Dataset Information

Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

ABSTRACT: Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-seq to provide a comprehensive map of transcriptome regulation upon TMZ in patient-derived GBM stem-like cells displaying different drug sensitivity. In a search for regulatory mechanisms, we integrated thousands of molecular associations stored in public databases to generate a background "RNA interactome". Our systems-level analysis uncovered a coordinated program of TMZ response reflected by regulatory circuits that involve transcription factors, mRNAs, miRNAs, and lncRNAs. We discovered 22 lncRNAs involved in regulatory loops and/or with functional relevance in drug response and prognostic value in gliomas. Thus, the investigation of TMZ-induced gene networks highlights novel RNA-based predictors of chemosensitivity in GBM. The computational modeling used to identify regulatory circuits underlying drug response and prioritizing gene candidates for functional validation is applicable to other datasets.

SUBMITTER: Fritah S

PROVIDER: S-EPMC7563839 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

Fritah Sabrina S Muller Arnaud A Jiang Wei W Mitra Ramkrishna R Sarmini Mohamad M Dieterle Monika M Golebiewska Anna A Ye Tao T Van Dyck Eric E Herold-Mende Christel C Zhao Zhongming Z Azuaje Francisco F Niclou Simone P SP

Cancers 20200910 9

Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-s ...[more]

PMID: 32927769

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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:BackgroundAccumulating evidence shows that long noncoding RNAs (lncRNAs) are important regulator molecules involved in diverse biological processes. Acquired drug resistance is a major challenge in the clinical treatment of glioblastoma (GBM), and lncRNAs have been shown to play a role in chemotherapy resistance. However, the underlying mechanisms by which lncRNA mediates TMZ resistance in GBM remain poorly characterized.MethodsQuantitative reverse transcription PCR (qRT-PCR) and fluorescence in situ hybridization assays were used to detect small nucleolar RNA host gene 12 (SNHG12) levels in TMZ-sensitive and TMZ-resistant GBM cells and tissues. The effects of SNHG12 on TMZ resistance were investigated through in vitro assays (western blots, colony formation assays, flow cytometry assays, and TUNEL assays). The mechanism mediating the high expression of SNHG12 in TMZ-resistant cells and its relationships with miR-129-5p, mitogen-activated protein kinase 1 (MAPK1), and E2F transcription factor 7 (E2F7) were determined by bioinformatic analysis, bisulfite amplicon sequencing, methylation-specific PCR, dual luciferase reporter assays, chromatin immunoprecipitation assays, RNA immunoprecipitation assays, immunofluorescence, qRT-PCR, and western blot. For in vivo experiments, an intracranial xenograft tumor mouse model was used to investigate SNHG12 function.ResultsSNHG12 was upregulated in TMZ-resistant cells and tissues. Overexpression of SNHG12 led to the development of acquired TMZ resistance, while knockdown of SNHG12 restored TMZ sensitivity. An abnormally low level of DNA methylation was detected within the promoter region of SNHG12, and loss of DNA methylation made this region more accessible to the Sp1 transcription factor (SP1); this indicated that methylation and SP1 work together to regulate SNHG12 expression. In the cytoplasm, SNHG12 served as a sponge for miR-129-5p, leading to upregulation of MAPK1 and E2F7 and endowing the GBM cells with TMZ resistance. Disinhibition of MAPK1 regulated TMZ-induced cell apoptosis and the G1/S cell cycle transition by activating the MAPK/ERK pathway, while E2F7 dysregulation was primarily associated with G1/S cell cycle transition. Clinically, SNHG12 overexpression was associated with poor survival of GBM patients undergoing TMZ treatment.ConclusionOur results suggest that SNHG12 could serve as a promising therapeutic target to surmount TMZ resistance, thereby improving the clinical efficacy of TMZ chemotherapy.

Project description:Background:LncRNAs have been shown to play essential roles in cancer therapeutic response. However, the detailed mechanism of lncRNAs in temozolomide (TMZ) resistance in glioblastoma (GBM) remain to be elucidated. Methods:To elucidate the mechanism maintaining TMZ resistance, we constructed two TMZ-resistant GBM cell lines (T98G-R/U118-R). LncRNAs from four public datasets were reanalyzed, and the candidate lncRNA ADAMTS9-AS2 was evaluated in TMZ-treated GBM patients and in vitro cell lines. Results:Reanalysis of lncRNA expression profiles identified ADAMTS9-AS2 as significantly overexpressed in TMZ-resistant GBM cells and as positively associated with the IC50 of TMZ in GBM cells. Overexpression of ADAMTS9-AS2 was also significantly associated with poor TMZ response and shorter progression-free survival (PFS) in TMZ-treated GBM patients. Knockdown of ADAMTS9-AS2 inhibited proliferation and attenuated the IC50 of TMZ, as well as mitigating invasion and migration in TMZ-resistant GBM cells. Subsequent investigations indicated that reduced expression of ADAMTS9-AS2 significantly suppressed expression of the FUS protein, which was predicted as a direct substrate of ADAMTS9-AS2. Expression trends of FUS were directly correlated with those of ADAMTS9-AS2, as shown by increasing concentrations and prolonged treatment with TMZ. RNA pull-down and RIP assays indicated that both endogenous and exogenous ADAMTS9-AS2 directly binds to the RRM and Znf_RanBP2 domains of FUS, consequently increasing FUS protein expression. Knockdown of ADAMTS9-AS2 reduced the half-life of FUS and decreased FUS protein stability via K48 ubiquitin degradation. Moreover, the E3 ubiquitin-protein ligase MDM2 interacts with and down regulates FUS, while the RRM and Znf_RanBP2 domains of FUS facilitate its binding with MDM2. ADAMTS9-AS2 decreased the interaction between MDM2 and FUS, which mediates FUS K48 ubiquitination. Additionally, knockdown of the ADAMTS9-AS2/FUS signaling axis significantly alleviated progression and metastasis in TMZ-resistant cells. Conclusion:ADAMTS9-AS2 possessed a novel function that promotes TMZ resistance via upregulating the FUS/MDM2 axis in GBM cells. The RRM or Znf_RanBP2 domains of FUS facilitate the combination of ADAMTS9-AS2 and FUS, competitively inhibiting MDM2-dependent FUS K48 ubiquitination and resulting in enhanced FUS stability and TMZ resistance. Our results suggest that the ADAMTS9-AS2/FUS/MDM2 axis may represent a suitable prognostic biomarker and a potential target in TMZ-resistant GBM therapy.

Dataset Information

Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

Publications

Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

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