cGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-dependent manner

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dc.contributor.advisorJae-Ho Lee-
dc.contributor.authorBASIT ABDUL-
dc.date.accessioned2022-11-29T02:32:28Z-
dc.date.available2022-11-29T02:32:28Z-
dc.date.issued2021-02-
dc.identifier.other30652-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/20008-
dc.description학위논문(석사)--아주대학교 일반대학원 :의생명과학과,2021. 2-
dc.description.tableofcontentsI. INTRODUCTION 1 II. MATERIALS AND METHODS 14 A. Antibodies 14 B. Cell culture 14 C. Synchronization and drug treatment 15 D. Plasmids and transfection experiments 16 E. Knock-down experiments 16 F. Immunoblotting 17 G. Immunocytochemistry 18 H. Time-lapse analysis 18 I. Mitotic index 19 J. Statistical analysis 19 III. RESULTS 20 A. Investigating cGAS/STING pathway to choose best suited cancer cell-line for my experimental setting 20 B. cGAS add-back can rescue cells from cGAS-depletion induced CIN 22 C. cGAS down-regulation augments chromosomal missegregations 26 D. Micronuclei formation is regulated by enzymatic activity of cGAS 29 E. STING plays mediating role to maintain chromosomal stability 32 F. Depletion of TBK1 or IRF3 also enhanced CIN phenotypes 35 G. p21 expression levels are dependent on cGAS/STING pathway 37 H. p21 as a principal regulator in maintaining chromosomal integrity via cGAS/STING pathway 42 IV. DISCUSSION 47 V. CONCLUSION 52 REFERENCES 53-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titlecGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-dependent manner-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2021. 2-
dc.description.degreeMaster-
dc.identifier.localId1202968-
dc.identifier.uciI804:41038-000000030652-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000030652-
dc.subject.keywordCell-cycle progression-
dc.subject.keywordChromosomal instability-
dc.subject.keywordInnate immunity-
dc.subject.keywordcGAS/STING/TBK1/IRF3 pathway-
dc.subject.keywordp21-
dc.description.alternativeAbstractChromosomal instability (CIN) in cancer cells has been reported to activate the cGAS-STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, converse effects of the cGAS/STING pathway per se on CIN have not yet been investigated. I addressed this issue using knockdown and add-back of each component of the cGAS/STING/TBK1/IRF3 pathway, and monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in an increased micronucleus phenotype and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK binding kinase-1), or IRF3 (interferon regulatory factor-3) also increased micronuclei formation. Moreover, transfection of cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1- and IRF3-knockdown cells in association with precocious G2/M transition and an enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated precocious G2/M transition, indicating that the decrease in p21 and subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN by defective cGAS/STING signaling.-
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Graduate School of Ajou University > Department of Biomedical Sciences > 3. Theses(Master)
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