Yanling Liu

Attenuating Ischemia-Reperfusion Injury in Kidney Transplantation by Perfusing Donor Organs With siRNA Cocktail Solution.

Background Ischemia-reperfusion (I/R) injury is the major cause of delayed renal graft function in kidney transplantation. To date, there are no effective therapeutic approaches for preventing I/R injury. We previously reported that treatment of animals with small interference RNA (siRNA) would prevent warm I/R injury in nontransplant models and cold I/R injury in heart transplantation. In the present study, we further explore the feasibility of protecting grafts from extended cold I/R injury as applied to kidney transplantation by downregulating I/R-associated genes using siRNA. Methods Donor kidneys were intra-arterially perfused with siRNA containing solution during donor excision and preserved in siRNA containing solution. The siRNA-treated donor organs were then implanted into syngeneic recipient mice, and the 2 original kidneys were removed from the recipient. The effect of siRNA solution on extended cold I/R injury was determined by assessing renal function, histopathological change, cell apoptosis, and inflammation. Results The perfused siRNA solution knocked down the expression of complement 3, RelB, and Fas in the kidney at the mRNA and protein levels. Administration of siRNA solution reduced the levels of blood urea nitrogen and serum creatinine as compared with control groups. The siRNA cocktail decreased cell apoptosis and histopathological changes in the kidney and prolonged graft survival. The siRNA cocktail also reduced the expression of proinflammatory cytokines, IL-6, and TNF&agr;. Conclusions In conclusion, this is the first demonstration that perfusing donor organs with an siRNA cocktail solution can induce gene silencing in the kidney and prevent kidneys from extended cold I/R injury in kidney transplantation, highlighting the promise of the clinical application of siRNA-based therapies in the preservation of donor organs.

Gene silencing of indoleamine 2,3-dioxygenase 2 in melanoma cells induces apoptosis through the suppression of NAD+ and inhibits in vivo tumor growth

Indoleamine 2,3-dioxygenase 2 (IDO2) is a newly discovered enzyme that catalyzes the initial and rate-limiting step in the degradation of tryptophan. As a homologous protein of IDO1, IDO2 plays an inhibitory role in T cell proliferation, and it is essential for regulatory T cell (Treg) generation in healthy conditions. Little is known about the immune-independent functions of IDO2 relevant to its specific contributions to physiology and pathophysiology in cancer cells. The purpose of this study was to assess the impact of IDO2 gene silencing as a way to inhibit B16-BL6 cancer cells in a murine model. Here, for the first time, we show that knockdown of IDO2 using small interfering RNA (siRNA) inhibits cancer cell proliferation, arrests cell cycle in G1, induces greater cell apoptosis, and reduces cell migration in vitro. Knockdown of IDO2 decreased the generation of nicotinamide adenine dinucleotide (NAD+) while increasing the generation of reactive oxygen species (ROS). We further demonstrate that cell apoptosis, induced by IDO2 downregulation, can be weakened by addition of exogenous NAD+, suggesting a novel mechanism by which IDO2 promotes tumor growth through its metabolite product NAD+. In addition to in vitro findings, we also demonstrate that IDO2 silencing in tumor cells using short hairpin RNA (shRNA) delayed tumor formation and arrested tumor growth in vivo. In conclusion, this study demonstrates a new non-immune-associated mechanism of IDO2 in vitro and IDO2 expression in B16-BL6 cells contributes to cancer development and progression. Our research provides evidence of a novel target for gene silencing that has the potential to enhance cancer therapy.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection

BackgroundWhile substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival.MethodVectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed.ResultAllogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10–16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4 + CD25 + FoxP3+ regulatory T cell differentiation in vitro.ConclusionThis study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

Synergic therapy of melanoma using GNRs-MUA-PEI/siIDO2-FA through targeted gene silencing and plasmonic photothermia

Correction for ‘Synergic therapy of melanoma using GNRs-MUA-PEI/siIDO2-FA through targeted gene silencing and plasmonic photothermia’ by Yujuan Zhang et al., RSC Adv., 2016, 6, 77577–77589.

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.

A new cancer immunotherapy via simultaneous DC-mobilization and DC-targeted IDO gene silencing using an immune-stimulatory nanosystem: Immunostimulation and mobilization of DCs for cancer therapy

The activity of negative immune regulatory molecules, such as indoleamine 2,3‐oxygenase (IDO), significantly attenuates DC (Dendritic cells)‐mediated immunotherapy. We have previously reported that knockdown of IDO using siRNA can reinstall anti‐tumor immunity. However, a DC‐targeted siRNA delivery system for in vivo mobilized DCs remains to be developed, while gene silencing in mobilized DCs for cancer immunotherapy has never been explored. In our study, we developed a novel DC‐targeted siRNA delivery system, man‐GNR‐siIDO, using as a nanocarrier of siRNA specific for IDO (siIDO) and mannose (man) as a guide molecule for targeting DCs. We explored the immunostimulatory man‐GNR‐siIDO nano‐construct in DCs mobilized by Flt3‐L, a receptor‐type tyrosine kinase ligand, for lung cancer immunotherapy. In vivo DC‐targeted gene silencing of IDO resulted in robust anti‐tumor immunity as evidenced by promoting DC maturation, up‐regulating tumor antigen‐specific T‐cell proliferation and enhancing tumor‐specific cytotoxicity. A combinatorial treatment for Lewis Lung Carcinoma (LLC)‐bearing mice, with man‐GNR‐siIDO and Flt3‐L, significantly attenuated tumor growth and delayed tumor formation, suggesting the treatment feasibility of the man‐GNR‐siIDO system in Flt3‐L mobilized DCs in the immunotherapy of lung cancer. Therefore, our study highlights a clinical potential for a first‐in‐class anti‐cancer immunotherapy through simultaneous DC‐mobilization and DC‐targeted gene silencing of IDO with man‐GNR‐siIDO and Flt3‐L treatments.

TdIF1: a putative oncogene in NSCLC tumor progression

TdT-interacting factor 1 (TdIF1) is a ubiquitously expressed DNA- and protein-binding protein that directly binds to terminal deoxynucleotidyl transferase (TdT) polymerase. Little is known about the functional role of TdIF1 in cancer cellular signaling, nor has it previously been identified as aberrant in any type of cancer. We report here for the first time that TdIF1 is abundantly expressed in clinical lung cancer patients and that high expression of TdIF1 is associated with poor patient prognosis. We further established that TdIF1 is highly expressed in human non-small cell lung cancer (NSCLC) cell lines compared to a normal lung cell line. shRNA-mediated gene silencing of TdIF1 resulted in the suppression of proliferation and anchorage-independent colony formation of the A549 adenocarcinoma cell line. Moreover, when these TdIF1-silenced cells were used to establish a mouse xenograft model of human NSCLC, tumor size was greatly reduced. These data suggest that TdIF1 is a potent regulator of lung tumor development. Several cell cycle-related and tumor growth signaling pathways, including the p53 and HDAC1/2 pathways, were identified as participating in the TdIF1 signaling network by in silico analysis. Microarray, transcriptome and protein-level analyses validated p53 and HDAC1/2 modulation upon TdIF1 downregulation in an NSCLC cellular model. Moreover, several other cell cycle regulators were affected at the transcript level by TdIF1 silencing, including an increase in CDKN1A/p21 transcripts. Taken together, these results indicate that TdIF1 is a bona fide tumor-promoting factor in NSCLC and a potential target for therapy.Immune protein involved in lung cancerA protein involved in the immune system also plays a role in the most common type of lung cancer. Weiping Min, of the University of Western Ontario in Canada, and international colleagues found, for the first time, that the protein TdIF1 is significantly upregulated in non-small cell lung cancer (NSCLC) tissues in patients. High expression levels of this protein were correlated with poor prognosis. NSCLC tumor tissues grown in mice where TdIF1 expression was ‘knocked down’ were significantly smaller than in those without TdIF1 knockdown. Further analyses showed the protein was involved in known cell signaling pathways with roles in NSCLC progression. The findings indicate TdIF1 should be further investigated as a biomarker of NSCLC or as a molecular target for its treatment.

Temperature-dependent cell death patterns induced by functionalized gold nanoparticle photothermal therapy in melanoma cells

Photothermal therapy (PTT) is a promising approach for cancer targeting therapy. However, the temperature-dependent killing of tumor cells in PTT remains unclear. In this study, we report necroptosis plays a role in the anti-tumor effects observed in gold nanorod (GNR)-mediated PTT in melanoma. We first synthesized gold nanorods with a targeting adaptor FA (GNRs-FA), which achieved high efficacy of targeted delivery to melanoma cells. We further demonstrated PTT, precipitated by GNRs-FA under the induction of near-infrared laser, was temperature-dependent. Furthermore, the photothermal killing of melanoma cells showed different patterns of cell death depending on varying temperature in PTT. In a lower temperature at 43 °C, the percentages of apoptosis, necroptosis and necrosis of tumor cells were 10.2%, 18.3%, and 17.6%, respectively, suggesting the cell killing is ineffective at lower temperatures. When the temperature increased to 49 °C, the cell death pattern switched to necrosis dominant (52.8%). Interestingly, when the PTT achieved a moderate temperature of 46 °C, necroptosis was significantly increased (35.1%). Additionally, GNRs-FA/PPT-mediated necroptosis was regulated by RIPK1 pathway. Taken together, this study is the first to demonstrate that temperature-dependent necroptosis is an important mechanism of inducing melanoma cell death in GNR-mediated PTT in addition to apoptosis and necrosis.

A novel GNRs-PEI/GNRs-PEI-folate for efficiently delivering siRNA

RNA interference (RNAi) employs double-stranded RNA or siRNA (small interfering RNA) to silence gene expression in cells. The widespread use of RNAi therapeutics requires the development of clinically suitable, safe and effective delivery vehicles. PEI (Poly(ethylene imine)) carrying the positive charges has attracted considerable attention for siRNA delivery. Gold nanorods (GNRs) exhibit specially localized surface plasmon resonance when excited by the visible and near-infrared laser, which is useful for photothermal therapy. However, the toxicity derived from a large amount of the surfactant cetyltrimethylammonium bromide (CTAB) during GNR synthesis severely limits their medical applications. Here, we report the synthesis of GNRs-PEI/GNRs-PEI-folate to improve biocompatibility, siRNA delivery and photothermal therapy of GNRs. Firstly, GNRs were synthesized according to the seed-mediated template-assisted protocol. The characterization results of GNRs showed: the size was length about 218 nm and width about 26.8 nm; the Zeta potential was +38.1 mV derived from CTAB on their surface; the dipole resonance extinction spectrum peak was 752 nm which is effective for photothermal therapy in vivo. Secondly, we synthesized PEI-MUA (Mercaptoundecanoic acid) and PEI-MUA-folate based on the chemical reaction between amino group of PEI and carboxyl group of MUA or Folate. PEI-MUA or PEI-MUA-folate to replace CTAB on GNRs obtained the GNRs-MUA-PEI system or the GNRs-MUA-PEI-folate system due to the solid conjugation between the thiol group of MUA and GNRs. The products were measured using the FTIR Spectrometer, and the spectra suggest MUA-PEI or PEI-MUA-folate has successfully replaced CTAB on the surface of GNRs. Finally, GNRs-MUA-PEI was incubated with siRNA-Cy3. The unbound siRNA-Cy3 was measured the intensity of fluorescence for calculating the uploaded amount of siRNA by GNRs-MUA-PEI, and the results indicate that the uploaded percentage of siRNA is about 70%. We conclude that the GNRs-MUA-PEI system is an effective siRNA loading vehicle.