Qian Mei

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.

Expression of Annexin A5 Is Associated With Higher Tumor Stage and Poor Prognosis in Colorectal Adenocarcinomas

Goals To gain an insight into the putative role of annexin A5 (ANXA5) in the tumor stage and its clinical outcome. Background ANXA5 is a calcium-binding protein, which has been implicated in the carcinogenesis of several carcinomas. However, the role of ANXA5 in colorectal cancer (CRC) is unclear. Study We investigated the expression of ANXA5 in colorectal adenocarcinoma. This study included 207 consecutive patients with sporadic CRC. Paired colorectal tissue samples and corresponding nonmalignant tissues were obtained by surgical resection. ANXA5 mRNA and protein expression in each tissue were assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Data were statistically correlated with pathologic parameters and clinical outcome. Results Real-time reverse transcriptase polymerase chain reaction showed that there is an up-regulation in the mRNA level of ANXA5 in tumors (P<0.001). Immunohistochemical study revealed that high ANXA5 expression was present in 40.58% (84 of 207) of tumors. Univariate analysis showed increased ANXA5 expression correlated with pT stage (P=0.008), liver metastasis (P=0.024), pathologic tumor-node–metastasis stage (P=0.015), Dukes stage (P=0.017), recurrence (P=0.024), cancer-related death (P=0.028), recurrence-free probability (P=0.003), and overall survival (P=0.005). Multivariate analysis showed that ANXA5 expression and liver metastasis significantly correlated with recurrence-free probability (P=0.039 and P=0.048, respectively) and overall survival (P=0.012 and P=0.021, respectively) independent of pT stage and pN stage. Conclusions From these findings ANXA5 expression seems to be related to the tumor stage and clinical outcome of CRC. Thus ANXA5 could serve as a prognostic marker for tumor progression.

Calcium-dependent proapoptotic effect of Taenia solium metacestodes annexin B1 on human eosinophils : A novel strategy to prevent host immune response

Annexins are a family of calcium-dependent phospholipid-binding proteins that have been proposed to be involved in a wide range of important biological processes. At present, only a few annexins have been identified in parasites, and the physiological roles of these annexins are obscure. Earlier, we cloned a novel annexin (annexin B1) from Taenia solium metacestodes and found that annexin B1 was detectable in the surrounding host-derived layer with granulomaous infiltration. The objective of this study was to investigate the secretion and physiological function of annexin B1. We expressed a green fluorescent protein-tagged annexin B1 (GFP-anxB1) in living SiHa cells and showed that it was secreted upon stimulation with dexamethasone (Dex). This secretion was not inhibited by brefeldin A but was blocked by pre-treatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Furthermore, we describe for the first time that annexin B1 can bind to the extracellular surface of human eosinophils and produce Ca(2+)-influx. The Ca(2+)-influx induced apoptosis in eosinophils, which was inhibited by pre-loading the Ca(2+) channel blocker 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-metho-xyphenethyl]-1H-imidazole, HCl (SKF-96365). In conclusion, these findings represent direct and substantial evidence for the secretion of annexin B1 by living cells; the apoptosis in eosinophil induced by annexin B1 might be a novel strategy for T. solium metacestodes to prevent the hosts immune attack.

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.

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.