoters of the genes encoding PIK3C3, HMGCR, and HMGCL, which are associated with autophagosome formation or tumor progression and metastasis, and regulate their transcription in vivo. The crystal structure of the vIRF2 DNA-binding domain (DBD) (referred to here as vIRF2DBD) showed variable loop conformations and positive-charge distributions different from those of vIRF1 and cellular IRFs that are associated with DNA-binding specificities. Structure-based mutagenesis revealed that Arg82 and Arg85 are required for the in vitro DNA-binding activity of vIRF2DBD and can abolish the transcription regulation function of vIRF2 on the promoter reporter activity of PIK3C3, HMGCR, and HMGCL. Collectively, our study provided unique insights into the DNA-binding potency of vIRF2 and suggested that vIRF2 could act as a transcription factor of its target genes in the host antiviral immune response. The oncogenic herpesvirus KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV has developed a unique mechanism to subvert the host antiviral immune responses by encoding four homologues of cellular interferon regulatory factors (vIRF1 to -4). However, none of their DNA-binding profiles in the human genome have been characterized until now, and the structural basis for their diverse DNA-binding properties remain poorly understood. In this study, we performed the first genome-wide vIRF2-binding site mapping in the human genome and found vIRF2 can bind to the promoter regions of 100 target cellular genes. X-ray structure analysis and functional studies provided unique insights into its DNA-binding potency and regulation of target gene expression. Our study suggested that vIRF2 could act as a transcription factor of its target genes and contribute to KSHV infection and pathogenesis through versatile functions. Show less

To identify the mutation in the disease gene and provide prenatal diagnosis for a hereditary multiple osteochondromas (HMO) family. The exons of EXT1 gene in the proband with HMO and his family members were amplified by PCR. The products were analyzed by direct sequencing. Prenatal genetic diagnosis was performed by amniocentesis sampling after genotyping the proband. In the family, the affected proband was heterozygous of the mutation of 1476₁₄₇₇delTC in the EXT1 gene, and the proband's father carried the same mutation in part of his somatic cells. No mutation was found in the EXT1 gene of the proband's mother and other 11 siblings of his father. METHODS for molecular diagnosis and prenatal diagnosis of HMO were established and applied to a family of HMO. Show less

The expression changes of liver X receptor alpha (LXRα), histone deacetylase 3 (HDAC3) and CCAAT/enhancer binding protein alpha (C/EBPα) were detected in liver tissues of our high-fat-diet E3 rat model. The aim of this study is to pinpoint the molecular mechanism of HDAC3 and C/EBPα to orchestrate LXRα expression in hepatocytes. We confirmed that LXRα and its target genes were negatively regulated by HDAC3 in stable expressed clones with pEGFP-Hdac3 or shRNA-Hdac3 vector. However, transient pEGFP-C/EBPα plasmid transfection showed an upregulation of LXRα expression and C/EBPα enhanced LXRα promoter activity in a dose-dependent manner in CBRH-7919 cells. By using 5'-serial deletion reporter analysis, we identified that fragment from -2881 to -1181bp of LXRα promoter was responsible for C/EBPα binding to the promoter, especially CBS1 and CBS4 were identified essentially by using ChIP and luciferase reporter assay. Co-IP, qRT-PCR and ChIP revealed that HDAC3 interacted with C/EBPα co-regulated LXRα expression. Sumoylation of C/EBPα at lysine 159 was detected in CBRH-7919 cells with transient overexpressed C/EBPα, and Co-IP assay detected that sumoylated C/EBPα interacted with more HDAC3 than C/EBPα K159L mutant. Luciferase reporter assay demonstrated that C/EBPα participated in HDAC3-repressed LXRα transcription, and HDAC3 was involved in sumoylated C/EBPα-inactivated LXRα activity. Luciferase reporter assay demonstrated that sumoylation of C/EBPα by SUMO-1 directly reversed the activation of C/EBPα on LXRα promoter. The results suggested that HDAC3 interacts with sumoylated C/EBPα to negatively regulate the LXRα expression. Show less

Liver X receptor α (LXRα) and sterol regulatory element binding protein-1c (SREBP-1c) were studied in rats with non-alcoholic steatohepatitis (NASH) induced by a high-fat diet. Forty 5-week-old rats were fed either a high-fat diet (n = 30) or a normal diet (n = 10) for 9, 13 or 17 weeks. The mRNA and protein levels for LXRα and SREBP-1c were measured at each time point, as was fatty acid synthase (FAS) activity and the serum levels of free fatty acid (FFA) and triglyceride (TG). The mRNA and protein levels for LXRα and SREBP-1c, FAS activity and serum levels of FFA and TG all significantly increased from week 9 in the high-fat diet rats versus controls. In conclusion, a high-fat diet upregulates LXRα which, in turn, upregulates SREBP-1c, increasing the activity of FAS and FFA and accumulation of TG in hepatocytes. Thus, LXRα and SREBP-1c contribute to the development of NASH. Show less

In the previous experiment, we found that there was a different response between E3 rats and DA.1U rats to high-fat-diet-induced metabolic syndrome (HFD-MetS). The aim of this study was to explore the cause and molecular mechanism of the genetic difference in susceptibility to metabolic syndrome in E3 rats as compared with DA.1U rats. Firstly, a 12-week HFD-MetS model in E3 and DA.1U rats was carried out and assessed. Then, the expression of key insulin signaling molecules, metabolic nuclear receptors, metabolic key enzymes and histone deacetylases (Hdacs) was determined by different methods. Finally, the effects of overexpression and disruption of Hdac3 on metabolic nuclear receptors were analyzed in CBRH-7919 cells and primarily-hepatic cells from DA.1U and E3 rats. We found that E3 rats were susceptible, while DA.1U rats were resisted to HFD-MetS. The expression of liver X receptor α,β (LXR-α,β), farnesoid X receptor (FXR), peroxisome proliferator activated receptor γ (PPAR-γ) and cholesterol 7α-hydroxylase (CYP7A1) increased markedly in DA.1U rat liver, whereas they decreased significantly in E3 rats. The expression of Hdac3 increased by HFD treatment in both E3 and DA.1U rat livers, but the constitutive Hdac3 expression was lower in DA.IU rat liver than in E3 rat liver. Importantly, overexpression of Hdac3 could downregulate the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from DA.IU rats. On the contrary, disruption of Hdac3 by shRNA upregulated the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from E3 rats. The results suggested that a high constitutive expression of Hdac3 inhibiting the expression of PPAR-γ, LXR-α and CYP7A1 in liver contributes to HFD-MetS in E3 rats. Show less