Near infrared light fluorescence imaging-guided biomimetic nanoparticles of extracellular vesicles deliver indocyanine green and paclitaxel for hyperthermia combined with chemotherapy against glioma.

BACKGROUND: We investigated the therapeutic effect of targeting extracellular vesicles (EVs) loaded with indocyanine green (ICG) and paclitaxel (PTX) on glioma. METHODS: Raw264.7 cells were harvested to extract EVs for the preparation of ICG/PTX@RGE-EV by electroporation and click chemistry. We evaluated the success of modifying Neuropilin-1 targeting peptide (RGE) on the EV membrane of ICG/PTX@RGE-EV using super-resolution fluorescence microscopy and flow cytometry. Spectrophotometry and high performance liquid chromatography (HPLC) were implemented for qualitative and quantitative analysis of the ICG and PTX loaded in EVs. Photothermal properties of the vesicles were evaluated by exposing to 808-nm laser light. Western blot analysis, cell counting kit 8 (CCK-8), Calcein Acetoxymethyl Ester/propidium iodide (Calcein-AM/PI) staining, and flow cytometry were utilized for assessing effects of vesicle treatment on cellular behaviors. A nude mouse model bearing glioma was established to test the targeting ability and anti-tumor action of ICG/PTX@RGE-EV in vivo. RESULTS: Under exposure to 808-nm laser light, ICG/PTX@RGE-EV showed good photothermal properties and promotion of PTX release from EVs. ICG/PTX@RGE-EV effectively targeted U251 cells, with activation of the Caspase-3 pathway and elevated apoptosis in U251 cells through chemotherapy combined with hyperthermia. The anti-tumor function of ICG/PTX@RGE-EV was confirmed in the glioma mice via increased accumulation of PTX in the ICG/PTX@RGE-EV group and an increased median survival of 48 days in the ICG/PTX@RGE-EV group as compared to 25 days in the PBS group. CONCLUSION: ICG/PTX@RGE-EV might actively target glioma to repress tumor growth by accelerating glioma cell apoptosis through combined chemotherapy-hyperthermia.

Glioma exosomal microRNA-148a-3p promotes tumor angiogenesis through activating the EGFR/MAPK signaling pathway via inhibiting ERRFI1.

BACKGROUND: Glioma is the most frequent and lethal primary brain malignancy. Amounting evidence has highlighted the importance of exosomal microRNAs (miRNAs or miRs) in this malignancy. This study aimed to investigate the regulatory role of exosomal miR-148a-3p in glioma. METHODS: Bioinformatics analysis was firstly used to predict the target genes of miR-148a-3p. Exosomes were then extracted from normal human astrocytes and glioma cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was applied to determine the expression patterns of miR-148a-3p and ERBB receptor feedback inhibitor 1 (ERRFI1). Dual-luciferase reporter gene assay was applied to verify the direct binding between miR-148a-3p and ERRFI1. Cell counting kit-8 and tube formation assays were further conducted to assess the proliferation and angiogenic properties of human umbilical vein endothelial cells (HUVECs) in the co-culture system with exosomes. Lastly, glioma tumor models were established in BALB/c nude mice to study the role of exosomal miR-148a-3p in vivo. RESULTS: miR-148a-3p was highly expressed, while ERRFI1 was poorly expressed in glioma. miR-148a-3p was found to be enriched in glioma cells-derived exosomes and could be transferred to HUVECs via exosomes to promote their proliferation and angiogenesis. ERRFI1 was identified as a target gene of miR-148a-3p. In addition, miR-148a-3p activated the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling pathway by inhibiting ERRFI1. In the co-culture system, our data demonstrated that glioma cells-derived exosomal miR-148a-3p down-regulated ERRFI1 and activated the EGFR/MAPK signaling pathway, so as to promote cell proliferation and angiogenesis. In vivo experimentation further demonstrated that this mechanism was responsible for the promotive role of exosomal miR-148a-3p in tumorigenesis and angiogenesis. CONCLUSION: Taken together, glioma-derived exosomal miR-148a-3p promoted tumor angiogenesis through activation of the EGFR/MAPK signaling pathway by ERRFI1 inhibition.

Knockdown of Rho-associated protein kinase 1 suppresses proliferation and invasion of glioma cells.

Rho-associated protein kinase 1 (ROCK1), a serine/threonine protein kinase, affects cell invasion and migration by changing the status of the cytoskeleton. In recent years, ROCK1 was found to be overexpressed in a variety of tumors. However, the information of ROCK1 in glioma still remains elusive. In our study, the expression of ROCK1 in glioma tissues was examined by real-time PCR and the relationship between ROCK1 expression and clinical characteristics of patients with glioma was also analyzed. With the inhibition of ROCK1 expression by RNAi, the effects of ROCK1 on biological behaviors of glioma cells including cell viability, cell cycle, and cell invasion were probed in the U251 cell line by methyl thiazolyl tetrazolium (MTT) assay, flow cytometer analysis, and Transwell invasion experiment. In addition, the effects of ROCK1 on the regulation of Ki67, cyclin D1, matrix metalloproteinases 9 (MMP9), and E-cadherin were also investigated. The results indicated that ROCK1 messenger RNA (mRNA) was increased significantly compared to that in the adjacent normal tissue (P < 0.05) and the expression level of ROCK1 mRNA in high-grade malignant glioma tissue was significantly higher than that in low-grade malignant glioma tissue (P < 0.05). MTT assay and flow cytometer analysis revealed that the cell viability and cell proliferation in the ROCK1 small interfering RNA (siRNA) transfection group were markedly lower than those in the blank or negative control group (P < 0.05), and no obvious differences were found between the blank group and negative control group. The Transwell invasion experiments showed that the invasive ability of U251 cells in the ROCK1 siRNA transfection group was obviously lower than that in the blank or negative control group (P < 0.05), and there were no visible differences between the blank group and negative control group. Western blot demonstrated that the protein levels of Ki67, cyclin D1, and MMP9 in the ROCK1 siRNA transfection group were distinctly lower than those in the blank or negative control group (P < 0.05) and that the protein level of E-cadherin displayed an opposite variation (P < 0.05). In summary, the expressions of ROCK1 in glioma tissue were visibly upregulated and the increase of ROCK1 had a positive correlation with the malignant grade of glioma. The results implied that the proliferation and metastasis of the glioma cell could be inhibited by suppressing the expression of ROCK1, and our findings would provide a new target for intervention and treatment of glioma.