RNA interference-mediated silencing of SOCS-1 via lentiviral vector promotes apoptosis of alveolar epithelial cells in vitro

Suppressor of cytokine signaling-1 (SOCS1) is a protein that negatively regulates cytokine and growth factor signaling. However, little is known regarding the precise role it plays in idiopathic pulmonary fibrosis. The aim of the present study was to construct a recombinant lentiviral vector for RNA...

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Veröffentlicht in:Molecular medicine reports 2012-02, Vol.5 (2), p.452-456
Hauptverfasser: Qian, Yan-Rong, Zhang, Qiu-Rui, Cheng, Ting, Wan, Huan-Ying, Zhou, Min
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creator Qian, Yan-Rong
Zhang, Qiu-Rui
Cheng, Ting
Wan, Huan-Ying
Zhou, Min
description Suppressor of cytokine signaling-1 (SOCS1) is a protein that negatively regulates cytokine and growth factor signaling. However, little is known regarding the precise role it plays in idiopathic pulmonary fibrosis. The aim of the present study was to construct a recombinant lentiviral vector for RNA interference targeting the SOCS1 gene and to detect the expression in human alveolar epithelial cells. A lentiviral vector-mediated RNA interference method was used to establish a SOCS1-negative cell line of alveolar origin (A549). Three pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the SOCS1 gene were designed, synthesized and inserted into the pPll3.7 vector. Packaged lentivirus particles were obtained after 48 h, and the supernatant was used to transfect the human alveolar epithelial cell line A549. The expression of the SOCS1 protein was detected by Western blotting. MTT assay was used to detect the cell proliferation of alveolar epithelial cells with SOCS1 knockdown. The recombinant plasmids were confirmed by sequencing. The lentivirus-containing supernatant effectively infected the A549 cell line, and the expression of SOCS1 protein was inhibited, which was confirmed by Western blotting in the target cells. MTT assay indicated the inhibition effect for cell proliferation of A549 cells in the SOCS1-RNA interference group, compared to the control group with no interference-mediated silencing of the SOCS1 gene. A lentiviral vector for RNA interference targeting the SOCS1 gene was successfully constructed, and cell survival tests showed that knockdown of the SOCS1 gene promotes the apoptosis of alveolar cells.
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However, little is known regarding the precise role it plays in idiopathic pulmonary fibrosis. The aim of the present study was to construct a recombinant lentiviral vector for RNA interference targeting the SOCS1 gene and to detect the expression in human alveolar epithelial cells. A lentiviral vector-mediated RNA interference method was used to establish a SOCS1-negative cell line of alveolar origin (A549). Three pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the SOCS1 gene were designed, synthesized and inserted into the pPll3.7 vector. Packaged lentivirus particles were obtained after 48 h, and the supernatant was used to transfect the human alveolar epithelial cell line A549. The expression of the SOCS1 protein was detected by Western blotting. MTT assay was used to detect the cell proliferation of alveolar epithelial cells with SOCS1 knockdown. The recombinant plasmids were confirmed by sequencing. The lentivirus-containing supernatant effectively infected the A549 cell line, and the expression of SOCS1 protein was inhibited, which was confirmed by Western blotting in the target cells. MTT assay indicated the inhibition effect for cell proliferation of A549 cells in the SOCS1-RNA interference group, compared to the control group with no interference-mediated silencing of the SOCS1 gene. A lentiviral vector for RNA interference targeting the SOCS1 gene was successfully constructed, and cell survival tests showed that knockdown of the SOCS1 gene promotes the apoptosis of alveolar cells.</description><identifier>ISSN: 1791-2997</identifier><identifier>EISSN: 1791-3004</identifier><identifier>DOI: 10.3892/mmr.2011.672</identifier><identifier>PMID: 22086204</identifier><language>eng</language><publisher>Greece: Spandidos Publications UK Ltd</publisher><subject>Adenocarcinoma, Bronchiolo-Alveolar - metabolism ; Adenocarcinoma, Bronchiolo-Alveolar - pathology ; Alveoli ; Apoptosis ; Base Sequence ; Cell growth ; Cell Line, Tumor ; Cell proliferation ; Cell survival ; Cellular biology ; Cytokines ; Epithelial cells ; Epithelial Cells - cytology ; Epithelial Cells - metabolism ; Fibrosis ; Gene Silencing ; Genetic Vectors - chemistry ; Genetic Vectors - metabolism ; Growth factors ; Humans ; Inflammation ; Kinases ; Lentivirus - genetics ; Lung diseases ; Molecular Sequence Data ; Oligonucleotides ; Plasmids ; Proteins ; Pulmonary fibrosis ; RNA Interference ; RNA, Small Interfering - metabolism ; RNA-mediated interference ; SOCS-1 protein ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins - antagonists &amp; inhibitors ; Suppressor of Cytokine Signaling Proteins - genetics ; Suppressor of Cytokine Signaling Proteins - metabolism ; Western blotting</subject><ispartof>Molecular medicine reports, 2012-02, Vol.5 (2), p.452-456</ispartof><rights>Copyright Spandidos Publications UK Ltd. 2012</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c356t-bb30696499148d543570513b245fe46878be04bb0fe90977d29ce8c3f544e67a3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22086204$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Qian, Yan-Rong</creatorcontrib><creatorcontrib>Zhang, Qiu-Rui</creatorcontrib><creatorcontrib>Cheng, Ting</creatorcontrib><creatorcontrib>Wan, Huan-Ying</creatorcontrib><creatorcontrib>Zhou, Min</creatorcontrib><title>RNA interference-mediated silencing of SOCS-1 via lentiviral vector promotes apoptosis of alveolar epithelial cells in vitro</title><title>Molecular medicine reports</title><addtitle>Mol Med Rep</addtitle><description>Suppressor of cytokine signaling-1 (SOCS1) is a protein that negatively regulates cytokine and growth factor signaling. However, little is known regarding the precise role it plays in idiopathic pulmonary fibrosis. The aim of the present study was to construct a recombinant lentiviral vector for RNA interference targeting the SOCS1 gene and to detect the expression in human alveolar epithelial cells. A lentiviral vector-mediated RNA interference method was used to establish a SOCS1-negative cell line of alveolar origin (A549). Three pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the SOCS1 gene were designed, synthesized and inserted into the pPll3.7 vector. Packaged lentivirus particles were obtained after 48 h, and the supernatant was used to transfect the human alveolar epithelial cell line A549. The expression of the SOCS1 protein was detected by Western blotting. MTT assay was used to detect the cell proliferation of alveolar epithelial cells with SOCS1 knockdown. The recombinant plasmids were confirmed by sequencing. 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subjects Adenocarcinoma, Bronchiolo-Alveolar - metabolism
Adenocarcinoma, Bronchiolo-Alveolar - pathology
Alveoli
Apoptosis
Base Sequence
Cell growth
Cell Line, Tumor
Cell proliferation
Cell survival
Cellular biology
Cytokines
Epithelial cells
Epithelial Cells - cytology
Epithelial Cells - metabolism
Fibrosis
Gene Silencing
Genetic Vectors - chemistry
Genetic Vectors - metabolism
Growth factors
Humans
Inflammation
Kinases
Lentivirus - genetics
Lung diseases
Molecular Sequence Data
Oligonucleotides
Plasmids
Proteins
Pulmonary fibrosis
RNA Interference
RNA, Small Interfering - metabolism
RNA-mediated interference
SOCS-1 protein
Suppressor of Cytokine Signaling 1 Protein
Suppressor of Cytokine Signaling Proteins - antagonists & inhibitors
Suppressor of Cytokine Signaling Proteins - genetics
Suppressor of Cytokine Signaling Proteins - metabolism
Western blotting
title RNA interference-mediated silencing of SOCS-1 via lentiviral vector promotes apoptosis of alveolar epithelial cells in vitro
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