Extracellular vesicles derived from hypoxic glioma stem-like cells confer temozolomide resistance on glioblastoma by delivering miR-30b-3p.

Rationale: Glioma stem-like cells (GSCs) contribute to temozolomide (TMZ) resistance in gliomas, although the mechanisms have not been delineated. Methods:In vitro functional experiments (colony formation assay, flow cytometric analysis, TUNEL assay) were used to assess the ability of extracellular vesicles (EVs) from hypoxic GSCs to promote TMZ resistance in glioblastoma (GBM) cells. RNA sequencing and quantitative Reverse Transcription-PCR were employed to identify the functional miRNA in hypoxic EVs. Chromatin immunoprecipitation assays were performed to analyze the transcriptional regulation of miRNAs by HIF1α and STAT3. RIP and RNA pull-down assays were used to validate the hnRNPA2B1-mediated packaging of miRNA into EVs. The function of EV miR-30b-3p from hypoxic GSCs was verified by in vivo experiments and analysis of clinical samples. Results: Hypoxic GSC-derived EVs exerted a greater effect on GBM chemoresistance than those from normoxic GSCs. The miRNA profiling revealed that miR-30b-3p was significantly upregulated in the EVs from hypoxic GSCs. Further, HIF1α and STAT3 transcriptionally induced miR-30b-3p expression. RNA immunoprecipitation and RNA-pull down assays revealed that binding of miR-30b-3p with hnRNPA2B1 facilitated its transfer into EVs. EV-packaged miR-30b-3p (EV-miR-30b-3p) directly targeted RHOB, resulting in decreased apoptosis and increased proliferation in vitro and in vivo. Our results provided evidence that miR-30b-3p in CSF could be a potential biomarker predicting resistance to TMZ. Conclusion: Our findings indicated that targeting EV-miR-30b-3p could provide a potential treatment strategy for GBM.

Yin J ，Ge X ，Shi Z ，Yu C ，Lu C ，Wei Y ，Zeng A ，Wang X ，Yan W ，Zhang J ，You Y ... -  《Theranostics》

Extracellular vesicles carry miR-27a-3p to promote drug resistance of glioblastoma to temozolomide by targeting BTG2.

Glioblastoma (GBM) is the most common central nervous system tumor. Temozolomide (TMZ) is a commonly used drug for GBM management. This study explored the mechanism of extracellular vesicles (EVs) regulating TMZ-resistance in GBM. LN229 cells were inducted into TMZ-resistant LN229r strain by stepwise induction. After the intervention of miR-27a-3p expression, cell viability of GBM cells treated with different concentrations of TMZ was detected by MTT and IC50 value was calculated. Cell proliferation and apoptosis were detected by colony formation and flow cytometry. EVs extracted from LN18 cells were identified and the internalization of EVs by LN229r cells was evaluated. The 100 μmol/L TMZ-treated LN229r cells were treated with EVs or EVs with downregulated miR-27a-3p to verify the effect of EVs-carried miR-27a-3p on TMZ resistance. The binding relation between BTG2 and miR-27a-3p was verified. miR-27a-3p and BTG2 expressions in GBM cells and EVs were detected by RT-qPCR. The BTG2 effect on TMZ-resistance in GBM was verified. The xenograft tumor nude mouse model was established by injecting LN229r cells and treated with EVs and 100 μmol/L TMZ. miR-27a-3p was highly expressed in LN229r cells. IC50 value and proliferation of LN229r cells with silenced miR-27a-3p were decreased and apoptosis was increased, indicating that miR-27a-3p silencing reduced the drug-resistant cell LN229r resistance to TMZ. LN18-derived EVs could be internalized by LN229r cells, and release its encapsulated miR-27a-3p into LN229r cells and increase miR-27a-3p expression. EV treatment increased LN229r cell proliferation and reduced apoptosis, while EVs with silenced miR-27a-3p showed the opposite trend. miR-27a-3p targeted BTG2. BTG2 overexpression reduced LN229r cell resistance to TMZ. In vivo, after EVs treatment, tumor volume and weight, Ki67-positive rate, and miR-27a-3p were increased, while BTG2 expression was decreased. GBM-derived EVs were internalized by GBM cells, released miR-27a-3p into GBM cells, upregulated miR-27a-3p expression, and targeted BTG2, thus promoting TMZ resistance.

Chen L ，Li Z ，Hu S ，Deng Q ，Hao P ，Guo S ... -  《-》

miR-126-3p sensitizes glioblastoma cells to temozolomide by inactivating Wnt/β-catenin signaling via targeting SOX2.

The acquired drug resistance has been regarded as a main barrier for the effective treatment of temozolomide (TMZ) in glioblastoma (GBM). MiR-126-3p is commonly down-regulated and exerts tumor-suppressive roles in kinds of human cancers, including GBM. This study was designed to investigate the functions and mechanisms of miR-126-3p in regulating TMZ resistance in GBM. qRT-PCR analysis was used to measure the expressions of miR-126-3p and SOX2 mRNA in GBM tissues and cells. Cell viability, colony forming ability and apoptosis were detected to evaluate the effect of miR-126-3p or SOX2 on TMZ resistance. Luciferase reporter experiments were applied to identify the target genes of miR-126-3p. Western blot analysis was performed to determine the protein levels associated with Wnt/β-catenin signaling. TOP/FOP Flash assays were conducted to determine the effects of miR-126-3p or SOX2 on Wnt/β-catenin signaling. miR-126-3p expression was decreased in TMZ-resistant GBM tissues and cells. High levels of miR-126-3p enhanced TMZ sensitivity by inhibiting cell viability, reducing colony forming potential and inducing apoptosis. Additionally, SOX2 was identified as a downstream target of miR-126-3p. On the contrary, SOX2 overexpression conferred TMZ resistance of GBM cells. Moreover, miR-126-3p-mediated TMZ sensitivity was reversed following increased expression of SOX2. Furthermore, miR-126-3p-induced inactivation of Wnt/β-catenin signaling was greatly abrogated by SOX2 up-regulation. MiR-126-3p sensitizes GBM cells to TMZ possibly by repressing SOX2 expression and blocking Wnt/β-catenin signaling. This study provides novel targets to overcome TMZ resistance in GBM chemotherapy.

Luo W ，Yan D ，Song Z ，Zhu X ，Liu X ，Li X ，Zhao S ... -  《-》

DNA-methylation-mediated activating of lncRNA SNHG12 promotes temozolomide resistance in glioblastoma.

Lu C ，Wei Y ，Wang X ，Zhang Z ，Yin J ，Li W ，Chen L ，Lyu X ，Shi Z ，Yan W ，You Y ... -  《Molecular Cancer》

Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma.

Chemoresistance blunts the effect of Temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Whether exosomal transfer of miRNAs derived from TMZ-resistant GBM cells could confer TMZ resistance remains to be determined. qPCR was used to determine miR-151a expression in two TMZ-resistant GBM cell lines. The direct targets of miR-151a were identified by microarray assays, bioinformatics and further RNA chromatin immunoprecipitation (RNA-ChIP) assay. We characterized exosomes from TMZ-resistant cell lines, serum and cerebrospinal fluid (CSF) and determined the effect of exosomes from TMZ-resistant cells on recipient GBM cells. miR-151a loss drove the acquisition of TMZ resistance. Restored miR-151a expression sensitized TMZ-resistant GBM cells via inhibiting XRCC4-mediated DNA repair. TMZ-resistant GBM cells conferred TMZ chemoresistance to recipient TMZ-sensitive cells in an exosomal miR-151a loss-dependent manner. Restoration of exosomal miR-151a from donor TMZ-resistant cells abolished the chemoresistance dissemination that was directed by donor TMZ-resistant cells. CSF-derived exosomes contained miRNA signatures reflective of the underlying chemoresistant status of GBMs in terms of miR-151a expression levels. Exosomal miR-151a is not only essentially a less-invasive 'liquid biopsy' that might predict chemotherapy response, but also represents a promising therapeutic target for therapy-refractory GBMs.

Zeng A ，Wei Z ，Yan W ，Yin J ，Huang X ，Zhou X ，Li R ，Shen F ，Wu W ，Wang X ，You Y ... -  《-》