miR-485-5p/HSP90 axis blocks Akt1 phosphorylation to suppress osteosarcoma cell proliferation and migration via PI3K/AKT pathway.

Osteosarcoma (OS) is closely related to the dysregulation of various intracellular signaling pathways, especially the PI3K/Akt signaling pathway. Reportedly, HSP90 was responsible for phospho-Akt stabilization, and both AKT1 and HSP90 were upregulated within osteosarcoma. Herein, we demonstrated that AKT1 and HSP90 mRNA and protein expression were upregulated within osteosarcoma tissues and cells; AKT1 knockdown significantly inhibited OS cell viability. HSP90 knockdown suppressed the phosphorylation of AKT1, decreased ki-67 and Vimentin protein levels, enhanced p21 and E-cadherin protein levels, and inhibited OS cell proliferation and migration; AKT1 overexpression exerted opposing effects and significantly attenuated the effects of HSP90 knockdown. miR-485-5p targeted AKT1 and HSP90 3'-UTR to inhibit AKT1 and HSP90 expression. miR-485-5p overexpression dramatically reduced AKT1, HSP90, and ki-67 proteins, increased E-cadherin protein levels, and inhibited OS cell proliferation and migration. In conclusion, HSP90 knockdown blocked the phosphorylation of AKT1 suppressing the proliferation and migration capacity of OS cells via the PI3K/AKT pathway; miR-485-5p binds to HSP90 and AKT1 in their 3'-UTR to inhibit HSP90 and AKT1 expression, therefore exerting a tumor suppressor function within osteosarcoma.

Enhanced osteoinductivity and corrosion resistance of dopamine/gelatin/rhBMP-2-coated ß-TCP/Mg-Zn orthopedic implants: An in vitro and in vivo study.

Magnesium-based biomaterials are attracting increasingly more attention for orthopedic applications based on their appropriate mechanical properties, biodegradability, and favorable biocompatibility. However, the high corrosion rate of these materials remains to be addressed. In this study, porous ß-Ca3(PO4)2/Mg-Zn (ß-TCP/Mg-Zn) composites were fabricated via a powder metallurgy method. The ß-TCP/Mg-Zn composites with 6% porosity exhibited optimal mechanical properties, and thus, they were selected for surface modification. A novel dopamine/gelatin/recombinant human bone morphogenetic protein-2 (rhBMP-2) coating with demonstrated stability was prepared to further improve the corrosion resistance of the composite and enhance early osteoinductivity. The homogeneously coated ß-TCP/Mg-Zn composite showed significantly improved corrosion resistance according to electrochemical and immersion tests. In addition, extracts from the dopamine/gelatin/rhBMP-2-coated ß-TCP/Mg-Zn composite not only facilitated cell proliferation but also significantly enhanced the osteogenic differentiation of Sprague-Dawley rat bone marrow-derived mesenchymal stem cells in vitro. Furthermore, in vivo experiments were performed to evaluate the biodegradation, histocompatibility, and osteoinductive potential of the coated composite. No obvious pathological changes in the vital visceral organs were observed after implantation, and radiography and hematoxylin-eosin staining showed strong promotion of new bone formation, matched composite degradation and bone regeneration rates, and complete absorption of the released hydrogen gas. Collectively, these results indicate that the dopamine/gelatin/rhBMP-2-coated ß-TCP/Mg-Zn composite offers improved corrosion resistance, favorable biocompatibility, and enhanced osteoinductive potential for use in the fabrication of orthopedic implants.

Mg2+ in ß-TCP/Mg-Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx.

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg-Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg-Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and ß-tricalcium phosphate (ß-TCP; manifesting process not shown), prepared Mg, Zn, and Mg-Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg-Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg-Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg-Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg2+ in ß-TCP/Mg-Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.

miR-342-5p inhibits osteosarcoma cell growth, migration, invasion, and sensitivity to Doxorubicin through targeting Wnt7b.

Osteosarcoma (OS) accounts for 9 percent of cancer-related deaths in young people. The PI3K/Akt signaling, a well-known carcinogenic signaling pathway in human cancer, cooperates with other signaling pathways such as Wnt signaling to promote cancer progression. Wnt7b, as a transforming member of the Wnt family, could activate mTORC1 through PI3K-AKT signaling and is upregulated in OS. In the present study, we found that miR-342-5p inhibits Wnt7b expression via direct binding to Wnt7b 3'-UTR. miR-342-5p overexpression remarkably suppressed the viability and invasion while enhanced the apoptosis of OS cells; meanwhile, Wnt7b, ß-catenin, c-myc, and cyclin D1 proteins were reduced while E-cadherin protein showed to be increased. Consistent with its expression pattern, Wnt7b exerted oncogenic effects on OS cells. Wnt7b could significantly attenuate the impacts of miR-342-5p. In conclusion, we demonstrated a miR-342-5p/Wnt7b axis that regulates the capacity of OS cells to proliferate and to invade through Wnt/ß-catenin signaling. The miR-342-5p/Wnt7b axis might be novel targets for OS targeted therapy, which needs further in vivo and clinical investigations.

