Hong-Li Yan

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.

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)

Methylation‐induced loss of miR‐484 in microsatellite‐unstable colorectal cancer promotes both viability and IL‐8 production via CD137L

Colorectal cancer (CRC) exhibiting MSI (microsatellite instability) represents a well‐defined subtype characterized by a deficient mismatch repair pathway and typical clinico‐pathological features. Our objective was to identify the entire miRNome and its molecular pathological roles in MSI CRCs. We profiled miRNA expression in MSI CRCs and compared it with MSS counterparts. Microarray and qRT‐PCR analysis identified eight miRNAs that could distinguish the MSI status of CRCs. MiR‐484 was the most significantly decreased miRNA in MSI CRCs, primarily mediated by the CpG island methylator phenotype. MiR‐484 functions as a tumour suppressor to inhibit MSI CRC cell viability in vitro and in vivo. Moreover, miR‐484 repressed CD137L expression and thereby attenuated IL‐8 production by MSI CRC cells. Our results contribute to a better understanding of the roles of dysregulated miRNAs in the distinct phenotypic features of MSI CRCs and indicate an option for early diagnosis and gene therapy for these patients. Copyright

Clinical features and mismatch repair genes analyses of Chinese suspected hereditary non-polypsis colorectal cancer: A cost-effective screening strategy proposal

China has the largest numbers of hereditary non‐polyposis colorectal cancer (HNPCC) patients based on its population of 1.4 billion. However, the clinical data and mismatch repair (MMR) gene analyses have been limited. Here we performed microsatellite instability (MSI) and immunohistochemistry (IHC) analyses on a series of patients with a high‐risk for HNPCC: 61 patients with family histories fulfilling Amsterdam criteria II (ACII‐HNPCC) or suspected HNPCC criteria (S‐HNPCC), and 106 early onset colorectal cancer (CRC) patients. Sixty late‐onset CRC patients were used as control. Methylation of the hMLH1 promoter was analyzed on tumors lacking hMLH1 expression. MMR germ‐line mutations were screened on patients with tumors classified as MSI‐H/L or negative for IHC. We identified 27 germ‐line MMR variants in the 167 patients with a high‐risk for HNPCC while only one germ‐line mutation in hMSH6 was found in the late‐onset CRC group. Of those, 23 were pathogenic mutations. The high incidence of gastric and hepatobiliary cancers coupled with the increasing number of small families in China reduces the sensitivity (43.5%, 30.4%) and positive predictive value (PPV) (45.5%, 17.9%) of the ACII‐ or S‐HNPCC criteria. MSI or IHC testing are highly sensitive in detecting pathogenic mutations (sensitivities = 91.3% and 95.6%, respectively), but the PPVs are quite low (25.6% and 27.8%, respectively). Considering that all 12 tumors with pathogenic mutations in hMLH1 also showed promoter unmethylation, the sensitivity of IHC in conjunction with hMLH1 promoter methylation analysis is not reduced, but the PPV was increased from 27.8% to 61.1%, and the total cost was greatly reduced. (Cancer Sci 2008; 99: 770–780)

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.

Germline hMSH2 promoter mutation in a Chinese HNPCC kindred: evidence for dual role of LOH.

Hereditary non‐polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer predisposition syndrome that is caused by germline mutations in mismatch repair genes. By screening the core promoters of hMSH2, hMLH1, and hMSH6 in 37 Chinese suspected HNPCC families, a novel germline mutation c.−78_−79delGT was found in the hMSH2 promoter. Its pathogenic effects were supported by the following findings: (a) it co‐segregated with HNPCC‐related cancers and was not present in the 220 control subjects, (b) tumors harboring the mutation lacked the expression of hMSH2 and showed high microsatellite instability, (c) it significantly decreased the promoter activity, and (d) it abolished the binding ability of the transcription factor E1A‐F. Loss of heterozygosity (LOH) was found in three of the tumors studied. Intriguingly, in the tumors from patients II:1 and III:1, LOH occurred in the wild‐type allele and agreed well with the traditional ‘two‐hit’ model. In contrast, in the tumor from patient III:3, LOH occurred in the mutant allele. A pathogenic somatic mutation (c.2210+1G>A) was also found in this tumor; therefore, we proposed that the ‘second hit’ was inactivated by somatic mutation, and the mutant allele was lost during tumor progression; this provided evidence for the new hypothesis for the dual role of LOH.

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family.

Abstract We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca2+. This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that α-helices are the main secondary structures of the protein. Ca2+ binding increases the α-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10°C. Functional Ca2+-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca2+ in each domain of annexin B1 singly or in combination, we found that Ca2+ binding in the first domain is more important than that at the other Ca2+ binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.

Potentials of the elevated circulating miR-185 level as a biomarker for early diagnosis of HBV-related liver fibrosis

Early diagnosis of liver fibrosis is critical for early intervention and prognosis of various chronic liver diseases. Conventional repeated histological assessment is impractical due to the associated invasiveness. In the current study, we evaluated circulating miR-185 as a potential biomarker to predict initiation and progression of liver fibrosis. We found that miR-185 was significantly up-regulated in blood specimens from patients with HBV-liver fibrosis and rats with liver fibrosis, the miR-185 levels were correlated with liver fibrosis progression, but not with the different viral loads in HBV-infected patients. miR-185 was observed in collagen deposition regions during advanced liver fibrosis. We found that differences in miR-185 levels facilitated the discrimination between early-staged or advanced-staged liver fibrosis and the healthy controls with high specificity, sensitivity, and likelihood ratio using receiver-operator characteristic analysis. miR-185 targeted SREBF1, and increased expression of COL1A1 and a-SMA genes that are hallmarks of liver fibrosis. Our data supported that circulating miR-185 levels could be used as potential biomarkers for the early diagnosis of liver fibrosis.

Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency.

To obtain a thrombus‐targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg‐Gly‐Asp‐Ser) peptide into the loop between domains II and III of the sequence‐deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1‐RGDS‐ScuPA by fusing ScuPA32k [low‐molecular‐mass single‐chain urokinase (32 kDa)] with the N‐terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion‐exchange and gel‐filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane‐binding activity and anti‐platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet‐targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.

Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host‐derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP—annexin B1 plasmid and transfected it into SiHa cells. We found that GFP—annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12‐myristate 13‐acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.