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Exosomal Circ-MEMO1 Promotes the Progression and Aerobic Glycolysis of Non-small Cell Lung Cancer Through Targeting MiR-101-3p/KRAS Axis.

ABSTRACT: Circular RNA mediator of cell motility 1 (circ-MEMO1) was identified as an oncogene in non-small cell lung cancer (NSCLC). Nevertheless, the working mechanism behind circ-MEMO1-mediated progression of NSCLC is barely known. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of circ-MEMO1, microRNA-101-3p (miR-101-3p), and KRAS proto-oncogene, GTPase (KRAS). Cell proliferation and aerobic glycolysis were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and glycolysis detection kits. Flow cytometry was used to evaluate cell cycle progression and apoptosis of NSCLC cells. Western blot assay was used to measure the protein expression of hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), KRAS, CD9, CD81, tumor susceptibility 101 (TSG101), and Golgi matrix protein 130 kDa (GM130). The target relationship between miR-101-3p and circ-MEMO1 or KRAS was predicted by StarBase software and confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA-pull down assay. In vivo tumor growth assay was conducted to assess the effect of circ-MEMO1 in vivo. Exosomes were isolated using the ExoQuick precipitation kit. Circ-MEMO1 was up-regulated in NSCLC, and high expression of circ-MEMO1 predicted poor prognosis in NSCLC patients. Circ-MEMO1 accelerated the proliferation, cell cycle progression, and glycolytic metabolism and inhibited the apoptosis of NSCLC cells. Circ-MEMO1 negatively regulated the expression of miR-101-3p through direct interaction, and si-circ-MEMO1-induced biological effects were attenuated by the introduction of anti-miR-101-3p. MiR-101-3p directly interacted with the 3' untranslated region (3' UTR) of KRAS messenger RNA (mRNA), and KRAS level was regulated by circ-MEMO1/miR-101-3p axis. Circ-MEMO1 silencing suppressed the NSCLC tumor growth in vivo. ROC curve analysis revealed that high expression of serum exosomal circ-MEMO1 (exo-circ-MEMO1) might be a valuable diagnostic marker for NSCLC. Circ-MEMO1 facilitated the progression and glycolysis of NSCLC through regulating miR-101-3p/KRAS axis.

SUBMITTER: Ding C

PROVIDER: S-EPMC7483554 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Exosomal Circ-MEMO1 Promotes the Progression and Aerobic Glycolysis of Non-small Cell Lung Cancer Through Targeting MiR-101-3p/KRAS Axis.

Ding Chengzhi C Xi Gaoyuan G Wang Guolei G Cui Dong D Zhang Binbin B Wang Hongtao H Jiang Gongqian G Song Jingchao J Xu Guanghui G Wang Jiao J

Frontiers in genetics 20200828

Circular RNA mediator of cell motility 1 (circ-MEMO1) was identified as an oncogene in non-small cell lung cancer (NSCLC). Nevertheless, the working mechanism behind circ-MEMO1-mediated progression of NSCLC is barely known. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of circ-MEMO1, microRNA-101-3p (miR-101-3p), and KRAS proto-oncogene, GTPase (KRAS). Cell proliferation and aerobic glycolysis were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-dip ...[more]

PMID: 33005174

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Project description:BackgroundColorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear.MethodsCRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils.ResultsOur study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis.ConclusionOur study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Project description:BackgroundExosomes are extracellular vesicles secreted by most cells to deliver functional cargoes to recipient cells. MicroRNAs (miRNAs) constitute a significant part of exosomal contents. The ease of diffusion of exosomes renders them speedy and highly efficient vehicles to deliver functional molecules. Cancer cells secrete more exosomes than normal cells. Reports have showed that exosomal miRNAs of cancer cells facilitate cancer progression. Yet the complexity of cancer dictates that many more functional exosomal miRNAs remain to be discovered.MethodsIn this study, we analyzed miRNA expression profiles of tissue and plasma exosome samples collected from 10 colorectal cancer (CRC) patients and 10 healthy individuals. We focused on hsa-miR-101-3p (101-3p), a profoundly up-regulated miRNA enriched in plasma exosomes of patients bearing CRC. We performed target analysis of 101-3p and pursued functional studies of this microRNA in two colorectal cancer cell lines, namely HCT116 and SW480.ResultsOur results indicated that inhibiting 101-3p slowed cell growth and retarded cell migration in vivo in two colorectal cancer cell lines. Target analysis showed that Homeodomain-interacting protein kinase (HIPK3) is a target of miR-101-3p. HCT116 and SW480 cells stably overexpressing HIPK3 showed increased level of phosphorylated FADD, as well as retarded cell growth, migration, and increased sensitivity to 5-FU. In-depth analysis revealed increased mitochondrial membrane potential upon HIPK3 overexpression along with increased production of reactive oxygen species, number of mitochondria, and expression of respiratory complexes. Measurements of glycolytic parameters and enzymes revealed decreased level of glycolysis upon HIPK3 overexpression in these two cell lines. Xenograft model further confirmed a profoundly improved potency of the synergistic treatment combining both 5-FU and 101-3p inhibitor compared to 5-FU alone.ConclusionThis study unraveled an oncogenic nature of the exosomal 101-3p and suggested a relationship between the 101-3p-HIPK3 axis and metabolic homeostasis in colorectal cancer. Expression level of 101-3p is positively correlated with glycolytic capacity in CRC and therefore 101-3p itself is an oncomiR. Combining 101-3p inhibitor with chemotherapeutic agents is an effective strategy against CRC.

Dataset Information

Exosomal Circ-MEMO1 Promotes the Progression and Aerobic Glycolysis of Non-small Cell Lung Cancer Through Targeting MiR-101-3p/KRAS Axis.

Publications

Exosomal Circ-MEMO1 Promotes the Progression and Aerobic Glycolysis of Non-small Cell Lung Cancer Through Targeting MiR-101-3p/KRAS Axis.

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