Browsing by Author "Subasinghe, Kumudu"
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Item EXOSOME PROFILING OF BRONCHIAL LAVAGE FLUID IN A MOUSE MODEL OF SURGERY RESECTION OF BREAST CANCER WITH LUNG METASTASIS(2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, NicoleThe lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).Item Imputation Accuracy of Apolipoprotein E ε Alleles in Genome-wide arrays and real-time SNP Genotyping assays(2022) Subasinghe, Kumudu; Garlotte, Isabelle; O'Bryant, Sid E.; Barber, Robert C.; Phillips, NicolePurpose: The vast majority of the established genetic-based risk for late-onset Alzheimer's disease (AD) is attributable to variation within the apolipoprotein E (APOE) gene. This gene, which encodes a protein implicated in various aspects of AD pathology, is characterized by two single nucleotide polymorphisms (SNPs; rs429358 and rs7412) that result in three distinct isoforms (ε4, ε3 and ε2). Most population-based genome-wide association studies to date have identified the APOE ε4 and ε2 alleles as the strongest genetic-based risk and protective factors for AD, respectively. APOE genotype is not only critical for determining disease risk and diagnosis, but also for developing individualized therapeutic strategies. Genotyping via real-time quantitative PCR (qPCR) is the gold standard for APOE isoform determination; however, if genome wide SNP data is available, imputation of APOE (i.e., probabilistic genotyping through inference) may eliminate the need for qPCR genotyping. In this project, we evaluate the concordance of APOE genotypes obtained via qPCR and a genome-wide SNP chip in non-Hispanic White and Mexican American individuals from the Health & Aging Brain among Latino Elders (HABLE) cohort. Method: DNA was extracted from buffy coat samples (n = 1650) on the Hamilton robotic system with the Mag-Bind Blood & Tissue DNA HDQ 96 Kit. qPCR was then performed using the TaqMan Genotyping Kit as per manufacturer's protocol. Results produced via qPCR were then compared to those imputed for rs429358 and directly typed for rs7412 on the Illumina Infinium Global Screening Arrays (GSA) and analyzed with Genome Studio 2.0. Samples with call rates less than 98% were repeated or excluded. Results: Concordance between the APOE genotypes obtained from qPCR and Infinium GSA was 99.32%. Discordance was likely due to poor sample quality and low-frequency imputation errors of rs429358, which may be corrected with more conservative thresholding of the imputed genotype confidence statistics. Conclusion: Genotype imputation from SNPs commonly typed in the APOE region is an effective method for APOE isoform determination, even in Mexican Americans who are more genetically heterogenous due to ancestral admixture; this method may be effectively implemented in large population-based studies of aging and AD.Item Longitudinal microRNA profiling of neuronal-enriched exosomes associated with cognitive function and decline(2024-03-21) Subasinghe, Kumudu; Hall, Courtney; Zhou, Zhengyang; Barber, Robert; Phillips, NicoleBackground. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that disproportionately affects several racial/ethnic groups, including Mexican Americans (MAs). Evidence suggests that early alterations in the AD brain can propagate to local and distal cells through small biological packages called exosomes. Exosomes secreted by neurons are capable of mediating cell-to-cell communication through their bioactive cargo, leading to metabolic and epigenetic reprogramming in target cells. Exosomes derived from neurons have been detected in plasma and isolated from other subpopulations using the neural cell adhesion molecule CD171. These neuronal-enriched exosomes (NEEs) cross the blood-brain barrier and thus represent an easily accessible derivative of otherwise inaccessible brain tissue in living humans. Small, non-coding RNAs called microRNAs (miRNA) are transcribed from nuclear DNA and function as strong intracellular expression regulators. miRNAs, which can be selectively packaged and transported by exosomes, have been shown to significantly alter the expression patterns of target cells. This project aims to identify the aberrant miRNA profiles that correlate with disease progression and key comorbidities (e.g., type 2 diabetes (T2D), hyperlipidemia, hypertension) in NEEs of plasma from MAs and Non-Hispanic Whites (NHWs). Hypothesis. We hypothesize that population-specific differences in NEE miRNA cargo will reflect cognitive function and decline. Methods. Longitudinal plasma samples (two time points, 2 years apart) received from the Texas Alzheimer’s Research and Care Consortium (TARCC) were processed using a two-step method that involves precipitation of total exosomes followed by NEE capture with a biotinylated antibody against the neuronal surface marker, CD171. After isolating RNA from NEEs, miRNAs were then profiled using next-generation sequencing. These profiles were then analyzed for differential miRNA expression in individuals with cognitive impairment compared to the normal control group. Results. Our preliminary quality control and sequencing data confirmed the successful isolation of miRNA from NEEs. We identified specific miRNA candidates that were differentially expressed in NEEs from cognitively impaired subjects compared to healthy controls. These miRNAs target gene networks that have been implicated in AD pathophysiology. Conclusion. This innovative workflow along with the unique sample type provides novel insight into the role of exosomal miRNA cargo in AD pathogenesis, identifying novel, population-specific targets for biomarker/diagnosis as well as therapeutic design. Further, this approach provides a conceptual framework for blood-based exosomal profiling in other complex diseases characterized by epigenetic dysregulation and systemic inflammation.Item Top Alzheimer's disease risk allele frequencies differ in HABS-HD Mexican- versus Non-Hispanic White Americans(John Wiley & Sons, Inc., 2024-01-02) Housini, Mohammad; Zhou, Zhengyang; Gutierrez, John; Rao, Sumedha; Jomaa, Rodwan; Subasinghe, Kumudu; Reid, Danielle M.; Silzer, Talisa; Phillips, Nicole; O'Bryant, Sid E.; Barber, Robert C.; Team, HABS-HD StudyINTRODUCTION: Here we evaluate frequencies of the top 10 Alzheimer's disease (AD) risk alleles for late-onset AD in Mexican American (MA) and non-Hispanic White (NHW) American participants enrolled in the Health and Aging Brain Study-Health Disparities Study cohort. METHODS: Using DNA extracted from this community-based diverse population, we calculated the genotype frequencies in each population to determine whether a significant difference is detected between the different ethnicities. DNA genotyping was performed per manufacturers' protocols. RESULTS: Allele and genotype frequencies for 9 of the 11 single nucleotide polymorphisms (two apolipoprotein E variants, CR1, BIN1, DRB1, NYAP1, PTK2B, FERMT2, and ABCA7) differed significantly between MAs and NHWs. DISCUSSION: The significant differences in frequencies of top AD risk alleles observed here across MAs and NHWs suggest that ethnicity-specific genetic risks for AD exist. Given our results, we are advancing additional projects to further elucidate ethnicity-specific differences in AD.