Xiaoling Lu

Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.

To the Editor: CRISPR-Cas systems have been developed as an efficient gene editing technology in cells and model organisms. Here we use a CRISPR-Cas system to induce genomic DNA fragment deletion in mice by coinjecting two single-guide RNAs (sgRNAs) targeting the Uhrf2 locus with Cas9 mRNA. Furthermore, we report the generation of a Mc3R and Mc4R double-gene knockout rat by means of a single microinjection. High germline-transmission efficiency was observed in both mice and rats. The clustered, regularly interspaced, short palindromic repeats (CRISPR)associated protein (Cas) system has evolved in bacteria and archaea as an RNA-based adaptive immune system against viral and plasmid invasion1. Based on gene conservation and locus organization, three major types of CRISPR systems have been identified2,3. In the type II systems, the complex of a CRISPR RNA (crRNA) annealed to a trans-activating crRNA (tracrRNA) is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to generate double-strand breaks in target DNA4. Previous studies established a strategy for multiplex genome engineering with the Cas9 RNA-guided endonuclease in mammalian cells5,6. Recently, efficient genome editing by the CRISPR-Cas system has been shown in multiple organisms, including zebrafish, mice and bacteria7–9. Several groups have demonstrated that compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas–mediated gene targeting has similar or greater efficiency in cells and zebrafish5–7,10. Although it has been demonstrated that multiple genes can be disrupted in individual mouse embryos using CRISPR-Cas–mediated systems9, germline transmission of Cas9-mediated mutations in animals has not yet been reported. In addition, whether long, specific, genomic DNA target fragments

Antitumor action of a novel histone deacetylase inhibitor, YF479, in breast cancer.

Accumulating evidence demonstrates important roles for histone deacetylase in tumorigenesis (HDACs), highlighting them as attractive targets for antitumor drug development. Histone deactylase inhibitors (HDACIs), which have shown favorable anti-tumor activity with low toxicity in clinical investigations, are a promising class of anticancer therapeutics. Here, we screened our compound library to explore small molecules that possess anti-HDAC activity and identified a novel HDACI, YF479. Suberoylanilide hydroxamic acid (SAHA), which was the first approved HDAC inhibitor for clinical treatment by the FDA, was as positive control in our experiments. We further demonstrated YF479 abated cell viability, suppressed colony formation and tumor cell motility in vitro. To investigate YF479 with superior pharmacodynamic properties, we developed spontaneous and experimental breast cancer animal models. Our results showed YF479 significantly inhibited breast tumor growth and metastasis in vivo. Further study indicated YF479 suppressed both early and end stages of metastatic progression. Subsequent adjuvant chemotherapy animal experiment revealed the elimination of local-regional recurrence (LRR) and distant metastasis by YF479. More important, YF479 remarkably prolonged the survival of tumor-bearing mice. Intriguingly, YF479 displayed more potent anti-tumor activity in vitro and in vivo compared with SAHA. Together, our results suggest that YF479, a novel HDACI, inhibits breast tumor growth, metastasis and recurrence. In light of these results, YF479 may be an effective therapeutic option in clinical trials for patients burdened by breast cancer.

Oridonin Inhibits Tumor Growth and Metastasis through Anti-Angiogenesis by Blocking the Notch Signaling

While significant progress has been made in understanding the anti-inflammatory and anti-proliferative effects of the natural diterpenoid component Oridonin on tumor cells, little is known about its effect on tumor angiogenesis or metastasis and on the underlying molecular mechanisms. In this study, Oridonin significantly suppressed human umbilical vascular endothelial cells (HUVECs) proliferation, migration, and apillary-like structure formation in vitro. Using aortic ring assay and mouse corneal angiogenesis model, we found that Oridonin inhibited angiogenesis ex vivo and in vivo. In our animal experiments, Oridonin impeded tumor growth and metastasis. Immunohistochemistry analysis further revealed that the expression of CD31 and vWF protein in xenografts was remarkably decreased by the Oridonin. Furthermore, Oridonin reinforced endothelial cell-cell junction and impaired breast cancer cell transendothelial migration. Mechanistically, Oridonin not only down-regulated Jagged2 expression and Notch1 activity but also decreased the expression of their target genes. In conclusion, our results demonstrated an original role of Oridonin in inhibiting tumor angiogenesis and propose a mechanism. This study also provides new evidence supporting the central role of Notch in tumor angiogenesis and suggests that Oridonin could be a potential drug candidate for angiogenesis related diseases.

Lgr4 Gene Deficiency Increases Susceptibility and Severity of Dextran Sodium Sulfate-induced Inflammatory Bowel Disease in Mice

Background: Lgr4 as a transmembrane receptor binds with R-spondins to modulate Wnt signaling. Results: Deletion of Lgr4 in mice leads to loss of epithelial barrier function and high susceptibility to inflammatory bowel disease. Conclusion: Lgr4-mediated Wnt/β-catenin signaling is required for intestinal homeostasis and regeneration. Significance: Lgr4 has potential implications for diagnosis or drug discovery for human colitis. Lgr4/Gpr48 is one of the newly identified R-spondins receptors and potentiates Wnt signaling, which regulates intestinal homeostasis. We used a hypomorphic mouse strain to determine the role of Lgr4 in intestinal inflammation and recovery. Intestinal inflammation was induced with dextran sulfate sodium (DSS) followed by a recovery period. Intestinal inflammation symptoms and molecular mechanisms were examined. We found that Lgr4−/− mice exhibited dramatically higher susceptibility to and mortality from DSS-induced inflammatory bowel disease than WT mice. Lgr4 deficiency resulted in greatly reduced numbers of either Paneth cells or stem cells in the intestine. During the intestinal regeneration process, cell proliferation but not apoptosis of intestinal epithelial cells was significantly impaired in Lgr4−/− mice. When Wnt/β-catenin signaling was reactivated by crossing with APCmin/+ mice or by treating with a GSK-3β inhibitor, the number of Paneth cells was partially restored and the mortality caused by DSS-induced inflammatory bowel disease was strikingly reduced in Lgr4-deficient animals. Thus, Lgr4 is critically involved in the maintenance of intestinal homeostasis and protection against inflammatory bowel disease through modulation of the Wnt/β-catenin signaling pathway.

Lgr4/Gpr48 Negatively Regulates TLR2/4-associated Pattern Recognition and Innate Immunity by Targeting CD14 Expression

Background: Lgr4/Gpr48 has been found to play an important role in development of different organs. Results: Overactivated innate immune response was found in Lgr4-deficient mice. Conclusion: Lgr4/Gpr48 could be a negative regulator in TLR2/4-associated immune responses. Significance: Our novel findings provide new insights into the physiological functions of Lgr4/Gpr48 in immune diseases. The recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is pivotal in both innate and adaptive immune responses. Here we demonstrate that deletion of Lgr4/Gpr48 (G-protein-coupled receptor 48), a seven-transmembrane glycoprotein hormone receptor, potentiates TLR2/4-associated cytokine production and attenuates mouse resistance to septic shock. The expression of CD14, a co-receptor for TLR2/4-associated pathogen-associated molecular patterns, is increased significantly in Lgr4-deficient macrophages, which is consistent with the increased immune response, whereas the binding activity of cAMP-response element-binding protein is decreased significantly in Lgr4-deficient macrophages, which up-regulate the expression of CD14 at the transcriptional level. Together, our data demonstrate that Lgr4/Gpr48 plays a critical role in modulating the TLR2/4 signaling pathway and represents a useful therapeutic approach of targeting Lgr4/Gpr48 in TLR2/4-associated septic shock and autoimmune diseases.

Serum retinol-binding protein 4 levels in patients with diabetic retinopathy.

Retinol-binding protein 4 (RBP4) has been reported to be involved in the development of insulin resistance and diabetes. This study was designed to investigate serum levels of RBP4 in patients with type 2 diabetes with and without diabetic retinopathy. Based on ophthalmological examination, 92 patients with type 2 diabetes were divided into three subgroups: those without diabetic retinopathy (NDR; n = 40); those with simple diabetic retinopathy (SDR; n = 37); and those with proliferative diabetic retinopathy (PDR; n = 15). The serum RBP4 level was significantly elevated in individuals with PDR compared with those with NDR or SDR. There was a significant positive correlation between serum RBP4 level and the urine albumin excretion rate (r = 0.219). This study showed that RBP4 may be involved in the process of diabetic retinopathy and may be a novel biomarker for its diagnosis and treatment in diabetic patients.

A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release.

LRP5/6 directly bind to Frizzled and prevent Frizzled-regulated tumour metastasis.

How Wnt signalling including canonical and non-canonical pathways are initiated at the cell surface is not completely understood. Here we report that Wnt receptor Frizzled (Frz) and theco-receptors LRP5 and LRP6 (LRP5/6) directly interact with each other and this interaction is regulated by the LRP6 ectodomain. Importantly, through direct binding to Frz, LRP5/6 are able to prevent Frz-regulated non-canonical pathway activation and further non-canonical pathway-mediated tumour metastasis. Knockdown of endogenous LRP5/6 promotes otherwise-nonaggressive tumour cells to migrate in vitro, whereas a soluble recombinant protein of LRP6 ectodomain suppresses migration and metastasis of otherwise-aggressive tumour cells in vitro and in vivo. Furthermore, the expression level of membrane LRP5/6 correlates inversely with metastasis in mouse and human breast cancer. Our study suggests a previously unrecognized mode of receptor interaction, revealing the mechanism of LRP5/6 in inhibition of non-canonical pathway, and a possible clinical use of the LRP6 ectodomain to impede metastasis.

Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein

TRIM28 regulates its target genes at both transcriptional and posttranscriptional levels. Here we report that a TRIM28-TWIST1-EMT axis exists in breast cancer cells and TRIM28 promotes breast cancer metastasis by stabilizing TWIST1 and subsequently enhancing EMT. We find that TRIM28 is highly expressed in both cancer cell lines and advanced breast cancer tissues, and the levels of TRIM28 and TWIST1 are positively correlated with the aggressiveness of breast carcinomas. Overexpression and depletion of TRIM28 up- and down-regulates the protein, but not the mRNA levels of TWIST1, respectively, suggesting that TRIM28 upregulates TWIST1 post-transcriptionally. Overexpression of TRIM28 in breast cancer cell line promotes cell migration and invasion. Knockdown of TRIM28 reduces the protein level of TWIST1 with concurrent upregulation of E-cadherin and downregulation of N-cadherin and consequently inhibits cell migration and invasion. Furthermore, Immunoprecipitation and GST pull-down assays demonstrated that TRIM28 interacts with TWIST1 directly and this interaction is presumed to protect TWIST1 from degradation. Our study revealed a novel mechanism in breast cancer cells that TRIM28 enhances metastasis by stabilizing TWIST1, suggesting that targeting TRIM28 could be an efficacious strategy in breast cancer treatment.

Folate-modified Chitosan Nanoparticles Containing the IP-10 Gene Enhance Melanoma-specific Cytotoxic CD8+CD28+ T Lymphocyte Responses

Background: Adoptive immunotherapy with cytotoxic T lymphocytes (CTLs) has great potential for the treatment of some malignant cancers. Therefore, augmenting the responses of tumor-specific CTLs is significant for the adoptive immunotherapy of melanoma. This study aimed to investigate the anti-tumor response of a combination therapy employing folate-modified chitosan nanoparticles containing IP-10 (interferon-γ-inducible protein-10) plus melanoma TRP2-specific CD8+CD28+ T cells. Methods: We prepared folate-modified chitosan nanoparticles containing the mouse IP-10 gene (FA-CS-mIP-10), and induced melanoma TRP2-specific CD8+CD28+ T cells by co-culturing them with artificial antigen-presenting cells. B16-bearing mice were treated with FA-CS-mIP-10, melanoma TRP2-specific CD8+CD28+ T cells, a combination of both, and the saline control. Tumor volumes and the survival time of mice were recorded. The proportion of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor microenvironment and regulatory T cells (Tregs) in the spleen was analyzed by flow cytometry. We also detected the proliferation and angiogenesis of tumors by immunohistochemistry and apoptosis by TUNEL. Results: The combination therapy inhibited the progression of melanoma in vivo. Compared with other treatments, it more efficiently inhibited tumor growth and increased the survival time of mice. After treatment with combination therapy, the proportion of MDSCs and Tregs decreased, while the percentage of CXCR3+CD8+ T cells increased. Furthermore, combination therapy inhibited proliferation and promoted apoptosis of tumor cells and significantly inhibited tumor angiogenesis in vivo. Conclusion: We describe a novel strategy for improving the anti-tumor response of CD8+CD28+ CTLs by combining them with FA-CS-mIP-10 nanoparticles.