Research on corrosion behavior and biocompatibility of a porous Mg-3%Zn/5%ß-Ca3(PO4)2 composite scaffold for bone tissue engineering.

BACKGROUND: Rapid corrosion rates are a major impediment to the use of magnesium alloys in bone tissue engineering despite their good mechanical properties and biodegradability. Zinc is a promising alloy element, and it is an effective grain refiner for magnesium. ß-Ca3(PO4)2 (ß-TCP) is widely used for bone regeneration because of its good biocompatibility, and it also has a similar chemical and crystal structure to human bone. METHODS: In this research, the magnesium alloy was reinforced by adding 3%Zn (wt.%) and 5%ß-TCP (wt.%) particles in order to improve the corrosion resistance and biocompatibility. Furthermore, the biomaterial was prepared through powder metallurgy technology using NH4HCO3 as space-holding particles to construct porous Mg-3%Zn/5%ß-TCP scaffolds. RESULTS: The results revealed that the magnesium-zinc phase and calcium phosphate phase were uniformly distributed in the α-magnesium matrix. Mechanical and corrosion tests indicated that the scaffolds had mechanical strengths similar to that of human bone, and their corrosion resistance decreased with an increase in the porosity. The scaffolds had cytotoxicity grades of 0-1 against MG63 cells, SaoS2 cells, and HK-2 cells, which suggested that they were appropriate for cellular applications. In addition, the scaffolds demonstrated excellent biocompatibility when tested in rabbits. CONCLUSIONS: These results indicate that porous Mg-3%Zn/5%ß-TCP scaffolds are promising biodegradable implants for bone tissue engineering.

A Novel Fusion Antibody Exhibits Antiangiogenic Activity and Stimulates NK Cell-mediated Immune Surveillance Through Fused NKG2D Ligand.

A single-chain variable fragment (scFv) targeting vascular endothelial growth factor receptor 2 was previously generated from a phage display library in our laboratory. However, it has shortened half-life and lacks Fc fragment for effector cell recognition. To address these challenges, a ligand of NK-cell receptor NKG2D was fused to the scFv and created a fusion protein scFv-major histocompatibility complex class I-related chain A (MICA), which is expected to recognize tumor cells through the scFv moiety and stimulate NK cells through the MICA. The fusion protein demonstrated specific binding to both vascular endothelial growth factor receptor 2 and NKG2D in protein-based and cell-based assays. In addition, it demonstrated antiangiogenic activities including restraining the proliferation, migration, transwell invasion, and tube formation of human umbilical vein endothelial cells. Furthermore, the fusion protein exhibited significant cytotoxicity on K562, MDA-MB-435, and B16F10 cells and triggered NK92 cell-mediated cytotoxicity on MDA-MB-435 cells by stimulating the release of significant cytokines. The fusion protein targeting strategy, therefore, provides a means to engage lymphocyte effector cells against tumor specific antigen overexpressing tumor cells.

Locally deformed-ring hybrid microlasers exhibiting stable unidirectional emission from a Si waveguide.

We propose and demonstrate a locally deformed-ring (LDR) hybrid microlaser to realize stable unidirectional emission from a silicon waveguide. The coupled modes eliminate the competition between clockwise (CW) and counter-clockwise (CCW) modes, and the highly unidirectional characteristics are achieved with an enhanced mode quality factor based on a locally deformed notch in the LDR resonator. Using a divinylsiloxane-benzocyclobutene bonding technique, a LDR hybrid microlaser is fabricated vertically coupled to a silicon waveguide with a radius of 20 µm, a ring width of 4 µm and a notch width of 500 nm. The threshold current is 7 mA, and single-mode lasing is achieved with the side-mode suppression ratio of 27 dB. Nearly total unidirectional output from the CCW direction of a Si waveguide is demonstrated with a unidirectional ratio of 0.053.

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer.

