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Title:	Pemprofilan transkriptom dan proteom eosinofil teraktif dalam asma
Authors:	Norziha Zainul Abidin (P34104)
Supervisor:	Roslan Harun, Prof Madya Dr.
Keywords:	Asma Dissertations, Academic -- Malaysia
Issue Date:	28-Jun-2014
Description:	Inflamasi eosinofil saluran pernafasan merupakan satu ciri asma. Peningkatan aras eosinofil dalam darah periferi dan mukosa bronkial telah dikaitkan dengan keterukan dan status kawalan asma. Walau bagaimanapun, mekanisme yang tepat bagaimana eosinofil menyumbang kepada fenotip asma yang berbeza sebahagian besarnya masih tidak diketahui. Objektif kajian ini adalah untuk (i) mengenalpasti profil transkriptom dan proteom berkaitan dengan pengaktifan eosinofil aruhan IL-5, (ii) menentukan tapak jalan molekul dan proses biologikal yang terlibat dalam pengaktifan eosinofil dan (iii) mengenalpasti profil pengekspresan gen dan tapak jalan molekul yang berkaitan dengan pelbagai fenotip klinikal asma. Eosinofil darah periferi telah diaruh dengan IL-5 selama 24 jam dan kemudiannya RNA jumlah dan protein telah diekstrak pada titik masa bersiri: awal (0j dan 1j) dan lewat (6j, 12j dan 24j) untuk pengekspresan gen menggunakan Illumina Sentrix® Human RefSeq-8 Beadchip v2 dan pemprofilan proteom bebas-label LC-MS/MS masing-masing. Analisis pengekspresan gen juga dilakukan ke atas eosinofil darah periferi yang dipencilkan daripada pesakit asma pelbagai fenotip (n = 40) dan subjek normal (n = 7). Kajian transkriptom in vitro mengenalpasti 301 gen yang mengalami perbezaan ekspresi (p ï‚£ 0.05, 2 FC) pada fasa awal pengaktifan eosinofil. Beberapa proses biologi dan tapak jalan penting telah dikenalpasti seperti pengisyaratan TCR, pengisyaratan TLR, pengisyaratan MAPK dan apoptosis. Walau bagaimanapun, hanya 67 protein telah dikenalpasti menunjukkan perbezaan ekspresi (p ï‚£ 0.05) di mana 20 daripadanya terlibat pada fasa awal pengaktifan eosinofil dan 12 protein lagi terlibat di fasa lewat. Beberapa tapak jalan seperti Notch, pintasan pentosa-fosfat dan metabolisme glukosa dan proses biologi seperti pergerakan komponen bersel, pemasangan berkas aktin filamen dan glikolisis didapati terlibat dalam pengaktifan eosinofil. Namun begitu, korelasi antara profil transkriptom dan proteom adalah lemah. Hanya 4 calon molekul iaitu ACTN4, ANXA1, PGK1 dan TKT telah menunjukkan korelasi yang signifikan antara ekspresi gen dan protein. Analisis mikroatur transkriptom telah mengenalpasti 498 (p ‰¤ 0.05), 428 (p ‰¤ 0.05), 1677 (p ‰¤ 0.01) dan 558 (p ‰¤ 0.01) gen berbeza ekspresi yang berkaitan dengan respon terhadap steroid, kawalan asma, keterukan asma dan pembentukan asma masing-masing. Analisa pengayaan set gen (GSEA) telah mengenalpasti beberapa tapak jalan seperti pengisyaratan VIPR->CREB/CEBP (respon terhadap steroid), pengisyaratan TNFRSF1A->AP-1/ATF/TP53 (kawalan asma) dan pengisyaratan IFNAR->STAT (pembentukan asma) yang berkaitan dengan respon inflamatori akibat eosinofil dalam asma. Ekspresi 11 gen iaitu NKX3-1, CCNH, RGS2, NAP1L4, CSNK2A1, NFE2L2, IRF1, RNASE1, ADM, IL1R2 and IL5RA yang berupaya meramal pelbagai fenotip asma telah disahkan oleh tindak balas berantai polimerase masa-nyata (qRT-PCR). Kesimpulannya, kajian ini telah mengenalpasti penanda molekul tertentu yang berkaitan dengan pengaktifan eosinofil dan memberi pemahaman mengenai perhubungan antara inflamasi eosinofilik dan fenotip penyakit asma.,Eosinophil inflammation of the airways is a characteristic feature of asthma. Elevation of eosinophil levels in peripheral blood and bronchial mucosa has been associated with asthma severity and control status. However the exact mechanisms on how the eosinophils contribute to the distinct asthma phenotypes are still largely unknown. The objectives of this study were to (i) identify transcriptome and proteome profiles associated with IL-5 induced eosinophil activation, (ii) determine the biological processes and molecular pathways involved in the activation of eosinophils (iii) identify gene expression profiles and molecular pathways associated with various clinical phenotypes of asthma. Peripheral blood eosinophils were stimulated with IL-5 for 24 hours and subsequently the total RNA and proteins were extracted at serial time points: early (0h and 1h) and late (6h, 12h and 24h) for the gene expression using Illumina Sentrix® Human RefSeq-8 Beadchip and label-free LC-MS/MS differential proteomic profiling respectively. In addition microarray gene expression analyses were also performed on peripheral blood eosinophils isolated from patients with various asthma phenotypes (n = 40) and normal subjects (n = 7). The in vitro trancriptomic study identified 301 early genes that were differentially expressed (p ‰¤ 0.05, 2 FC) in the early phase of eosinophil activation. Several important biological processes and pathways were identified such as TCR signaling pathway, TLR signaling pathway, MAPK signaling pathway and apoptosis. However, only 67 proteins were differentially expressed (p ‰¤ 0.05) whereby 20 of them were involved in the early phase of eosinophil activation and 12 proteins at the late phase. Several pathways such as Notch, pentose-phosphate shunt and glucose metabolism and biological processes such as cellular component of movement, actin filament bundle assembly and glycolysis were found to be involved in the activation of eosinophils. However, the correlation between transcriptome and proteome profiles was poor. Only 4 molecular candidates i.e. ACTN4, ANXA1, PGK1 and TKT showed significant correlation between gene and protein expression. Transcriptomic microarray analyses had identified 498 (p ‰¤ 0.05), 428 (p ‰¤ 0.05), 1677 (p ‰¤ 0.01) and 558 (p ‰¤ 0.01) differentially expressed genes that were associated with response to steroid, asthma control, asthma severity and asthma development respectively. Gene set enrichment analyses (GSEA) identified several key pathways such as VIPR->CREB/CEBP signaling (response to steroid), TNFRSF1A->AP-1/ATF/TP53 signaling (asthma control) and IFNAR->STAT signaling (asthma development) that were associated with eosinophil mediated inflammatory response in asthma. The expression of 11 genes i.e. NKX3-1, CCNH, RGS2, NAP1L4, CSNK2A1, NFE2L2, IRF1, RNASE1, ADM, IL1R2 and IL5RA that were able to predict various phenotypes of asthma were confirmed by real-time PCR. In conclusion, this study has identified specific molecular signature associated with eosinophil activation and provides insights about the relationships between eosinophilic inflammation and disease phenotypes of asthma.,PhD
Pages:	310
Publisher:	UKM, Bangi
Appears in Collections:	UKM Medical Molecular Biology Institute / Institut Perubatan Molekul (UMBI)

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