Jinzhong Chen

MicroRNA‐7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3‐kinase/Akt pathway in hepatocellular carcinoma

MicroRNAs (miRNAs) are known to be involved in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Recently, microRNA‐7 (miR‐7) has been proven to play a substantial role in glioblastoma and breast cancer, but its functions in the context of HCC remain unknown. Here, we demonstrate that miR‐7 inhibits HCC cell growth and metastasis invitro and in vivo. We first screened and identified a novel miR‐7 target, phosphoinositide 3‐kinase catalytic subunit delta (PIK3CD). Overexpression of miR‐7 would specifically and markedly down‐regulate its expression. miR‐7‐overexpressing subclones showed significant cell growth inhibition by G0/G1‐phase cell‐cycle arrest and significant impairment of cell migration in vitro. To identify the mechanisms, we investigated the phosphoinositide 3‐kinase (PI3K)/Akt pathway and found that Akt, mammalian target of rapamycin (mTOR), and p70S6K were down‐regulated, whereas 4EBP1 was up‐regulated in miR‐7‐overexpressing subclones. We also identified two novel, putative miR‐7 target genes, mTOR and p70S6K, which further suggests that miR‐7 may be a key regulator of the PI3K/Akt pathway. In xenograft animal experiments, we found that overexpressed miR‐7 effectively repressed tumor growth (3.5‐fold decrease in mean tumor volume; n = 5) and abolished extrahepatic migration from liver to lung in a nude mouse model of metastasis (n = 5). The number of visible nodules on the lung surface was reduced by 32‐fold. A correlation between miR‐7 and PIK3CD expression was also confirmed in clinical samples of HCC. Conclusion: These findings indicate that miR‐7 functions as a tumor suppressor and plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR‐signaling pathway in vitro and in vivo. By targeting PIK3CD, mTOR, and p70S6K, miR‐7 efficiently regulates the PI3K/Akt pathway. Given these results, miR‐7 may be a potential therapeutic or diagnostic/prognostic target for treating HCC. (HEPATOLOGY 2012;55:1852–1862)

Four microRNAs promote prostate cell proliferation with regulation of PTEN and its downstream signals in vitro.

Background Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro. Methodology/Principal Findings We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3’ UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1. Conclusions These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer.

Cloning and Identification of a Novel cDNA Coding Thioredoxin-Related Transmembrane Protein 2

Thioredoxin plays an important role in various cellular processes through redox regulation. Here we report the molecular cloning and characterization of one member of the thioredoxin superfamily, designated as TMX2. The TMX2 cDNA consists of 1644 nucleotides and contains an open reading frame encoding a protein of 372 amino acids with a predicted molecular mass of 42.5 kDa and an isoelectric point of 8.94 . The TMX2 protein may possess an N-terminal signal peptide, a potential transmembrane domain, an Myb DNA-binding domain repeat signature, a thioredoxin consensus pattern, an endoplasmic reticulum (ER) membrane retention signal (KKXX-like motif), and a dileucine motif in the tail. Northern blot analysis shows it is widely expressed in human tissues.

DAXX interacts with phage ΦC31 integrase and inhibits recombination

Phage ΦC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between ΦC31 integrase and cellular proteins have never been investigated. Using pLexA-ΦC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 106 independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and ΦC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for ΦC31 binding. Hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of ΦC31 is responsible for the interaction with DAXX. This tetramer is also necessary for ΦC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with ΦC31 integrase in a HEK293-derived ΦC31 integrase activity reporter cell line significantly reduced the ΦC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with ΦC31 causing a mild inhibition in the integration efficiency. This is the first time that ΦC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

A global genomic view of MIF knockdown-mediated cell cycle arrest.

Macrophage immigration inhibitory factor (MIF) is a ubiquitously expressed proinflammatory mediator that has also been implicated in cell growth, cell cycle and carcinogenesis. In this study, we demonstrate siRNA-mediated knockdown of MIF results in G0/G1 cell cycle arrest in HEK293 cells. To elucidate the molecular mechanism of cell cycle perturbation following MIF knockdown, we employed microarray to investigate the genome-wide expression profile in MIF-deficient cells and normal cells. Quantitative real-time PCR were used to confirm the differential expression patterns of ~50 transcripts involved in cell cycle and signaling identified by microarray. The dynamic model of MIF-dependent signaling pathways linked to cell cycle machinery were constructed through analyzing gene expression data in the context of known biological pathways. The results demonstrate that knockdown of MIF could inhibit G1/S transition through inhibition of MAPK, PI3K/Akt, NF-κB, c-Myc-dependent pathway and activation of TGF-β, p53-dependent pathway. Meanwhile, inhibition of E2F-, NFκB, c-Myc, and Ap-1-mediated transcription and stimulation of p53 and FoxO4 transcriptional activity will lead to differential expression of cell cycle regulators and subsequent cell cycle arrest in G0/G1 phase in MIF-knockdown cells. This study provides novel insights into the pleiotropic activities of endogenous MIF, especially its essential and crucial role in cell proliferation and the cell cycle.

More Vaccine Efficacy Studies on the Recombinant Bacille Calmette-Guerin Co-expressing Ag85B, Mpt64190–198 and Mtb8.4

The immunogenicity of the recombinant Bacille Calmette‐Guerin: rBCG‐Ag85B‐Mpt64190–198‐Mtb8.4 (rBCG‐AMM) was evaluated in our previous study. This paper compares the protective efficacy of rBCG‐AMM, rBCG‐A which overexpresses Ag85B and BCG in C57BL/6 mice. There was no significant difference in proliferation characteristics among rBCG‐AMM, rBCG‐A and BCG. The growth characteristics of rBCG‐AMM in host tissue were identical to control BCG, suggesting the improved protective efficacy was directly related to the expression of the Ag85B‐Mpt64190–198‐Mtb8.4 fusion protein. The protective experiment demonstrated that rBCG‐AMM could confer similar or even better protective efficacy against Mycobacterium tuberculosis infection compared with BCG or rBCG‐A as evaluated by bacterial organ loads, lung histopathology and net weight gain or loss. The results suggested that the recombinant BCG: rBCG‐Ag85B‐Mpt64190–198‐Mtb8.4 is a potential vaccine candidate for further study.

Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

AbstractUracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase-EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism.

Chimaeric protein improved immunogenicity compared with fusion protein of Ag85B and ESAT-6 antigens of Mycobacterium tuberculosis

Antigen 85B (Ag85B) and ESAT‐6 are important immunodominant antigens of Mycobacterium tuberculosis, and both are very promising vaccine candidate molecules. In this study, we relied on the T‐cell epitopes of Ag85B and ESAT‐6 to design a chimaeric protein by inserting ESAT‐6 into Ag85B from the amino acids 167–182. We found the ratio of IgG2b/IgG1 and the secretion of interferon (IFN)‐γ in the mice vaccinated with the new protein with adjuvant MPL and TDM were higher than the mice immunized with fusion protein Ag85B‐ESAT‐6, which have been reported and could induce levels of protective immunity similar to BCG in the mouse model of tuberculosis (TB) infection. These results suggest that the chimaeric protein Ag85BN‐ESAT‐6‐Ag85BC is a strong candidate for further study and the T‐cell epitopes of the antigens should be considered when we design the subunit vaccine.

Evaluation of a new Recombinant BCG which Contains Mycobacterial Antigen ag85B–mpt64190–198–mtb8.4 in C57/BL6 Mice

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be one of the major public health problems in the world. The eventual control of this disease will require the development of a safe and effective vaccine. Bacille Calmette‐Guerin (BCG), the only vaccine against TB, is not perfect for its limited ability to protect against the adult form of TB. Some improvements of TB vaccines relied to strengthening the immunogenicity and/or persistence of genetically modified recombinant BCG (rBCG) strain. Antigen 85B (Ag85B) and Mtb8.4 are importantly immunodominant antigens of M. tuberculosis, and both are very promising vaccine candidate molecules. MPT64190–198, is presented to CD8+ T cells during mycobacterial infections. In this study, we combined these above genes into one recombinant gene of ag85B–mpt64190–198–mtb8.4. Then we constructed the new rBCG containing this united gene. This rBCG can induce an increased Th1‐type immune response in mice, characterized by an elevated level of interferon‐γ in antigen‐stimulated splenocyte culture and a strong IgG2a antibody response. Also, it can elicit longer immune responses than BCG. The results show that this rBCG is a promising candidate for further study.

ΦC31 integrase interacts with TTRAP and inhibits NFκB activation

Phage ΦC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes. To evaluate its safety in human cells, it is important to understand the interactions between this integrase and cellular proteins. Here we show that ΦC31 integrase interacts with TTRAP as presented by yeast two-hybrid and co-immunoprecipitation assays. Reducing the expression of endogenous TTRAP can increase the efficiency of ΦC31 integrase-mediated integration. A possible effect of interaction between ΦC31 integrase and TTRAP was highlighted by the fact that ΦC31 integrase inhibited the NFκB activation mediated by IL-1 in a dose-dependent manner. Because low dose of ΦC31 integrase can mediate considerable recombination events, we suggest that low dose of ΦC31 integrase be used when this integrase is applied in human cells.