AKT Axis, miR-21, and RECK Play Pivotal Roles in Dihydroartemisinin Killing Malignant Glioma Cells

Wang

et al. 2020

Cancer Cell Int

Background: Growing studies have focused on the role of microRNA-21 (miR-21) in glioma, thus our objective was to discuss the effect of M2 bone marrow-derived macrophage (BMDM)-derived exosomes (BMDM-Exos) shuffle miR-21 on biological functions of glioma cells by regulating paternally expressed gene 3 (PEG3). Methods: Seventy-one cases of human glioma tissues and 30 cases of non-tumor normal brain tissues were collected and stored in liquid nitrogen. PEG3 and miR-21 expression in glioma tissues was tested. The fasting venous blood of glioma patients and healthy control was collected and centrifuged, and then the supernatant was stored at − 80 °C refrigerator. The contents of interferon (IFN)-γ and transforming growth factor-β1 (TGF-β1) in serum were tested by ELISA. Glioma cells and normal glial cells were cultured to screen the target cells for further in vitro experiments. BMDM-Exos was obtained by ultra-high speed centrifugation and then was identified. BMDM-Exos was cocultured with U87 cells to detect the biological functions. The fasting venous blood of glioma patients was extracted and treated with ethylene diamine tetraacetic acid-K2 anti-freezing, and then CD8 + T cells were isolated. CD8 + T cells were co-cultured with U87 cells to detect the CD8 + T proliferation, cell cytotoxic activity, U87 cell activity, as well as IFN-γ and TGF-β1 levels. Moreover, BALB/c-nu/nu mice was taken, and the human-nude mouse glioma orthotopic transplantation model was established with U87 cells, and then mice were grouped to test the trends in tumor growth. The brain of mice (fixed by 10% formaldehyde) was sliced to detect the expression of Ki67 and proliferating cell nuclear antigen (PCNA). The spleen of mice was taken to prepare single-cell suspension, and the percentage of T lymphocytes in spleen to CD8 + T cells was detected. Results: PEG3 expression was decreased and miR-21 expression was increased in glioma cells and tissues. Depleting miR-21 or restoring PEG3 suppressed growth, migration and invasion as well as accelerated apoptosis of glioma cells, also raised CD8 + T proliferation, cell cytotoxic activity, and IFN-γ level as well as decreased U87 cell activity and TGF-β1 level. BMDM-Exos shuttle miR-21 promoted migration, proliferation and invasion as well as suppressed apoptosis of glioma cells by reducing PEG3. Exosomes enhanced the volume of tumor, Ki67 and PCNA expression, reduced the percentage of CD8 + T cells in glioma mice.

Section: Discussionsupporting

confidence: 91%

M2 bone marrow-derived macrophage-derived exosomes shuffle microRNA-21 to accelerate immune escape of glioma by modulating PEG3

Wang

et al. 2020

Cancer Cell Int

“…Elevated miR-21 expression leads to increased glioma cell proliferation, invasion and also chemo-resistance, which indicates poor prognosis and tumor recurrence of glioma patients [ 7 – 10 ]. On the other hand, downregulation of miR-21 leads to repression of antiapoptotic capacity, reduction of migratory, and invasion, as well as increase of chemical-induced death in glioma cells [ 8 , 11 – 13 ]. All of these make miR-21 not only a potential glioma marker for diagnosis and prognosis but a target for novel therapeutic intervention.…”

Section: Introductionmentioning

confidence: 99%

Double-Targeted Knockdown of miR-21 and CXCR4 Inhibits Malignant Glioma Progression by Suppression of the PI3K/AKT and Raf/MEK/ERK Pathways

Liu

BioMed Research International

2020

Currently, miR-21 and CXCR4 are being extensively investigated as two key regulators in glioma malignancy. In this study, we investigated the combined effects of these two factors on glioma progression. Herein, the expression of miR-21 and CXCR4 was increased in tumor tissues and cell lines. Inhibition of miR-21, CXCR4, and miR-21 and CXCR4 together all reduced the migration, invasiveness, proliferation, and enhanced apoptosis in glioma cells, as well as reduced tumor volume and mass in xenograft model. The inhibition effect was strongest in double-targeted knockdown of miR-21 and CXCR4 group, whose downstream pathways involved in AKT axis and ERK axis activation. In conclusion, our findings reported that double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion, and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways.

