Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis
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| Language: | English |
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University of Toledo Health Science Campus / OhioLINK
2017
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=mco1499964056117743 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-mco1499964056117743 |
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English |
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Biomedical Research Genetics Molecular Biology Physiology lncRNAs circRNAs miRNAs genetics CRISPR-Cas9 blood pressure hypertension QT-interval tumorigenesis rat models |
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Biomedical Research Genetics Molecular Biology Physiology lncRNAs circRNAs miRNAs genetics CRISPR-Cas9 blood pressure hypertension QT-interval tumorigenesis rat models Cheng, Xi Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| author |
Cheng, Xi |
| author_facet |
Cheng, Xi |
| author_sort |
Cheng, Xi |
| title |
Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| title_short |
Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| title_full |
Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| title_fullStr |
Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| title_full_unstemmed |
Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis |
| title_sort |
evidence for non-coding rnas as inherited factors influencing cardiovascular disease, renal disease and tumorigenesis |
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University of Toledo Health Science Campus / OhioLINK |
| publishDate |
2017 |
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http://rave.ohiolink.edu/etdc/view?acc_num=mco1499964056117743 |
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AT chengxi evidencefornoncodingrnasasinheritedfactorsinfluencingcardiovasculardiseaserenaldiseaseandtumorigenesis |
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1719452592795287552 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-mco14999640561177432021-08-03T07:03:22Z Evidence for Non-Coding RNAs as Inherited Factors Influencing Cardiovascular Disease, Renal Disease and Tumorigenesis Cheng, Xi Biomedical Research Genetics Molecular Biology Physiology lncRNAs circRNAs miRNAs genetics CRISPR-Cas9 blood pressure hypertension QT-interval tumorigenesis rat models This dissertation is comprised of three projects. Projects 1 and 2 are focused on a GWAS locus for cardiovascular disease (short QT-interval) on human chromosome 17. We have previously validated and high resolution mapped the homologous genomic segment of this human locus to <42.5 kb on rat chromosome 10. This <42.5 kb segment in rats regulates both QT-interval and blood pressure and contains a single protein-coding gene, rififylin (Rffl). The expression of Rffl in the cardiac and renal tissues is differential between Dahl S and S.LEW congenic rats, which are the strains used for mapping this locus. Our previous study points to altered rate of endocytic recycling as the underlying mechanism, through which Rffl operates to control both QT-interval and blood pressure. Interestingly, Rffl also contributes to tumorigenesis by repressing caspases and tumor suppressor genes. Moreover, the expression of Methyl-CpG Binding Domain Protein 2 (Mbd2) in the cardiac and renal tissues is also higher in the S.LEW congenic strain than the background (control) Dahl S strain. Mbd2 can repress methylated tumor suppressor genes. Therefore, Project 1 proposed the hypothesis that the S.LEW congenic strain was more susceptible to tumorigenesis. To test this hypothesis, the S and S.LEW strains were compared for susceptibility to azoxymethane-induced colon tumors. The number of colon tumors was significantly higher in the S.LEW congenic strain compared with the S rat. Transcriptomic analysis confirmed that the chemical carcinogenesis pathway was significantly up-regulated in the congenic strain. These studies provide evidence for a GWAS-validated genomic segment on rat chromosome 10 as being important for the regulation of both cardiovascular function and tumorigenesis.The evidence that there is no exonic variant located in Rffl further drew our attention to the non-coding regions with total 171 variants in this <42.5 k QTL region. Our laboratory previously reported a first-generation catalog of rat lncRNAs involved in BP regulation, through which we identified a novel lncRNA, named Rffl-lnc1, located within Rffl 5’utr intronic region, containing a continuous 19bp sequence variation in the Dahl S rat (+19bp) and the S.LEW congenic strain (-19bp), which were used for mapping this QTL. Project 2 further continued the precise high resolution mapping from the <42.5 kb QTL to the 19bp within Rffl-lnc1 and tested whether these 19bp served as quantitative trait nucleotides (QTL) for cardiovascular regulation. Characterization of this novel lncRNA using rapid amplification of cDNA ends (RACE) provided evidence for the presence of more than a single isoform of Rffl-lnc1. To assess the role of this novel lncRNA, a panel of CRISPR/Cas9 based gene-edited disruption models of Rffl-lnc1 was developed on the genomic background of the Dahl S rat. These models harbored varied disruptions around the critical 19bp region. The disruption of Rffl-lnc1 significantly elevated blood pressure and shortened QT-interval, demonstrating the significance of Rffl-lnc1 in cardiovascular regulation. To further evaluate the 19bp polymorphism within Rffl-lnc1, 19bp were introduced into the genomic background of the S.LEW congenic strain through the CRISPR/Cas9 technology. The 19bp knock-in successfully rescued the S.LEW congenic strain by lowering its hypertension and normalizing QT-intervals. Our further study showed that these critical 19bp served as decisive variants in cardiovascular regulation compared to other variants in the <42.5 kb QTL region. To our knowledge, this is the first demonstration of a CRISPR/Cas9 based targeted disruption and knock-in approach applied to precisely characterize the quantitative trait nucleotides located in a long non-coding RNA for cardiovascular regulation.Circular RNAs (circRNAs) have emerged as an important new class of genomic regulatory molecules contributing to the development of various diseases, but their relevance to the development and progression of hypertension remains largely unknown. A major impediment to begin studying circRNAs in rat models of inherited hypertension is that the rat as a valuable model of human diseases lags far behind mouse and human for knowledge on circRNAs. In Project 3, a genome-wide circRNA profiling was performed from 4 rat strains which are widely used in hypertension research: the Dahl salt-sensitive rat (S), the Dahl salt-resistant rat (R), the spontaneously hypertensive rat (SHR) and the Wistar Kyoto rat (WKY). Combined hybridization data obtained from these 4 strains allowed for the identification of 12,846 circRNAs as being expressed in the rat kidneys. Out of these, 318 and 110 circRNAs were differentially expressed with a fold change > 1.5 (p < 0.05) in S versus R and SHR versus WKY, respectively. Using these circRNAs, circRNA/microRNA interaction was predicted as a feature occurring in rats. This is in-line with the knowledge that circRNAs serve as microRNA sponges to sequester microRNAs. Several circRNAs were further validated using quantitative real-time PCR. To our knowledge, our study is the first to profile circRNAs in renal tissue and illustrates that circRNAs could be candidate genetic factors controlling blood pressure. 2017 English text University of Toledo Health Science Campus / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=mco1499964056117743 http://rave.ohiolink.edu/etdc/view?acc_num=mco1499964056117743 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |