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MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy.

ABSTRACT: Temozolomide (TMZ) is one of the most commonly used drugs for the clinical treatment of glioblastomas. However, it has been reported that treatment with TMZ can induce autophagy, which leads to tumor resistance and increases the survival of tumor cells. MicroRNA-30a (miR-30a) has been found to have inhibitory effects on autophagy by directly targeting beclin 1. However, the exact role of miR-30a in TMZ-treated glioblastoma cells has not been studied previously. The present study aimed to investigate whether miR-30a increased the cytotoxicity of TMZ to glioblastoma U251 cells, as well as the underlying mechanism. MTT and flow cytometry assay results showed that treatment with TMZ inhibited the proliferation of U251 cells while inducing cell apoptosis in a dose-dependent manner. Western blotting data showed that the expression levels of LC3-II and beclin 1 as well as the ratio of LC3-II to LC3-I were markedly increased in TMZ-treated U251 cells compared with the untreated control cells, indicating that treatment with TMZ induced autophagy. Moreover, reverse transcription-quantitative polymerase chain reaction data showed that treatment with TMZ led to a significant reduction in miR-30a levels in a dose-dependent manner in U251 cells. Elevation of the miR-30a level significantly inhibited TMZ-induced autophagy, demonstrated by the decreased LC3-II and beclin 1 levels and ratio of LC3-II to LC3-I, accompanied by the reduced proliferation and increased apoptosis in TMZ-treated U251 cells. Furthermore, luciferase reporter assay data indicated that beclin 1 was a direct target of miR-30a in U251 cells. In summary, this study demonstrated that miR-30a increases the chemosensitivity of glioblastoma U251 cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy. Therefore, autophagy may be a promising target for the treatment of TMZ-resistant tumors.

SUBMITTER: Xu J

PROVIDER: S-EPMC5952084 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy.

Xu Jing J Huang He H Peng Renjun R Ding Xiping X Jiang Bing B Yuan Xianrui X Xi Jian J

Experimental and therapeutic medicine 20180329 6

Temozolomide (TMZ) is one of the most commonly used drugs for the clinical treatment of glioblastomas. However, it has been reported that treatment with TMZ can induce autophagy, which leads to tumor resistance and increases the survival of tumor cells. MicroRNA-30a (miR-30a) has been found to have inhibitory effects on autophagy by directly targeting beclin 1. However, the exact role of miR-30a in TMZ-treated glioblastoma cells has not been studied previously. The present study aimed to investi ...[more]

PMID: 29805498

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Project description:BackgroundThe regulatory roles of long non-coding RNA (lncRNA) CRNDE in temozolomide (TMZ) chemoresistance to glioblastoma multiforme (GBM) are still poorly understood. Therefore, the function, characteristics, and possible mechanism of CRNDE in TMZ-induced chemoresistance to GBM were explored.MethodsFirstly, the expression level of CRNDE in 58 cases of glioma tissue specimens and 30 cases of normal brain tissues were tested by qRT-PCR. Meanwhile, the correlation between CRNDE expression level, the clinicopathological characteristics, and survival time of patients with glioma were analyzed. Then, the CRNDE expression in various glioma cell lines was detected, and CRNDE knockdown cell models were constructed. Subsequently, to explore the effect of CRNDE on chemosensitivity to TMZ, cell viability was detected by the CCK-8 assay and IC50 values, and cell proliferation was detected by cell clone assay and EdU assay, as well as cell survival was detected by apoptosis with flow cytometry under TMZ treatment. Further, the expression of drug-resistance protein ABCG2, autophagy related proteins, and PI3K/Akt/mTOR pathway were measured by western blot or qRT-PCR in TMZ-treated glioma cells. Finally, the mouse tumor xenograft model was established and the tumor volume and weight were measured, and ABCG2 expression was conducted by immunohistochemistry assay.ResultsThe integrated results demonstrated lncRNA CRNDE was a poor prognosis factor for GBM patient, which was upregulated in patients who were resistant to TMZ, and closely associated with chemotherapeutic response status to TMZ treatment. Further, functional assays revealed that knockdown of CRNDE could notably reduce glioma cell viability and proliferation, and elevate cell apoptosis to enhance the chemosensitivity to TMZ in vitro and in vivo. Mechanistically, the depression of CRNDE could diminish the expression of LC3 II/I, Beclin1 and Atg5 and increase the p62 expression level to inhibit autophagy due to the activation of PI3K/Akt/mTOR pathway as well as highly correlated with ABCG2 expression.ConclusionsOverall, the study provided that lncRNA CRNDE is a reliable clinical predictor of outcome and prognosis and a potential biomarker for predicting TMZ treatment response in GBM by modulating the autophagy through PI3K/Akt/mTOR pathway and ABCG2 expression which may be a novel therapeutic target for regulating TMZ sensitivity to GBM.

Project description:BackgroundGlioblastoma (GB) is the most common and highly malignant brain tumor characterized by aggressive growth and resistance to alkylating chemotherapy. Autophagy induction is one of the hallmark effects of anti-GB therapies with temozolomide (TMZ). However, the non-classical form of autophagy, autophagy-based unconventional secretion, also called secretory autophagy and its role in regulating the sensitivity of GB to TMZ remains unclear. There is an urgent need to illuminate the mechanism and to develop novel therapeutic targets for GB.MethodsCancer genome databases and paired-GB patient samples with or without TMZ treatment were used to assess the relationship between HMGB1 mRNA levels and overall patient survival. The relationship between HMGB1 protein level and TMZ sensitivity was measured by immunohistochemistry, ELISA, Western blot and qRT-PCR. GB cells were engineered to express a chimeric autophagic flux reporter protein consisting of mCherry, GFP and LC3B. The role of secretory autophagy in tumor microenvironment (TME) was analyzed by intracranial implantation of GL261 cells. Coimmunoprecipitation (Co-IP) and Western blotting were performed to test the RAGE-NFκB-NLRP3 inflammasome pathway.ResultsThe exocytosis of HMGB1 induced by TMZ in GB is dependent on the secretory autophagy. HMGB1 contributed to M1-like polarization of tumor associated macrophages (TAMs) and enhanced the sensitivity of GB cells to TMZ. Mechanistically, RAGE acted as a receptor for HMGB1 in TAMs and through RAGE-NFκB-NLRP3 inflammasome pathway, HMGB1 enhanced M1-like polarization of TAMs. Clinically, the elevated level of HMGB1 in sera may serve as a beneficial therapeutic-predictor for GB patients under TMZ treatment.ConclusionsWe demonstrated that enhanced secretory autophagy in GB facilitates M1-like polarization of TAMs to enhance TMZ sensitivity of GB cells. HMGB1 acts as a key regulator in the crosstalk between GB cells and tumor-suppressive M1-like TAMs in GB microenvironment and may be considered as an adjuvant for the chemotherapeutic agent TMZ.

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MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy.

Publications

MicroRNA-30a increases the chemosensitivity of U251 glioblastoma cells to temozolomide by directly targeting beclin 1 and inhibiting autophagy.

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