Binding of MHC class I-related chain molecules A and B (MICA/B) to the natural killer (NK) cell receptor NK group 2, member D (NKG2D) is thought critical for activating NK-mediated immunosurveillance. Angiogenesis is important for tumor growth and interfering with angiogenesis using the fully human IgG1 anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (mAb04) can be effective in treating malignancy. In an effort to make mAb04 more effective we have generated a novel antibody fusion protein (mAb04-MICA) consisting of mAb04 and MICA. We found that mAb04-MICA maintained the anti-angiogenic and antineoplastic activities of mAb04, and also enhanced immunosurveillance activated by the NKG2D pathway. Moreover, in human breast tumor-bearing nude mice, mAb04-MICA demonstrated superior anti-tumor efficacy compared to combination therapy of mAb04 + Docetaxel or Avastin + Docetaxel, highlighting the immunostimulatory effect of MICA. In conclusion, mAb04-MICA provided new inspiration for anti-tumor treatment and had prospects for clinical application.

Tumoral NKG2D alters cell cycle of acute myeloid leukemic cells and reduces NK cell-mediated immune surveillance.

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor, initially discovered and expressed mostly on natural killer (NK) cells, T cells and natural killer T cells, can promote tumor immune surveillance. However, with increasing tumor grade, tumors themselves express NKG2D to self-stimulate oncogenic pathways. To confirm that cancer cells themselves express NKG2D, we have now investigated the role of the tumoral NKG2D in NK cell-mediated immune surveillance. Both anti-NKG2D and shRNA to that down-regulated tumoral NKG2D increased the number of cells in G1 phase and S phase, increased the expression of cyclin E-CDK2 and decreased P21. In addition, CD107a, IFN-γ and TNF-α increased when the cells were treated with anti-NKG2D which suggests that blocking tumoral NKG2D could augment tumor surveillance of NK cells. Altogether, tumoral NKG2D stimulates cell propagation and immune escape in acute myeloid leukemia cells.

A bispecific protein rG7S-MICA recruits natural killer cells and enhances NKG2D-mediated immunosurveillance against hepatocellular carcinoma.

MHC class I-related chain A (MICA) is a principal immunoligand of the natural killer (NK) cell receptor NK group 2, member D (NKG2D) and plays a key role in NK cell-mediated immune recognition. Shedding of MICA from tumor cells leads to immunosuppression. To reconstitute the immunosurveilance function of NK cells, we constructed a fusion protein rG7S-MICA and explored its potential anti-tumor activity against hepatocellular carcinoma (HCC). rG7S-MICA consists of human MICA and a single-chain antibody fragment (scFv) targeting the tumor-associated antigen cluster of differentiation 24 (CD24). In vitro, rG7S-MICA engaged both NK cells and CD24(+) human HCC cells, and triggered NK cell-mediated cytolysis. Furthermore, in CD24(+) HCC-bearing nude mice, rG7S-MICA specifically targeted to the tumor tissue, where it effectively recruited NK cells and induced the release of cytokines, and showed superior anti-tumor activity. In conclusion, rG7S-MICA provides a new approach for HCC-targeting immunotherapy and has attracting potentials for clinical applications.

Hybrid spiral-ring microlaser vertically coupled to silicon waveguide for stable and unidirectional output.

A hybrid spiral-ring laser vertically coupled to a silicon waveguide is demonstrated to achieve stable and unidirectional output theoretically and experimentally. The mode competition between clockwise (CW) and counter-clockwise (CCW) modes is eliminated due to the mode coupling in a spiral resonator. The simulation results indicate that the CCW and CW direction traveling waves are dominant components, respectively, for the spiral resonator without and with an output waveguide. A hybrid AlGaInAs/Si spiral-ring laser is designed and fabricated vertically coupled to a silicon waveguide. For a spiral-ring laser with a radius of 30 µm and a ring width of 5 µm, the continuous-wave lasing threshold of 9.5 mA is obtained with the threshold current density of 1.1 kA/cm(2) at a temperature of 285 K. The output power fluctuations due to the mode competition between CW and CCW modes are eliminated. The output power from CCW direction is five times that from CW direction.

Mode selection in square resonator microlasers for widely tunable single mode lasing.

Mode selection in square resonator semiconductor microlasers is demonstrated by adjusting the width of the output waveguide coupled to the midpoint of one side. The simulation and experimental results reveal that widely tunable single mode lasing can be realized in square resonator microlasers. Through adjusting the width of the output waveguide, the mode interval of the high-Q modes can reach four times of the longitudinal mode interval. Therefore, mode hopping can be efficiently avoided and the lasing wavelength can be tuned continuously by tuning the injection current. For a 17.8-µm-side-length square microlaser with a 1.4-µm-width output waveguide, mode-hopping-free single-mode operation is achieved with a continuous tuning range of 9.2 nm. As a result, the control of the lasing mode is realized for the square microlasers.

Efficient In Vitro Refolding and Characterization of Major Histocompatibility Complex Class I-Related Chain Molecules A (MICA) and Natural Killer Group 2 Member D (NKG2D) Expressed in E. coli.

Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.