Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of normalized signal intensities of various miRNAs in the exsosomes isolated by Exoquick or ultracentrifugation

ABSTRACT: MicroRNAs (miRNAs), small (18–22 nucleotides) non-coding RNAs, suppress the translation of target mRNAs by binding to their 3′ untranslated region. Evidence suggests that miRNAs are key regulators of several cellular processes, including angiogenesis. Recent findings indicate that Secreted miRNAs enclosed in exosomes also play an important role in cell–cell communication. To elucidate whether miRNAs secreted from neoplastic cells transfer into endothelial cells and are functionally active in the recipient cells, we investigated the interaction of a leukemia cell line and human umbilical vein endothelial cells (HUVECs). The culture medium was collected and centrifuged at 3000 × g for 15 min. The supernatant was filtered through a 0.22-µm PVDF filter (Millipore). The appropriate volume of Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA) was added to the filtered culture medium and mixed well by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1,500 × g for 30 min and all supernatant was removed by aspiration. Exosome pellets were resuspended with 500 µl of the serum-free AIM V medium (Invitrogen). K562 cells or K562/Cy3-miR-92a cells (K562 cells transfected with Cy3-labeled pre-miR-92a) were pre-cultured in serum-free AIM V medium (Invitrogen). The cells were cultured in 200 ml of AIM V medium (twenty 100 mm culture dishes). For exosome preparation, the culture media were collected and centrifuged at 2,000 × g for 10 min at room temperature, and the supernatant was centrifuged again at 12,000 × g for 30 min at room temperature, then the supernatant was ultracentrifuged at 110,000 × g for 70 min at 4 ºC. The pellets were washed with PBS, after ultracentrifugation, they were used for Microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Tomohiro Umezu

PROVIDER: E-GEOD-35256 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, KMS-11-HR, derived from KMS-11 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used KMS-11 cells and KMS-11-HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from KMS-11 cells (normoxia or hypoxia) and exosomes derived from KMS-11-HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the KMS-11-HR cells significantly increased tube formation of HUVECs than those from KMS-11 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in KMS-11 cells and KMS-11-HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). KMS-11 cells and KMS-11-HR cells were cultured for 24 hours under hypoxic conditions (1% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, U266HR, derived from U266 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used U266 cells and U266HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from U266 cells (normoxia or hypoxia) and exosomes derived from U266HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the U266HR cells significantly increased tube formation of HUVECs than those from U266 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in U266 cells and U266HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). U266 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, RPMI8226HR, derived from RPMI8226 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used RPMI8226 cells and RPMI8226HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from RPMI8226 cells (normoxia or hypoxia) and exosomes derived from RPMI8226HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the RPMI8226HR cells significantly increased tube formation of HUVECs than those from RPMI8226 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in RPMI8226 cells and RPMI8226HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). RPMI8226 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

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Comparison of normalized signal intensities of various miRNAs in the exsosomes isolated by Exoquick or ultracentrifugation

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