Jinglun Xue

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.

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.

Establishment of an AAV Reverse Infection-Based Array

Background The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.

Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo

1. It has been demonstrated that the hydrodynamics‐based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics‐based gene delivery into mouse liver in vivo.

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.

TAT-phiC31 integrase mediates DNA recombination in mammalian cells

Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.

Preparation of rAAV/hFlX by HSV/AAV hybrid helper virus and evaluation of its safety

The recombinant adeno-associated viral vector with human coagulation Factor IX minigene which was regulated by CMV promoter was constructed. Large quantity of recombinant adeno-associated viral particles (rAAV/ hFIX) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6X1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by an intramuscular injection of viral vectors (7.5X1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor IX expression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor IX expression curve. The QC-PCR method is easier and more accurate than traditional dot hybridization for determination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing in produced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.

Molecular cloning and expression of two closely related GTP-binding proteins from zebrafish.

Developmentally regulated GTP-binding proteins (DRGs) are a subclass of GTP-binding proteins that have been discovered recently. Here we report two zebrafish DRG cDNA clones closely related to human and mouse DRG genes. The two DRG sequences showed a high degree of similarity (55% identity, 72% similarity) at the amino acids level. Whole mount in situ hybridization revealed expression of zebrafish DRGs maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic. The expression of these two genes in different tissues follows a similar pattern, suggesting that they may serve a similar function.

Gene therapy for hemophilia B with liver-specific element mediated by Rep-RBE site-specific integration system.

Abstract: Adeno-associated virus (AAV) is a nonpathogenic virus capable of targeting human chromosome 19 for integration at AAVS1 site, and a 16 bp Rep binding element (RBE) sequence of AAV was sufficient for mediating this specific integration in the presence of AAV regulation proteins (Rep). Previously, we cotransduced 2 plasmids, pRBE-CMV-hFIX and pRC, into the AAVS1 transgenic mice by hydrodynamic injection, and a long-term expression of human coagulation Factor IX (hFIX) was observed. The corresponding AAVS1 locus site-specific integrations were verified by nested polymerase chain reaction. In this study, we established a novel hFIX expression plasmid, pRBE-HCR-hAAT-hFIX, driven by a liver-specific promoter by replacing the CMV promoter of pRBE-CMV-hFIX with a humanized promoter consisting of HCR-hAAT. The expression of hFIX in vitro was almost the same in transient transfection of pRBE-CMV-hFIX or pRBE-HCR-hAAT-hFIX. AAVS1-specific integrations were identified both in mice transfected with pRC/pRBE-CMV-hFIX cocktail and pRC/pRBE-HCR-hAAT-hFIX cocktail. However, the expression of hFIX of pRBE-HCR-hAAT-hFIX mice was higher and persisted longer. It achieved more than 1% of normal plasma hFIX concentration and maintained for 240 days. The result suggested that RBE-HCR-hAAT element could improve the expression of hFIX and present potential usage of Rep-RBE site-specific integration in gene therapy for hemophilia B.