“…We further investigated the potential downstream pathways that might be responsible for the repressive effect of anti-miR-21 + sh-CXCR4 on tumor progression. In this study, we focused on two pathways 1) PI3K/AKT and 2) Raf/ MEK/ ERK, which both played a vital role in glioma cell fate, and were aberrant in the miR-21 and/or CXCR4 overexpression environment (Ching and Hansel 2010, Guo et al, 2013, Dongfeng et al, 2014, Du et al, 2015, Han et al, 2016, Shao et al 2017). The results from western blot analysis presented no statistically difference in AKT, ERK1/2 among different treatment groups both in glioma cells and xenografts (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Evidence shows that miR-21 is overexpressed in gliomas and glioma cells compared to normal tissues, and its expression level is positively correlated with glioma grade (Tao et al, 2013, Lei et al, 2014). Elevated miR-21 expression leads to increased glioma cell proliferation invasion, and also chemo-resistance, which indicates poor prognosis and tumor recurrence of glioma patients (Gabriely et al, 2008, Kwak et al, 2011, Melnik 2015, Hermansen et al, 2016), On the other hand, downregulation of miR-21 leads to repression of anti-apoptotic capacity, reduction of migratory and invasion, as well as increase of chemical-induced death in glioma cells (Gabriely et al 2008, Loges et al, 2012, Luo et al, 2017, Shao et al, 2017). All of these make miR-21 not only a potential glioma marker for diagnosis and prognosis, but a target for novel therapeutic intervention.…”

Section: Introductionmentioning

confidence: 99%

“…Double-targeted knockdown generated a significantly enhanced inhibition effect on tumorigenicity even compared to single-targeted knockdown of miR-21 or CXCR4 alone. Also, we found that decrease of tumor progression was related to PI3K/AKT and Raf/MEK/ERK pathways which are responsible for glioma growth, invasiveness, chemo- and radio-therapy resistance, and recurrence (Loges et al 2012, Shao et al 2017). Thus, this study might also pave ways for further research on glioma pathology and physiology.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Double-targeted knockdown of miR-21 and CXCR4 inhibits malignant glioma progression by suppression of the PI3K/AKT and Raf/MEK/ERK pathways

Liu

2020

Preprint

Background: Currently, miR-21 and CXCR4 are being extensively investigated as two unrelated key regulators in glioma malignancy. In this study, we investigated the combined effect of these two factors on glioma progression. Methods:We confirmed the expression of miR-21 and CXCR4 in malignant glioma tissue and glioma cells with qRT-PCR and western blotting. Single-targeted knockdown of miR-21 and CXCR4, as well as double-targeted knockdown of miR-21 and CXCR4 lentiviral vectors were constructed and they were transfected to U87 and U251 cells. Cell proliferation, apoptosis, invasion, and migration from different treatment groups were assessed by MTT assay, Flow Cytometry analysis, Transwell analysis, and Scratch assay, respectively. U87 xenograft mice were constructed to detect roles and potential mechanisms of miR-21 and CXCR4 in malignant glioma tumor growth. Results:The expression of miR-21 and CXCR4 was increased in tumor tissues and cell lines. Inhibition of miR-21, CXCR4, and miR-21 and CXCR4 together all reduced the migration, invasiveness, proliferation and enhanced apoptosis in glioma cells, as well as reduced tumor volume and mass in xenograft model. The inhibition effect was strongest in double-targeted knockdown of miR-21 and CXCR4 group, whose downstream pathways involved in AKT axis and ERK axis activation. Conclusions:Our findings reported that double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways.