Unique Circulating microRNA Profiles in Epidemic Kaposi’s Sarcoma

Unique Circulating microRNA Profiles in Epidemic Kaposi’s Sarcoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Unique Circulating microRNA Profiles in Epidemic Kaposi’s Sarcoma Haruna Muwonge, Hassan Kasujja, Carolyne Atugonza, Josephine Kasolo, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-116856/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2022 Read the published version in Non-coding RNA Research → Version 1 posted You are reading this latest preprint version Abstract Background The Human herpesvirus 8 (HHV-8), causes Kaposi's sarcoma (KS). Kaposi sarcoma in HIV/AIDS patients is referred to as epidemic KS, and is the most common HIV-related malignancy worldwide. Lack of a diagnostic assay to detect latent and early stage disease has increased disease morbidity and mortality. Serum miRNAs have previously been used as potential biomarkers of normal physiology and disease. In the current study, we profiled the unique serum miRNAs in patients with epidemic KS to generate baseline data to aid in developing a miRNA-based non-invasive biomarker assay for Epidemic KS. Methods This was a comparative cross-sectional study involving 27 patients with epidemic KS, and 27 HIV-positive adults with no prior diagnosis, or clinical manifestation of KS. DNA and RNA were isolated from blood and serum collected from study participants respectively. Nested PCR for circulating HHV-8 DNA was performed on the isolated DNA, whereas miRNA library preparation and sequencing for circulating miRNA was performed on the RNA samples. The miRge2 pipeline and EdgeR were used to analyze the sequencing data. Results Fifteen out of the 27 epidemic KS positive subjects (55.6%) tested positive for HHV-8 DNA, whereas only 3 (11.1%) out of the 27 HIV positive, KS negative subjects tested positive for HHV-8 DNA. Additionally, we found a unique miRNA expression signature in 49 circulating miRNAs in epidemic KS subjects compared to subjects with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs down regulated. Subjects with latent KS infection had a differential upregulation of circulating miR-193a compared to HIV-positive, KS negative subjects for whom circulating HHV-8 DNA was not detected. Further analysis of serum from epidemic KS patients revealed a miRNA signature according to KS tumor status and time since first HIV diagnosis. Conclusions This study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have crucial roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection. Cancer Biology Oncology microRNA circulating microRNA Kaposi’s Sarcoma biomarker blood Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Kaposi sarcoma (KS) is the most common HIV-related malignancy worldwide and the 3 rd most frequently diagnosed cancer amongst men in Sub-Saharan Africa. Uganda has one of the highest rates of KS in the world, partly due to the HIV pandemic, with the incidence of KS in women surpassing that of cervical cancer as the most common female malignancy (Phipps et al., 2010). Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), is the etiological agent of Kaposi's sarcoma, and in vivo, it mainly infects B cells and endothelial cells, impairing the expression of pro and anti-cancer genes in these cells. In Majority of infected individuals, the virus may remain latent for years or decades, only to be activated by compromised immunity, such as seen in Human Immune Virus (HIV) infection and organ transplantation. Consequently, Epidemic Kaposi’s sarcoma is only found in patients who have HIV acquired immune deficiency syndrome (HIV/AIDS). In Africa, KS is typically aggressive, and patients with Epidemic KS often present late with widely disseminated and rapidly progressive disease (Mosam et al., 2012), and despite standard treatment with highly active antiretroviral therapy (HAART) and chemotherapy, about one-third of these patients succumb to the disease after 2 years (Freeman et al., 2016; Okuku et al., 2017). This relatively high disease morbidity and mortality partly results from delayed diagnosis arising from a lack of a reliable diagnostic assay for detection of latent or early stage disease. Nevertheless, the identification of reliable biomarkers for KS remains a challenge to-date. In resource-limited settings, diagnosis of KS is usually clinical, based on the presence of cutaneous or mucosal KS lesions, as well as associated symptoms. The challenge with the clinical approach for KS diagnosis is the fact that KS skin lesions are usually mimicked by other non-KS lesions (Yaqub et al., 2019). Moreover, even though histopathological analysis is considered the gold standard diagnostic test for KS (Corey Casper, Martin S Hirsch, & Bloom, 2019), this test is wrought with challenges, such as the prior requirement of a tissue sample acquired from an invasive tissue-biopsy procedure, as well as the subjective interpretation of test results leading to inter-observer variability among pathologists (Amerson et al., 2016). Additionally, histopathological diagnosis can only be performed for patients who have manifested KS like lesions on the skin or mucosa membranes, and thus may not apply to patients with sub-clinical or latent disease. Similarly, ELISA-based assays used to augment other tests during KS detection have limitations of low specificity and cross-reactivity with other antigens, such as Epstein Bar Virus (EBV) proteins (Ablashi, Chatlynne, Whitman, & Cesarman, 2002). PCR-based techniques that detect HHV-8 DNA in tissues and serum, albeit highly sensitive, are also limited by low specificity (Auten, Kim, Bradley, & Rosado, 2017), particularly in the viral latent phase. A novel class of molecules called microRNAs (miRNAs) have recently gained traction in healthcare management for their potential as biomarkers for human diseases (Dave et al., 2019). miRNAs are small (19-24 nucleotides), evolutionary conserved, endogenous non-coding RNA molecules that bind to the 3’UTR of target mRNA transcripts with partial or perfect sequence complementarity, resulting in translational repression and/or mRNA destabilization (O'Brien, Hayder, Zayed, & Peng, 2018). During the past decade, our knowledge about the role of miRNAs in human diseases has grown exponentially (Gilad et al., 2012; O'Brien et al., 2018). Abnormal expression of a single miRNA can have implications on the activity of multiple genes (Dave et al., 2019; O'Brien et al., 2018). It’s not surprising therefore that changes in miRNA expression have been found to contribute to a wide variety of human disease states and disorders such as cancer, cardiovascular, autoimmune, neurodegenerative, hepatic, and inflammatory diseases (Dave et al., 2019). The utility of miRNA’s in diagnostics is derived from their specificity for a particular type of tissue or cell, their abundance, as well as their remarkable stability in body tissues and fluids such as serum, urine, saliva, milk and cerebrospinal fluid (Dave et al., 2019; Gustafson, Tyryshkin, & Renwick, 2016). These characteristics demonstrate a remarkable potential for microRNAs to be used as biomarkers for disease. miRNAs have been successfully used to classify cancer, identify cancer tissue origin, determine prognosis and disease progression, predict resistance to chemotherapy, monitor therapy, and screen for disease (Ahmed et al., 2009; Calin et al., 2005; Eitan et al., 2009; Gustafson et al., 2016; Ji et al., 2009). HHV8 infection or HHV8-related malignancies can induce a unique signature of human and viral intracellular and extracellular miRNAs (Qin, Peruzzi, Reiss, & Dai, 2014), and this has been confirmed in vitro studies (Hoshina et al., 2016). These data though highly suggestive, cannot be extrapolated to epidemic KS, due to the additive effects of HIV-infection on cellular miRNA signaling. This study aimed at profiling the unique miRNAs found in serum of patients with epidemic KS, by comparing circulating miRNAs in patients with epidemic KS to those of a comparative group of patients with no KS. These data will provide baseline data that will aid in developing a miRNA-based minimally invasive PCR assay for Epidemic Kaposi’s Sarcoma, which would greatly facilitate the detection and management of a disease that has a high rate of recurrence and mortality. Methods Study setting and population; This was a comparative cross-sectional study conducted from August 2018 – July 2019. Whole blood samples were obtained from 27 HIV positive adult patients with epidemic Kaposi’s sarcoma, confirmed by histopathology at the Uganda Cancer Institute (UCI) and 27 HIV adult patients without clinical manifestation of KS at Makerere Joint AIDs Program (MJAP) clinic for comparison. The Skin Cancer clinic at UCI provides care for ambulatory patients with skin cancer. The MJAP clinic is an outpatient facility for ambulatory patients seeking continued HIV care services, and provides comprehensive HIV prevention, treatment, care, and support. The ISS clinic provides general care to over 15,000 HIV infected patients and opens 5 days a week with daily attendance of >300 patients. The pregnant, severely sick, and HIV-positive, KS negative individuals with mucosal and skin lesions that could mimic KS were excluded. The total number of study participants was fifty-four (54), and study participants were purposively sampled. The sample size was determined using the Fleiss’s equation (1980) with continuity correction, commonly used for comparative studies. All patients were taking anti-retroviral therapy (ART). Of the 50 participants who had available information regarding their ART medication, 34 were taking combined antiretroviral therapy medicines (cART) consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) in combination with a non-nucleoside reverse transcriptase inhibitors (NNRTI), 6 were taking cART consisting a two NRTIs and a protease inhibitors (PI), whereas 7 received an Integrase Strand Transfer Inhibitor (INSTI) therapy based ART. The proportion of participants taking cART was similar among Epidemic KS and none-KS participants. A questionnaire was used to obtain participants demographic information, including age, gender, as well as base line clinical characteristics, such as KS lesion morphotype, presence of edema, disease comorbidities and treatments received at presentation or after KS diagnosis. Five (5) milliliters of venous blood were collected from study participants in specialized vacutainer tubes, of which 2mls were stored at 4 0 C for DNA isolation, whereas 3mls were left to stand on ice for 30 minutes, centrifuged for 20 minutes at 1300g to collect serum that was then aliquoted in 2ml Eppendorf tubes containing RNA later (Thermo Fisher Scientific), and stored at -80 0 C for further analysis. DNA extraction DNA was extracted from whole blood using the QIAamp DNA midi blood kit (QIAGEN) in accordance with the manufacturer's instructions. Briefly, 100μl of the Protease enzyme was pipetted into a 15ml Centrifuge tube and 200μl of the sample, 800μl PBS, and 200μl of Buffer AL added. The resulting mixture was vortexed for 15 seconds, and then incubated at 70°C for 10 minutes to lyse the cells. 1 ml of absolute ethanol was then added to the mixture and vortexed to allow DNA precipitation. The resulting mixture was then added to the QIAamp Midi spin column, centrifuged at 8000 rpm for 1 minute and the supernatant discarded. 2ml of Wash buffer AW1 was added to the column and the centrifuged at 5000rpm for 1 minute and the supernatant discarded. Then, 2ml of Wash buffer AW2 was added to the column, centrifuged at 5000rpm for 15 minute and the supernatant was discarded. DNA was eluted in 200μl Buffer AE. The DNA quality was assessed using the NanoDrop TM 2000C spectrophotometer (Thermo Scientific, Wilmington, DE, USAs) Nested PCR for HHV-8 DNA Two amplifications were done by the nested PCR technique. The first amplification targeted a region in the open reading frame (ORF)-26 of HHV-8 as was done previously (Machado, Farias, Pereira, Freitas, & Fonseca, 2015). Briefly, for the first run, a total reaction mixture of 25μl was prepared from a mixture of the following; 12.5µl 1X Master Mix (BioLabs, New England), 1.25μl of 10pMol of each sense (5'AGCCGAAAGGATTCCACCAT-3’) and antisense (5'-TCCGTGTTGTCTACGTCCAG-3') primers (Euro films Genomics, Vienna), 7μl nuclease free water and 3μl sample DNA. During this run, 35 cycles of PCR amplification of HHV-8 DNA was done on a SimpliAmp TM Thermal cycler (Thermo Fisher Scientific, Applied Biosystems, Singapore). The PCR conditions were as follows; initial denaturation at 95 °C for 5 minutes, followed by further denaturation at 94 °C for 30 seconds, annealing at 56 °C for 30 seconds, extension at 68°C for 45 seconds and a final extension step at 68 °C for 10 minutes. The second PCR run targeted a 211bp region in the first amplicon. The nested PCR amplification mixture contained 5μl of the first RCR mixture, 25µl of the 1X master mix, 15μl of nuclease free water and 2.5μl of 10pMol of each of the internal forward primer (5'TTCCACCATTGTGCTCGAAT-3') and reverse primer (5'-TACGTCCAGACGATATGTGC-3'). During the run, 35 cycles of amplification were done under the following conditions; initial denaturation at 95°C for 5 minutes, further denaturation at 94 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 68 °C for 30 seconds and a final extension of 10 minutes at 68°C. Five microlitres (5μl) of the final PCR product were loaded into a 1.2% agarose gel, and electrophoresed at 120V for 90 minutes. Positive reactions yielded an amplicon of 211 bp, which was easily viewed on a UV trans-illuminator after ethidium bromide staining. As a positive control, confirmed HHV-8 DNA was used, as well as a set of negative controls of nuclease free and negative clinical samples for HHV-8 DNA were used. RNA extraction and quality control Serum total RNA was extracted from 200µl of serum samples using the Qiagen miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA, USA) according to manufacturer’s instructions (QIAGEN; Haldane, Germany). RNA abundance and integrity were determined after isolation using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only samples with an RNA integrity number (RIN) >9 were subjected to subsequent library preparation Library preparation and sequencing First, small RNA fragments between 18 to 30 nucleotides were extracted from total RNA on a PAGE gel. Then, a 5-adenylated, 3-blocked single-stranded DNA adapter was ligated to the 3’end of RNA fragments. Thereafter, reverse transcription (RT) primers with unique molecular identifiers (UMIs) were added to the resulting mixture to enable hybridization to the ligated 3’adaptors in RNA and dissociative 3’adapters. Next, 5’adapters were linked to the 5’end of the resulting ligation product, followed by one-strand cDNA synthesis with RT primers. The resulting cDNA was then amplified by PCR. Fragments between 110bp to 130bp of the cDNA library were selected by PAGE electrophoresis, quantified, and then pooled with libraries from other samples. The cDNA libraries were validated using the Agilent Technologies 2100 bioanalyzer, and thereafter subjected to high throughput DNA nanoball sequencing using Phi29 DNA polymerase. Sequencing data analysis After Sequencing, data was analyzed using the miRge2 pipeline (Lu, Baras, & Halushka, 2018) for alignment and quantification of miRNAs, whereas all statistical analyses of differentially expressed miRNAs were conducted in EdgeR (Robinson, McCarthy, & Smyth, 2010). In brief, Fastq files underwent quality control, removal of adapters sequences, and formation of unique reads. Specifically, unique reads were annotated to mature miRNAs, mRNA hairpins, and other RNA types by alignment toward miRbase v.22 (Griffiths-Jones, Grocock, van Dongen, Bateman, & Enright, 2006). Only quantified mature miRNAs were used in subsequent data analysis. Low read counts were filtered out setting at least five reads as the minimum cut-off. Filtered counts were then normalized and transformed to log2 counts per million (log CPM) using R package EdgeR. miRNAs with an FC > 1 and FDR < 0.05 were considered upregulated and those with an FC < 1 and FDR <0.05 downregulated. Differentially expressed miRNAs were visualized on volcano plots. Unsupervised clustering was performed by using log2 transformed values and using Euclidian distances between samples. All statistical analysis was conducted in R environment (version 3.6.3). Ethics: The study was approved by the Research and Ethics Committee of the School of Biomedical Sciences (SBS-REC) with study number SBS SBS-519, and also registered with the Uganda National Council for Science and Technology (UNCST); (study number: HS 2405). Participants’ confidentiality was maintained at all times. Results Patient demographics ; A total of 54 participants participated in this study. Half of the participants (27) had epidemic KS, whereas the other half were HIV-positive with no confirmed diagnosis of KS. All the 27 KS diseased patients had cutaneous lesions, with most of them localized to the lower extremities. A few of the participants had further manifestations of ulcerated tumors, swollen and painful lymph nodes and palate lesions. Details of the Sociodemographic and Clinical characteristics of KS patients in this study are shown in table 1. Table 1. Sociodemographic and Clinical characteristics of study participants Characteristic Frequency n (%) Gender Male Female 46 (85) 8 (15) Age in years (n = 54) 21-30 31-40 41-50 51-80 5(19) 14(52) 6(22) 2(7) Nature of residence (n = 54) Urban Rural 43(80) 11 (20) Occupation (n = 54) Employed Unemployed 34 (62) 20 (38) Educational level (n = 54) Primary Secondary Tertiary Non-formal 24 (44) 20 (37) 7 (13) 3 (6) Duration on ART (n=54) 120 months 3 (5) 10 (19) 22 (41) 12 (22) 7 (13) Duration from index KS diagnosis (n = 24) 12months 7 (29) 2 (8) 6 (25) 9 (38) Form of KS (n = 27) Skin Other 27 (100) 0 (0) No. of Chemotherapy cycles completed (n=20) One cycle Two cycles Three cycles Four cycles Five cycles Six cycles 2 (10) 6 (30) 3 (15) 4 (20) 2 (10) 3 (15) Positive HHV-8 DNA (n=54) KS (n=27) Non-KS (n=27) 15 (55.6) 3 (11.1) Tumor appearance (n = 27) Ulcerated 5 (19) Lymph node involvement (n = 27) Yes 8 (24) Palate involvement (n = 27) Yes 6 (22) Differential expression of miRNA To determine whether patients with Epidemic Kaposi’s sarcoma differentially express miRNAs relative to HIV patients without Kaposi’s sarcoma, we performed differential expression testing using EdgeR, using normalized read counts. We found that 49 miRNAs were differentially expressed, with 8 miRNAs downregulated and 41 miRNAs upregulated (adjusted p-value <0.05) ( Table 2, volcano plot in figure 2, and heatmap in figure 3 ). Table 2: Differentially expressed miRNAs in patients with Epidemic KS compared to HIV patients with no KS miRNA Fold change (log10) Up/Down regulation p-value Adjusted p-value hsa-miR-4446-3p 4.84849 Up 2.50E-05 0.00317 hsa-miR-451a -2.003 Down 3.72E-05 0.00317 hsa-miR-99b-5p 1.24184 Up 4.50E-05 0.00317 hsa-miR-411-5p 4.18601 Up 7.14E-05 0.00376 hsa-miR-1304-3p 5.29579 Up 9.94E-05 0.00419 hsa-miR-4732-3p -1.9894 down 0.00017 0.00445 hsa-miR-6819-3p 4.98167 UP 0.0002 0.00445 hsa-miR-654-5p 4.60077 Up 0.0002 0.00445 hsa-miR-93-5p -1.4618 Down 0.00022 0.00445 hsa-miR-1908-5p 4.45843 Up 0.00023 0.00445 hsa-miR-337-3p 3.05436 Up 0.00023 0.00445 hsa-miR-431-5p 4.28894 Up 0.0003 0.00535 hsa-miR-223-5p 3.92606 Up 0.0004 0.00637 hsa-miR-139-3p 1.44008 Up 0.00044 0.00637 hsa-miR-654-3p 3.76475 Up 0.00047 0.00637 hsa-let-7e-5p 1.86566 Up 0.00048 0.00637 hsa-miR-487a-5p 4.14846 Up 0.00052 0.00649 hsa-miR-379-5p 4.16306 Up 0.00068 0.00758 hsa-miR-486-5p -4.0858 Down 0.0007 0.00758 hsa-miR-369-5p 1.97987 Up 0.00075 0.00758 hsa-miR-222-3p 1.27732 Up 0.00075 0.00758 hsa-miR-191-3p 3.75839 Up 0.0009 0.00863 hsa-miR-382-5p 3.87227 Up 0.00097 0.00885 hsa-miR-409-3p 2.61324 Up 0.00122 0.01071 hsa-miR-584-5p 2.56752 Up 0.00176 0.01487 hsa-miR-154-5p 3.4633 Up 0.00244 0.01972 hsa-miR-1307-3p 1.04218 Up 0.00252 0.01972 hsa-miR-221-3p 3.34441 Up 0.00314 0.02296 hsa-miR-4433b-5p 1.73024 Up 0.00316 0.02296 hsa-miR-185-5p -1.7193 Down 0.00346 0.02396 hsa-let-7e-3p 3.45092 Up 0.00352 0.02396 hsa-miR-625-3p 3.57103 Up 0.00428 0.0274 hsa-miR-323b-3p 3.10619 Up 0.00429 0.0274 hsa-miR-328-3p 1.67445 Up 0.00491 0.02964 hsa-miR-199a-3p/199b-3p 0.81156 Up 0.00499 0.02964 hsa-miR-375-3p -1.3701 Down 0.00506 0.02964 hsa-miR-139-5p 0.97288 Up 0.00561 0.03199 hsa-miR-485-5p 2.36411 Up 0.00666 0.03602 hsa-miR-126-3p 0.70315 Up 0.00678 0.03602 hsa-miR-25-3p -1.0357 Down 0.00683 0.03602 hsa-miR-424-3p 3.15035 Up 0.00719 0.03698 hsa-miR-146a-5p 0.83632 Up 0.00815 0.04088 hsa-miR-224-5p 2.16699 Up 0.00833 0.04088 hsa-miR-7-5p -0.9513 Down 0.00933 0.04475 hsa-miR-340-3p 2.71832 Up 0.00981 0.04559 hsa-miR-323a-5p 3.26012 Up 0.00994 0.04559 hsa-miR-323a-3p 3.20691 Up 0.01045 0.04619 hsa-miR-425-3p 2.72079 Up 0.01051 0.04619 hsa-miR-127-3p 2.05625 Up 0.0108 0.04651 Relationship between miRNA expression and presence or absence of KSHV DNA. Changes in the levels of circulating miRNAs in the serum of humans and animals have been detected as a result of infection with a variety of viruses (Stenfeldt et al., 2017). Because Epidemic KS is caused by HHV-8, in addition to the fact that HHV-8 DNA has been detected in the blood of Epidemic KS patients, we examined the effect of presence of circulating HHV-8 DNA on the serum expression profile of circulating miRNAs. A nested PCR targeting a region in the open reading frame (ORF)-26 of HHV-8 DNA was conducted on DNA extracted from the blood of both epidemic KS patients and the comparative group. Analysis of samples from patients that tested positive for circulating HHV-8 DNA compared to those that tested negative revealed a differential expression of 4 miRNAs that were downregulated and only one miRNA that was up regulated ( table 3 and figure 4 ). Further stratification with regards to epidemic KS status did not reveal any significant differences in the differential expression of circulating miRNA between epidemic KS patients who tested positive for circulating HHV-8 DNA compared to epidemic KS patients that tested negative for HHV-8 DNA ( table 4 ). Table 3: Differential circulating miRNA expression analysis in participants testing positive or negative for HHV-8 DNA miRNA Fold change (log10) Up/Down regulation p-value Adjusted p-value hsa-miR-451a 1.930507 Up 2.72E-04 0.02273 hsa-miR-6819-3p -5.610377 Down 1.50E-05 0.00316 hsa-miR-654-3p -3.967188 Down 3.35E-04 0.02273 hsa-miR-139-3p -1.411567 Down 4.31E-04 0.02273 hsa-miR-1304-3p -4.57006 Down 1.06E-03 0.04470 Table 4: Differentially expressed circulating miRNAs in epidemic KS patients testing positive or negative for HHV-8 DNA miRNA Fold change (log10) Up/Down regulation p-value Adjusted p-value hsa-miR-150-5p -1.9696311 n.s 0.00314924 0.6644893 hsa-miR-16-5p 4.4026519 n.s 0.00901365 0.9509403 hsa-miR-1301-3p 4.1115834 n.s 0.01907221 0.9774167 hsa-miR-363-3p 3.8036035 n.s 0.01995521 0.9774167 hsa-miR-486-5p -3.6520209 n.s 0.02860547 0.9774167 hsa-miR-15b-5p 3.5675304 n.s 0.03616976 0.9774167 hsa-miR-25-3p 3.3827188 n.s 0.04592067 0.9774167 hsa-miR-532-5p 0.9929975 n.s 0.05097679 0.9774167 hsa-miR-451a 2.5280154 n.s 0.05651414 0.9774167 hsa-miR-29a-3p -1.2541687 n.s 0.05970539 0.9774167 Differential miRNA expression in latent HHV-8 infection. Three participants from the comparative group (HIV-positive, KS negative) had tested positive for HHV-8 DNA on nested PCR, indicating that they could be latently infected with KS. Consequently, serum miRNAs from these patients was compared to that of HIV-positive, KS negative patients who tested negative for HHV-8 DNA. Analysis for differential miRNA expression revealed a significant upregulation of miR-193a-5p in HHV-8 latently infected patients ( table 5 ). Table 5: Differentially expressed circulating miRNAs in KS negative patients testing positive or negative for HHV-8 DNA miRNA Fold change (log10) Up/Down regulation p-value Adjusted p-value hsa-miR-193a-5p* 6.565646 Up 2.13E-06 0.00026667 hsa-miR-10a-5p 2.786356 n.s 1.25E-03 0.05386547 hsa-miR-99a-5p 3.72719 n.s 1.42E-03 0.05386547 hsa-miR-143-3p 4.498237 n.s 1.72E-03 0.05386547 hsa-miR-375-3p 2.964285 n.s 2.64E-03 0.06611451 hsa-miR-483-3p 4.872215 n.s 6.32E-03 0.1217676 hsa-miR-942-5p -8.418699 n.s 6.82E-03 0.1217676 hsa-miR-122-5p 2.75821 n.s 2.59E-02 0.37944146 hsa-miR-10b-5p 3.901264 n.s 2.73E-02 0.37944146 hsa-miR-100-5p 3.984967 n.s 3.10E-02 0.38552523 *Indicates significantly dysregulated miRNA, n.s- Not significant Differential miRNA expression and duration from first HIV Diagnosis Participants were stratified in to four (4) groups from when they were diagnosed with HIV (i.e., 0-2 years, 2-5 years, 5-10 years, and above 10 years). Consequently, differential expression of circulating miRNAs was compared between these different groups. Analysis revealed significant differences in miRNA expression between patients diagnosed HIV positive 0-2 years and 2-5 years ago ( table 6 ). Between these two groups, 4 circulating miRNAs were down regulated, whereas 20 circulating miRNAs where up regulated (volcano plot in figure 5 ). There were no significant differences in the circulating miRNA expression when the remaining categories were compared. Table 6: Number of differentially upregulated or downregulated miRNAs at different time points from index HIV diagnosis Time since HIV Diagnosis (Years) Expression 0-2 Vs 2-5 Expression 0-2 Vs 2-5 Expression 0-2 Vs 2-5 Expression 0-2 Vs 2-5 Down 4 Down 4 Down 4 Down 4 Not significant 187 Not significant 187 Not significant 187 Not significant 187 up 20 up 20 up 20 up 20 Discussion Serum miRNAs have been proposed as potential biomarkers of normal physiology and disease, and serum microRNA signatures are currently used as diagnostic and prognostic markers of disease in prostate cancer, renal cell carcinoma, and lung cancer. In the current study, we profiled the unique miRNAs found in serum of patients with epidemic KS, by comparing circulating miRNAs in patients with epidemic KS to those of a comparative group of patients with no KS. Additionally, we analyzed the associations between the level of expression of dysregulated miRNAs and participant clinical data. We found a unique miRNA expression signature in 49 miRNAs when comparing serum collected from patients with epidemic KS to those with no epidemic KS. Moreover, three participants from the comparative group who tested positive for HHV-8 DNA (latent KS infection) differentially upregulated circulating levels of miR-193a compared to their counterparts (HIV-positive, HHV-8 DNA negative). Further analysis of serum of epidemic KS patients revealed a miRNA signature according to KS tumor status (1 downregulated miRNA) and time since first HIV diagnosis (1 upregulated and 4 down regulated miRNAs). However, no differential expression was found when comparing chemotherapeutic cycles, nature of KS lesions and duration since the first KS diagnosis in the epidemic KS patient group. miRNAs are prime candidates for use as non-invasive biomarkers in molecular diagnostics of disease (Blondal et al., 2013). The utility of miRNA’s in diagnostics is derived from their specificity for a particular type of tissue or cell, their abundance, as well as their remarkable stability in body tissues and fluids such as serum, urine, saliva, milk and cerebrospinal fluid (Dave et al., 2019; Gustafson et al., 2016). Even though the precise roles of circulating miRNA are still largely unknown, they have been found to be stable and survive conditions such as extreme variations in pH, boiling, multiple freeze thaw cycles, and extended storage, as well as degradation from RNAse enzymes. (Blondal et al., 2013; Wang, Peng, Wang, Qin, & Xue, 2018). In contrast to miRNAs, common RNA species like messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) are degraded within several seconds after being placed in a nuclease rich extracellular environment (Chen et al., 2008; Turchinovich, Samatov, Tonevitsky, & Burwinkel, 2013). In certain cancers, such as non-small cell lung cancer, differential expression of circulating miRNAs has been observed at different stages of disease, thereby contributing to diagnosis, treatment and prognosis (Wang et al., 2018; Yu, Guan, Cuk, Zhang, & Brenner, 2019). In the current study, we determined the differential expression profile of circulating miRNA by comparing the serum miRNA expression profiles of patients with Epidemic KS to that of a comparative group of HIV-positive patients with no KS. We found a unique miRNA expression signature in 49 miRNAs in patients with epidemic KS compared to those with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs down regulated. The top 10 significantly upregulated miRNAs were miR-4446-3p, miR-411-5p, miR-1304-3p, miR-6819-3p, miR-654-5p, miR-1908-5p, miR-431-5p, miR-223-5p, miR487a-5p, and miR-379-5p. The significantly downregulated miRNAs were miR-451a, miR-4732-3p, miR-93-5p, miR-486-5p, miR-185-5p, miR-375-3p, miR-25-3p, and miR-7-5p. These findings imply that the above patterns of differential expression of circulating miRNAs could be used to diagnose epidemic KS in HIV infected individuals. The life cycle of HHV-8 consists of latent and lytic replication phases. During the latent phase of the infection, only a limited number of HHV-8 genes are expressed in a bid to promote persistent infection, evade host immune responses, and induce HHV-8-related malignancies, such as KS (Ye, Lei, & Gao, 2011). In vitro studies conducted in cell lines derived from HHV-8 associated tumors have implicated the HHV-8 viral miRNA, miR-k12-11 as a key player in reprograming naïve B-cells towards supporting long-term latency. In the current study, three out of the 29 KS negative, HIV positive subjects in the comparison group tested positive for HHV-8 DNA, pointing to possible latent HHV-8 infection. Differential profiling of circulating miRNA in the latently infected individuals in our study revealed a significant upregulation in the expression of miR-193a-5p compared to HIV positive, HHV-8 PCR negative participants. This could imply that circulating levels of miR-193a-5p could be used as a none-invasive biomarker of latent epidemic KS infection. Indeed miR-193a-5p has been reported to be upregulated in primary lymphatic endothelial cells with in 72hrs of HHV-8 infection (Lagos et al., 2010), further strengthening the fact that they could be used as early disease biomarkers in KS. miR-193a-5p has also been reported to be upregulated in other cancers, such as prostate (Yang et al., 2017) and hepatocellular carcinoma (Ji et al., 2009), as well as during hypoxic conditions (Viollet et al., 2017). miRNAs are actively secreted via "exosomes". Exosomes are small vesicles released from cells, into which miRNAs are specifically sorted and accumulated. Exosomes help to protect circulating miRNA from degradation by RNases in the extracellular environment (Sohel, 2016). Host-encoded miRNAs have previously been found in exosomes released from KSHV-infected lymphoma cell lines (Hoshina et al., 2016). Indeed, some of the dysregulated miRNA in patients with epidemic KS in the current study have been reported to be expressed in KS endothelial tumor cells. Specifically, miR-146a-5p, miR-199a-3p, and miR-126-3p that we found to be upregulated in serum collected from patients with epidemic KS in our study, were also upregulated in tumor specimens collected from patients with both classical and epidemic KS in earlier studies (Punj et al., 2010; Wu et al., 2015), implying that they originated in the KS tumor lesions. Regarding the roles of these miRNA in KS pathophysiology, KSHV infection activates the transcription factor nuclear factor kappa B (NFκB), which is believed to up-regulate the expression of miR-146. miR-146a then down-regulates the chemokine ligand CXCR4, which promotes the release of KSHV-infected endothelial cells into the circulation (Hussein & Akula, 2017). miR-126a is believed to promote growth of KS by inducing the expression of Vascular Endothelial Growth Factor -A (VEGF-A), whereas miR-199a promotes KS development by inducing the proliferation and survival of endothelial cells (Shatseva, Lee, Deng, & Yang, 2011). During human immunodeficiency virus type 1 (HIV-1) infection, host miRNA profiles are altered either as a host response against the virus or as a mechanism for the virus to facilitate viral replication and infection, or to maintain latency (Su et al., 2018). Moreover, it has been shown that HIV-infected subjects have unique circulating miRNA profiles compared to HIV-uninfected persons. However, what is currently unknown is whether this pattern of differential expression stays constant during the lifecycle of HIV infection. In the current study, we compared the differential miRNA expression patterns at different time points from index HIV diagnosis up to a period of more than 10 years after index HIV diagnosis. We found an upregulation of 20 differentially expressed miRNAs, as well as down regulation of four miRNAs in patients diagnosed with in 0-2 years of the study compared to 2-5 years from index HIV diagnosis at the time of the study. The 10 most significantly upregulated miRNA were miR-1307-3p, miR-151a-5p, miR-151b-5p, miR490-5p, miR-222-3p, miR-6819-3p, miR-487a-5p, miR-154-5p, miR-431-5p, and miR-224-5p, whereas miR-451a, miR-486-5p, miR-150-5p, and miR-4732-3p were significantly downregulated. From amongst the upregulated miRNAs, miR-151 has previously been reported as an early biomarker of HIV-1 infection in a panel of circulating miRNAs that distinguished HIV-1 infected individuals from health HIV-negative controls (Qi et al., 2017). Conversely, downregulation of circulating miR-150 as described in the current study was previously reported to predict HIV/AIDS disease progression and therapy. miR-150 is a key regulator of immune cell differentiation and activation and is expressed in monocytes, as well as in mature and resting B and T lymphocytes (Munshi, Panda, Holla, Rewari, & Jameel, 2014). Additionally, differential co-expression of miR-150 in plasma has been used to identify cognitive impairment in HIV-infected patients (Kadri et al., 2016). These results add to the existing findings that HIV infection alters the differential expression of circulating miRNAs. Use of highly active antiretroviral therapy (HAART) reduces the incidence of epidemic KS in HIV positive, KS negative individuals. In individuals with epidemic KS, use of HAART alone can lead to resolution of KS (Stebbing, Portsmouth, & Gazzard, 2003). In addition to HAART, patients with epidemic KS at the Uganda Cancer Institute are treated with chemotherapy consisting of a combination of bleomycin and vincristine for varying treatment cycles, depending on the patients’ response. Although miRNAs have previously been used to predict response to chemotherapy in pancreatic ductal adenocarcinoma, colorectal cancer, and osteosarcoma (Review by (Wang et al., 2018)), we did not find any differential circulating miRNA expression when we compared epidemic KS patients who had received only three or less cycles of chemotherapy, compared to epidemic KS patients who had received more than 3 cycles of chemotherapy. Taken together, these results imply that circulating miRNAs are poor predictors of chemo-response or chemo-resistance in epidemic KS. Our results are encouraging as they advance the potential of circulating miRNAs as biomarkers for diagnosis of epidemic Kaposi’s sarcoma. As it is with all observational studies, causality cannot be inferred from this study. Thus, further work is needed, using a larger, controlled prospective study to fully validate the differential circulating miRNA expression patterns observed in the current study. Moreover, the differential fold expression results for circulating miRNAs in the current study have to be validated by real-time polymerase chain reaction (RT-PCR). Additionally, the methodology adopted for the current study does not provide insights into the causes of the differential expression of the circulating miRNAs observed, which could have resulted from factors other than epidemic KS. Conclusions In conclusion, this study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have potential roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection. Additionally, duration from index HIV diagnosis also significantly contributed to alterations in the differential expression of circulating miRNAs in all study subjects. Abbreviations 3’UTR three prime untranslated region AIDS Acquired Immune Deficiency Syndrome ART Anti-retroviral therapy cART Combined Antiretroviral Therapy cDNA Complementary DNA DNA deoxyribonucleic acid EBV Epstein Bar Virus ELISA Enzyme-linked Immunosorbent Assay FC Fold change FDR False discovery rate HAART Highly Active Antiretroviral Therapy HHV-8 Human Herpes Virus 8 HIV Human Immune Virus INSTI Integrase Strand Transfer Inhibitor ISS clinic Immune suppression syndrome clinic KS Kaposi’s Sarcoma KSHV Kaposi's Sarcoma-associated Herpesvirus log CPM log2 counts per million miRNA microRNA mRNA messenger RNA NFκB nuclear factor kappa B NNRTI Non-nucleoside Reverse Transcriptase Inhibitors NRTIs Nucleoside Reverse Transcriptase Inhibitors ORF Open Reading Frame PAGE polyacrylamide gel electrophoresis PCR Polymerase Chain Reaction PI Protease Inhibitors RIN RNA Integrity Number RNA ribonucleic acid rRNA ribosomal RNA RT primers Reverse Transcription primers SBS-REC School of Biomedical Sciences Research Ethics Committee tRNA transfer RNA UCI Uganda Cancer Institute UMIs Unique Molecular Identifiers () UNCST Uganda National Council for Science and Technology VEGF-A Vascular Endothelial Growth Factor -A Declarations Ethics approval and consent to participate This research was carried out in compliance with research principles adopted from the Helsinki Declaration, and approval to conduct the study was obtained from the Institutional Review Board of the Makerere University, College of Health Sciences, School of Biomedical Sciences Research and Ethics Committee (SBS-REC) (Protocol number: SBS-519 ) . Written informed consent was obtained from all study participants. Consent for publication Not applicable Availability of data and material The datasets used are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work is part of a research fellowship supported by Grant Number D43TW010132 supported by Office of the Director, National Institutes of Health (OD), National Institute of Dental & Craniofacial Research (NIDCR), National Institute of Neurological Disorders and Stroke (NINDS), National Heart, Lung, And Blood Institute (NHLBI), Fogarty International Center (FIC), National Institute on Minority Health and Health Disparities (NIMHD), as well as the Microbiology and Immunology Training in HIV and related infection in Uganda (MITHU) fellowship under the Uganda Case Western Research collaboration. The funding agencies were not involved in the design, data collection, analysis, and interpretation of data; and in writing the manuscript. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the supporting offices. Author’s contributions HM, HK, DN, DPK, and FB contributed to study conceptualization and design. HM, HK and FN conducted the laboratory analysis. CA conducted bioinformatics analysis on sequencing data for the study, and contributed to manuscript writing and revision. HM wrote the first draft of the manuscript. JK, AL, IK, JN, SLF, AMD, DN, DPK, FB, and FS contributed critical revisions and important intellectual content to the manuscript. All authors read and approved the final manuscript. Acknowledgments The authors deeply appreciate the study subjects for agreeing and volunteering time to participate in this study. 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Mechanisms of Kaposi's Sarcoma-Associated Herpesvirus Latency and Reactivation. Adv Virol, 2011 . doi:10.1155/2011/193860 Yu, H., Guan, Z., Cuk, K., Zhang, Y., & Brenner, H. (2019). Circulating MicroRNA Biomarkers for Lung Cancer Detection in East Asian Populations. Cancers (Basel), 11 (3). doi:10.3390/cancers11030415 Supplementary Files STROBEchecklistKSmanuscript.docx Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2022 Read the published version in Non-coding RNA Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Damalie","middleName":"","lastName":"Nakanjako","suffix":""},{"id":5421599,"identity":"e601717e-4b85-4a4d-8bca-cb3ec5674ded","order_by":11,"name":"David Patrick Kateete","email":"","orcid":"","institution":"Makerere University College of Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"Patrick","lastName":"Kateete","suffix":""},{"id":5421600,"identity":"0f823a56-5d00-493f-9e7a-fdb44790c361","order_by":12,"name":"Freddie Bwanga","email":"","orcid":"","institution":"Makerere University College of Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Freddie","middleName":"","lastName":"Bwanga","suffix":""}],"badges":[],"createdAt":"2020-11-27 00:07:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-116856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-116856/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.ncrna.2022.02.002","type":"published","date":"2022-04-01T01:55:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3987090,"identity":"3339e6d7-e6ef-4c93-acde-cdefb38c3033","added_by":"auto","created_at":"2020-12-03 15:58:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50973,"visible":true,"origin":"","legend":"Gel electrophoresis image after amplification for the HHV 8 DNA.\nLegend: Lanes labeled L - 1000bp DNA ladder. Lane NC - negative control, while lane PC positive control. Lanes 1, 2, 3, 5, 7, 11, and 12 – are from samples positive for HHV 8 DNA. Lanes 4, 6, 8, 9, and 10 – are from samples negative for HHV 8 DNA.","description":"","filename":"Figure1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/3cf53d0447c7cfe1660ab25e.JPG"},{"id":3987091,"identity":"b7f6910e-635d-4640-ab8a-11b397190e39","added_by":"auto","created_at":"2020-12-03 15:58:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70523,"visible":true,"origin":"","legend":"Volcano plot of miRNA differentially expressed in patients with KS verse those no KS.\nLegend: The plot is depicted with vertical and horizontal lines indicating the threshold for a relative expression fold change of 2 0r -2 compared with no KS (controls), at a 0.05 P-value. Down regulated miRNAs are depicted in red, whereas upregulated miRNAs are depicted in green.","description":"","filename":"Figure2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/508251971512ad6d3dc815c8.JPG"},{"id":3987092,"identity":"442869b4-8744-45c1-9004-44b4b52af8b0","added_by":"auto","created_at":"2020-12-03 15:58:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137071,"visible":true,"origin":"","legend":"The most variable circulating miRNAs expressed in patients with and without Epidemic KS.\nLegend: Z-score expression heat map of the most variable circulating miRNAs expressed in patients with and without Epidemic KS. Patients with Epidemic KS are denoted as uci whereas the comparative group of HIV-positive patients with no KS are denoted as iss","description":"","filename":"Figure3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/cdab3a806898e2dc3d78bda8.JPG"},{"id":3987093,"identity":"a839e00b-4d5a-4370-b496-a4a06c6db984","added_by":"auto","created_at":"2020-12-03 15:58:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38093,"visible":true,"origin":"","legend":"Differential circulating miRNAs expressed in participants testing positive or negative for HHV-8 DNA\nLegend: Volcano plot showing differentially expressed miRNAs in patients testing positive or negative for HHV-8 DNA. miRNAs differentially expressed with an adjusted p-value \u003c 0.005 are depicted in red for down regulation and green for up regulation.","description":"","filename":"Figure4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/5a11db0db3c23791b1158ad3.JPG"},{"id":3987095,"identity":"1f986e1d-d23f-4c94-b461-c1b675b4d3c4","added_by":"auto","created_at":"2020-12-03 15:58:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64990,"visible":true,"origin":"","legend":"Differential miRNA expression in patients 0-2 years versus 2-5 years after index HIV diagnosis.\nLegend: Volcano plot showing differential miRNA expression in patients 0-2 years versus 2-5 years after index HIV diagnosis. miRNAs differentially expressed with an adjusted p-value \u003c 0.005 are depicted in red for down regulation and green for up regulation.","description":"","filename":"Figure5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/e78df21163cd375997b898c7.JPG"},{"id":20155226,"identity":"077ac945-318c-4123-9bfc-4db215105179","added_by":"auto","created_at":"2022-04-10 01:55:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":933311,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/5b7c817a-d854-4902-a952-0554bac8da02.pdf"},{"id":3987094,"identity":"d807dfb8-8355-439e-b466-f9d6599bf7c6","added_by":"auto","created_at":"2020-12-03 15:58:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32313,"visible":true,"origin":"","legend":"","description":"","filename":"STROBEchecklistKSmanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-116856/v1/64133f028be69591e5c1a6bc.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eUnique Circulating microRNA Profiles in Epidemic Kaposi’s Sarcoma\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eKaposi sarcoma (KS) is the most common HIV-related malignancy worldwide and the 3\u003csup\u003erd\u003c/sup\u003e most frequently diagnosed cancer amongst men in Sub-Saharan Africa. Uganda has one of the highest rates of KS in the world, partly due to the HIV pandemic, with the incidence of KS in women surpassing that of cervical cancer as the most common female malignancy (Phipps et al., 2010). Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), is the etiological agent of Kaposi's sarcoma, and in vivo, it mainly infects B cells and endothelial cells, impairing the expression of pro and anti-cancer genes in these cells. In Majority of infected individuals, the virus may remain latent for years or decades, only to be activated by compromised immunity, such as seen in Human Immune Virus (HIV) infection and organ transplantation. Consequently, Epidemic Kaposi\u0026rsquo;s sarcoma is only found in patients who have HIV acquired immune deficiency syndrome (HIV/AIDS). In Africa, KS is typically aggressive, and patients with Epidemic KS often present late with widely disseminated and rapidly progressive disease (Mosam et al., 2012), and despite standard treatment with highly active antiretroviral therapy (HAART) and chemotherapy, about one-third of these patients succumb to the disease after 2 years (Freeman et al., 2016; Okuku et al., 2017).\u003c/p\u003e\n\u003cp\u003eThis relatively high disease morbidity and mortality partly results from delayed diagnosis arising from a lack of a reliable diagnostic assay for detection of latent or early stage disease. Nevertheless, the identification of reliable biomarkers for KS remains a challenge to-date. In resource-limited settings, diagnosis of KS is usually clinical, based on the presence of cutaneous or mucosal KS lesions, as well as associated symptoms. The challenge with the clinical approach for KS diagnosis is the fact that KS skin lesions are usually mimicked by other non-KS lesions (Yaqub et al., 2019). Moreover, even though histopathological analysis is considered the gold standard diagnostic test for KS (Corey Casper, Martin S Hirsch, \u0026amp; Bloom, 2019), this test is wrought with challenges, such as the prior requirement of a tissue sample acquired from an invasive tissue-biopsy procedure, as well as the subjective interpretation of test results leading to inter-observer variability among pathologists (Amerson et al., 2016). Additionally, histopathological diagnosis can only be performed for patients who have manifested KS like lesions on the skin or mucosa membranes, and thus may not apply to patients with sub-clinical or latent disease. Similarly, ELISA-based assays used to augment other tests during KS detection have limitations of low specificity and cross-reactivity with other antigens, such as Epstein Bar Virus (EBV) proteins (Ablashi, Chatlynne, Whitman, \u0026amp; Cesarman, 2002). PCR-based techniques that detect HHV-8 DNA in tissues and serum, albeit highly sensitive, are also limited by low specificity (Auten, Kim, Bradley, \u0026amp; Rosado, 2017), particularly in the viral latent phase.\u003c/p\u003e\n\u003cp\u003eA novel class of molecules called microRNAs (miRNAs) have recently gained traction in healthcare management for their potential as biomarkers for human diseases (Dave et al., 2019). miRNAs are small (19-24 nucleotides), evolutionary conserved, endogenous non-coding RNA molecules that bind to the 3\u0026rsquo;UTR of target mRNA transcripts with partial or perfect sequence complementarity, resulting in translational repression and/or mRNA destabilization (O'Brien, Hayder, Zayed, \u0026amp; Peng, 2018). During the past decade, our knowledge about the role of miRNAs in human diseases has grown exponentially (Gilad et al., 2012; O'Brien et al., 2018). Abnormal expression of a single miRNA can have implications on the activity of multiple genes (Dave et al., 2019; O'Brien et al., 2018). It\u0026rsquo;s not surprising therefore that changes in miRNA expression have been found to contribute to a wide variety of human disease states and disorders such as cancer, cardiovascular, autoimmune, neurodegenerative, hepatic, and inflammatory diseases (Dave et al., 2019). The utility of miRNA\u0026rsquo;s in diagnostics is derived from their specificity for a particular type of tissue or cell, their abundance, as well as their remarkable stability in body tissues and fluids such as serum, urine, saliva, milk and cerebrospinal fluid (Dave et al., 2019; Gustafson, Tyryshkin, \u0026amp; Renwick, 2016). These characteristics demonstrate a remarkable potential for microRNAs to be used as biomarkers for disease. miRNAs have been successfully used to classify cancer, identify cancer tissue origin, determine prognosis and disease progression, predict resistance to chemotherapy, monitor therapy, and screen for disease (Ahmed et al., 2009; Calin et al., 2005; Eitan et al., 2009; Gustafson et al., 2016; Ji et al., 2009). HHV8 infection or HHV8-related malignancies can induce a unique signature of human and viral intracellular and extracellular miRNAs (Qin, Peruzzi, Reiss, \u0026amp; Dai, 2014), and this has been confirmed in vitro studies (Hoshina et al., 2016). These data though highly suggestive, cannot be extrapolated to epidemic KS, due to the additive effects of HIV-infection on cellular miRNA signaling.\u003c/p\u003e\n\u003cp\u003eThis study aimed at profiling the unique miRNAs found in serum of patients with epidemic KS, by comparing circulating miRNAs in patients with epidemic KS to those of a comparative group of patients with no KS. These data will provide baseline data that will aid in developing a miRNA-based minimally invasive PCR assay for Epidemic Kaposi\u0026rsquo;s Sarcoma, which would greatly facilitate the detection and management of a disease that has a high rate of recurrence and mortality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy \u003c/strong\u003e\u003cstrong\u003esetting and population; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a comparative cross-sectional study conducted from August 2018 \u0026ndash; July 2019. Whole blood samples were obtained from 27 HIV positive adult patients with epidemic Kaposi\u0026rsquo;s sarcoma, confirmed by histopathology at the Uganda Cancer Institute (UCI) and 27 HIV adult patients without clinical manifestation of KS at Makerere Joint AIDs Program (MJAP) clinic for comparison. The Skin Cancer clinic at UCI provides care for ambulatory patients with skin cancer. The MJAP clinic is an outpatient facility for ambulatory patients seeking continued HIV care services, and provides comprehensive HIV prevention, treatment, care, and support. The ISS clinic provides general care to over 15,000 HIV infected patients and opens 5 days a week with daily attendance of \u0026gt;300 patients. The pregnant, severely sick, and HIV-positive, KS negative individuals with mucosal and skin lesions that could mimic KS were excluded. The total number of study participants was fifty-four (54), and study participants were purposively sampled. The sample size was determined using the Fleiss\u0026rsquo;s equation (1980) with continuity correction, commonly used for comparative studies. All patients were taking anti-retroviral therapy (ART). Of the 50 participants who had available information regarding their ART medication, 34 were taking combined antiretroviral therapy medicines (cART) consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) in combination with a non-nucleoside reverse transcriptase inhibitors (NNRTI), 6 were taking cART consisting a two NRTIs and a protease inhibitors (PI), whereas 7 received an Integrase Strand Transfer Inhibitor (INSTI) therapy based ART. The proportion of participants taking cART was similar among Epidemic KS and none-KS participants. A questionnaire was used to obtain participants demographic information, including age, gender, as well as base line clinical characteristics, such as KS lesion morphotype, presence of edema, disease comorbidities and treatments received at presentation or after KS diagnosis.\u003c/p\u003e\n\u003cp\u003eFive (5) milliliters of venous blood were collected from study participants in specialized vacutainer tubes, of which 2mls were stored at 4\u003csup\u003e0\u003c/sup\u003eC for DNA isolation, whereas 3mls were left to stand on ice for 30 minutes, centrifuged for 20 minutes at 1300g to collect serum that was then aliquoted in 2ml Eppendorf tubes containing RNA later (Thermo Fisher Scientific), and stored at -80\u003csup\u003e0\u003c/sup\u003eC for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA \u003c/strong\u003e\u003cstrong\u003eextraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from whole blood using the QIAamp DNA midi blood kit (QIAGEN) in accordance with the manufacturer's instructions. Briefly, 100\u0026mu;l of the Protease enzyme was pipetted into a 15ml Centrifuge tube and 200\u0026mu;l of the sample, 800\u0026mu;l PBS, and 200\u0026mu;l of Buffer AL added. The resulting mixture was vortexed for 15 seconds, and then incubated at 70\u0026deg;C for 10 minutes to lyse the cells. 1 ml of absolute ethanol was then added to the mixture and vortexed to allow DNA precipitation. The resulting mixture was then added to the QIAamp Midi spin column, centrifuged at 8000 rpm for 1 minute and the supernatant discarded. 2ml of Wash buffer AW1 was added to the column and the centrifuged at 5000rpm for 1 minute and the supernatant discarded. Then, 2ml of Wash buffer AW2 was added to the column, centrifuged at 5000rpm for 15 minute and the supernatant was discarded. DNA was eluted in 200\u0026mu;l Buffer AE. The DNA quality was assessed using the NanoDrop\u003csup\u003eTM\u003c/sup\u003e 2000C spectrophotometer (Thermo Scientific, Wilmington, DE, USAs)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNested PCR for HHV-8 DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo amplifications were done by the nested PCR technique. The first amplification targeted a region in the open reading frame (ORF)-26 of HHV-8 as was done previously (Machado, Farias, Pereira, Freitas, \u0026amp; Fonseca, 2015). Briefly, for the first run, a total reaction mixture of 25\u0026mu;l was prepared from a mixture of the following; 12.5\u0026micro;l 1X Master Mix (BioLabs, New England), 1.25\u0026mu;l of 10pMol of each sense (5'AGCCGAAAGGATTCCACCAT-3\u0026rsquo;) and antisense (5'-TCCGTGTTGTCTACGTCCAG-3') primers (Euro films Genomics, Vienna), 7\u0026mu;l nuclease free water and 3\u0026mu;l sample DNA. During this run, 35 cycles of PCR amplification of HHV-8 DNA was done on a SimpliAmp\u003csup\u003eTM \u003c/sup\u003eThermal cycler (Thermo Fisher Scientific, Applied Biosystems, Singapore). The PCR conditions were as follows; initial denaturation at 95 \u0026deg;C for 5 minutes, followed by further denaturation at 94 \u0026deg;C for 30 seconds, annealing at 56 \u0026deg;C for 30 seconds, extension at 68\u0026deg;C for 45 seconds and a final extension step at 68 \u0026deg;C for 10 minutes. The second PCR run targeted a 211bp region in the first amplicon. The nested PCR amplification mixture contained 5\u0026mu;l of the first RCR mixture, 25\u0026micro;l of the 1X master mix, 15\u0026mu;l of nuclease free water and 2.5\u0026mu;l of 10pMol of each of the internal forward primer (5'TTCCACCATTGTGCTCGAAT-3') and reverse primer (5'-TACGTCCAGACGATATGTGC-3'). During the run, 35 cycles of amplification were done under the following conditions; initial denaturation at 95\u0026deg;C for 5 minutes, further denaturation at 94 \u0026deg;C for 30 seconds, annealing at 58 \u0026deg;C for 30 seconds, extension at 68 \u0026deg;C for 30 seconds and a final extension of 10 minutes at 68\u0026deg;C. Five microlitres (5\u0026mu;l) of the final PCR product were loaded into a 1.2% agarose gel, and electrophoresed at 120V for 90 minutes. Positive reactions yielded an amplicon of 211 bp, which was easily viewed on a UV trans-illuminator after ethidium bromide staining.\u003c/p\u003e\n\u003cp\u003eAs a positive control, confirmed HHV-8 DNA was used, as well as a set of negative controls of nuclease free and negative clinical samples for HHV-8 DNA were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quality control \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum total RNA was extracted from 200\u0026micro;l of serum samples using the Qiagen miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA, USA) according to manufacturer\u0026rsquo;s instructions (QIAGEN; Haldane, Germany). RNA abundance and integrity were determined after isolation using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only samples with an RNA integrity number (RIN) \u0026gt;9 were subjected to subsequent library preparation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLibrary preparation and sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, small RNA fragments between 18 to 30 nucleotides were extracted from total RNA on a PAGE gel. Then, a 5-adenylated, 3-blocked single-stranded DNA adapter was ligated to the 3\u0026rsquo;end of RNA fragments. Thereafter, reverse transcription (RT) primers with unique molecular identifiers (UMIs) were added to the resulting mixture to enable hybridization to the ligated 3\u0026rsquo;adaptors in RNA and dissociative 3\u0026rsquo;adapters. Next, 5\u0026rsquo;adapters were linked to the 5\u0026rsquo;end of the resulting ligation product, followed by one-strand cDNA synthesis with RT primers. The resulting cDNA was then amplified by PCR. Fragments between 110bp to 130bp of the cDNA library were selected by PAGE electrophoresis, quantified, and then pooled with libraries from other samples. The cDNA libraries were validated using the Agilent Technologies 2100 bioanalyzer, and thereafter subjected to high throughput DNA nanoball sequencing using Phi29 DNA polymerase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter Sequencing, data was analyzed using the miRge2 pipeline (Lu, Baras, \u0026amp; Halushka, 2018) for alignment and quantification of miRNAs, whereas all statistical analyses of differentially expressed miRNAs were conducted in EdgeR (Robinson, McCarthy, \u0026amp; Smyth, 2010). In brief, Fastq files underwent quality control, removal of adapters sequences, and formation of unique reads. Specifically, unique reads were annotated to mature miRNAs, mRNA hairpins, and other RNA types by alignment toward miRbase v.22 (Griffiths-Jones, Grocock, van Dongen, Bateman, \u0026amp; Enright, 2006). Only quantified mature miRNAs were used in subsequent data analysis. Low read counts were filtered out setting at least five reads as the minimum cut-off. Filtered counts were then normalized and transformed to log2 counts per million (log CPM) using R package EdgeR. miRNAs with an FC \u0026gt; 1 and FDR \u0026lt; 0.05 were considered upregulated and those with an FC \u0026lt; 1 and FDR \u0026lt;0.05 downregulated. Differentially expressed miRNAs were visualized on volcano plots. Unsupervised clustering was performed by using log2 transformed values and using Euclidian distances between samples. All statistical analysis was conducted in R environment (version 3.6.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics:\u003c/strong\u003e The study was approved by the Research and Ethics Committee of the School of Biomedical Sciences (SBS-REC) with study number SBS SBS-519, and also registered with the Uganda National Council for Science and Technology (UNCST); (study number: HS 2405). Participants\u0026rsquo; confidentiality was maintained at all times.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient demographics\u003c/strong\u003e; A total of 54 participants participated in this study. Half of the participants (27) had epidemic KS, whereas the other half were HIV-positive with no confirmed diagnosis of KS. All the 27 KS diseased patients had cutaneous lesions, with most of them localized to the lower extremities. A few of the participants had further manifestations of ulcerated tumors, swollen and painful lymph nodes and palate lesions. Details of the Sociodemographic and Clinical characteristics of KS patients in this study are shown in table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Sociodemographic and Clinical characteristics of study participants\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"387\"\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristic \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency n (%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e46 (85)\u003c/p\u003e\n\u003cp\u003e8 (15)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eAge in years (n = 54)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e21-30\u003c/p\u003e\n\u003cp\u003e31-40\u003c/p\u003e\n\u003cp\u003e41-50\u003c/p\u003e\n\u003cp\u003e51-80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e5(19)\u003c/p\u003e\n\u003cp\u003e14(52)\u003c/p\u003e\n\u003cp\u003e6(22)\u003c/p\u003e\n\u003cp\u003e2(7)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eNature of residence (n = 54)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eUrban\u003c/p\u003e\n\u003cp\u003eRural\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e43(80)\u003c/p\u003e\n\u003cp\u003e11 (20)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eOccupation (n = 54)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eEmployed\u003c/p\u003e\n\u003cp\u003eUnemployed\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e34 (62)\u003c/p\u003e\n\u003cp\u003e20 (38)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eEducational level (n = 54)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003ePrimary\u003c/p\u003e\n\u003cp\u003eSecondary\u003c/p\u003e\n\u003cp\u003eTertiary\u003c/p\u003e\n\u003cp\u003eNon-formal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e24 (44)\u003c/p\u003e\n\u003cp\u003e20 (37)\u003c/p\u003e\n\u003cp\u003e7 (13)\u003c/p\u003e\n\u003cp\u003e3 (6)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eDuration on ART (n=54)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e\u0026lt;6 months\u003c/p\u003e\n\u003cp\u003e6-24 months\u003c/p\u003e\n\u003cp\u003e24-60 months\u003c/p\u003e\n\u003cp\u003e60-120 months\u003c/p\u003e\n\u003cp\u003e\u0026gt;120 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e3 (5)\u003c/p\u003e\n\u003cp\u003e10 (19)\u003c/p\u003e\n\u003cp\u003e22 (41)\u003c/p\u003e\n\u003cp\u003e12 (22)\u003c/p\u003e\n\u003cp\u003e7 (13)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eDuration from index KS diagnosis (n = 24)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e\u0026lt;3months\u003c/p\u003e\n\u003cp\u003e3-6months\u003c/p\u003e\n\u003cp\u003e6-12months\u003c/p\u003e\n\u003cp\u003e\u0026gt;12months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e7 (29)\u003c/p\u003e\n\u003cp\u003e2 (8)\u003c/p\u003e\n\u003cp\u003e6 (25)\u003c/p\u003e\n\u003cp\u003e9 (38)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eForm of KS (n = 27)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eSkin\u003c/p\u003e\n\u003cp\u003eOther\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e27 (100)\u003c/p\u003e\n\u003cp\u003e0 (0)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eNo. of Chemotherapy cycles completed (n=20)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eOne cycle\u003c/p\u003e\n\u003cp\u003eTwo cycles\u003c/p\u003e\n\u003cp\u003eThree cycles\u003c/p\u003e\n\u003cp\u003eFour cycles\u003c/p\u003e\n\u003cp\u003eFive cycles\u003c/p\u003e\n\u003cp\u003eSix cycles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e2 (10)\u003c/p\u003e\n\u003cp\u003e6 (30)\u003c/p\u003e\n\u003cp\u003e3 (15)\u003c/p\u003e\n\u003cp\u003e4 (20)\u003c/p\u003e\n\u003cp\u003e2 (10)\u003c/p\u003e\n\u003cp\u003e3 (15)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003ePositive HHV-8 DNA (n=54)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eKS (n=27)\u003c/p\u003e\n\u003cp\u003eNon-KS (n=27)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e15 (55.6)\u003c/p\u003e\n\u003cp\u003e3 (11.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eTumor appearance (n = 27)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eUlcerated\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e5 (19)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003eLymph node involvement (n = 27)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e8 (24)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"245\"\u003e\n\u003cp\u003e\u003cstrong\u003ePalate involvement (n = 27) \u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"135\"\u003e\n\u003cp\u003e6 (22)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential expression of miRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether patients with Epidemic Kaposi\u0026rsquo;s sarcoma differentially express miRNAs relative to HIV patients without Kaposi\u0026rsquo;s sarcoma, we performed differential expression testing using EdgeR, using normalized read counts. We found that 49 miRNAs were differentially expressed, with 8 miRNAs downregulated and 41 miRNAs upregulated (adjusted p-value \u0026lt;0.05) (\u003cstrong\u003e\u003cem\u003eTable 2, \u003c/em\u003e\u003c/strong\u003evolcano plot in\u003cstrong\u003e\u003cem\u003e figure 2, \u003c/em\u003e\u003c/strong\u003eand heatmap in \u003cstrong\u003e\u003cem\u003efigure 3\u003c/em\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Differentially expressed miRNAs in patients with Epidemic KS compared to HIV patients with no KS\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e\u003cstrong\u003eFold change (log10)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e\u003cstrong\u003eUp/Down regulation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e\u003cstrong\u003eAdjusted p-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-4446-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.84849\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e2.50E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00317\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-451a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-2.003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e3.72E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00317\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-99b-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.24184\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e4.50E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00317\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-411-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.18601\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e7.14E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00376\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-1304-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e5.29579\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e9.94E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00419\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-4732-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-1.9894\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003edown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-6819-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.98167\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-654-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.60077\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-93-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-1.4618\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00022\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-1908-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.45843\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-337-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.05436\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-431-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.28894\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00535\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-223-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.92606\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0004\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00637\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-139-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.44008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00044\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00637\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-654-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.76475\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00047\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00637\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-let-7e-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.86566\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00048\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00637\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-487a-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.14846\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00052\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00649\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-379-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e4.16306\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00068\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00758\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-486-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-4.0858\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00758\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-369-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.97987\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00075\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00758\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-222-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.27732\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00075\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00758\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-191-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.75839\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00863\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-382-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.87227\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00097\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.00885\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-409-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.61324\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00122\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.01071\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-584-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.56752\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00176\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.01487\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-154-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.4633\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00244\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.01972\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-1307-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.04218\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00252\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.01972\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-221-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.34441\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00314\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02296\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-4433b-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.73024\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00316\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02296\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-185-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-1.7193\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00346\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02396\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-let-7e-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.45092\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00352\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02396\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-625-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.57103\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00428\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.0274\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-323b-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.10619\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00429\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.0274\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-328-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e1.67445\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00491\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02964\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-199a-3p/199b-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e0.81156\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00499\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02964\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-375-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-1.3701\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00506\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.02964\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-139-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e0.97288\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00561\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.03199\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-485-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.36411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00666\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.03602\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-126-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e0.70315\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00678\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.03602\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-25-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-1.0357\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00683\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.03602\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-424-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.15035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00719\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.03698\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-146a-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e0.83632\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00815\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04088\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-224-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.16699\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00833\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04088\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-7-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e-0.9513\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00933\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04475\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-340-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.71832\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00981\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04559\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-323a-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.26012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.00994\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04559\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-323a-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e3.20691\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.01045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04619\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-425-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.72079\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.01051\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04619\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"124\"\u003e\n\u003cp\u003ehsa-miR-127-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"134\"\u003e\n\u003cp\u003e2.05625\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e0.0108\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e0.04651\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eRelationship between miRNA expression and presence or absence of KSHV DNA. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in the levels of circulating miRNAs in the serum of humans and animals have been detected as a result of infection with a variety of viruses (Stenfeldt et al., 2017). Because Epidemic KS is caused by HHV-8, in addition to the fact that HHV-8 DNA has been detected in the blood of Epidemic KS patients, we examined the effect of presence of circulating HHV-8 DNA on the serum expression profile of circulating miRNAs. A nested PCR targeting a region in the open reading frame (ORF)-26 of HHV-8 DNA was conducted on DNA extracted from the blood of both epidemic KS patients and the comparative group. Analysis of samples from patients that tested positive for circulating HHV-8 DNA compared to those that tested negative revealed a differential expression of 4 miRNAs that were downregulated and only one miRNA that was up regulated (\u003cstrong\u003e\u003cem\u003etable 3 \u003c/em\u003e\u003c/strong\u003eand\u003cem\u003e\u003cstrong\u003efigure 4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e). \u003c/strong\u003eFurther stratification with regards to epidemic KS status did not reveal any significant differences in the differential expression of circulating miRNA between epidemic KS patients who tested positive for circulating HHV-8 DNA compared to epidemic KS patients that tested negative for HHV-8 DNA (\u003cstrong\u003e\u003cem\u003etable 4\u003c/em\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u003c/strong\u003e\u003cstrong\u003e3: \u003c/strong\u003eDifferential circulating miRNA expression analysis in participants testing positive or negative for HHV-8 DNA\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e\u003cstrong\u003eFold change (log10)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e\u003cstrong\u003eUp/Down regulation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e\u003cstrong\u003eAdjusted p-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003ehsa-miR-451a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e1.930507\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eUp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e2.72E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.02273\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003ehsa-miR-6819-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e-5.610377\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e1.50E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.00316\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003ehsa-miR-654-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e-3.967188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e3.35E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.02273\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003ehsa-miR-139-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e-1.411567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e4.31E-04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.02273\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"126\"\u003e\n\u003cp\u003ehsa-miR-1304-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"157\"\u003e\n\u003cp\u003e-4.57006\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e1.06E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.04470\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: \u003c/strong\u003eDifferentially expressed circulating miRNAs in epidemic KS patients testing positive or negative for HHV-8 DNA\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eFold change (log10)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eUp/Down regulation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eAdjusted \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-150-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e-1.9696311\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.00314924\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.6644893\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-16-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e4.4026519\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.00901365\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9509403\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-1301-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e4.1115834\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.01907221\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-363-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e3.8036035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.01995521\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-486-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e-3.6520209\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.02860547\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-15b-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e3.5675304\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.03616976\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-25-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e3.3827188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.04592067\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-532-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9929975\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05097679\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-451a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e2.5280154\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05651414\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-29a-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e-1.2541687\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05970539\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.9774167\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential miRNA expression in latent HHV-8 infection. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree participants from the comparative group (HIV-positive, KS negative) had tested positive for HHV-8 DNA on nested PCR, indicating that they could be latently infected with KS. Consequently, serum miRNAs from these patients was compared to that of HIV-positive, KS negative patients who tested negative for HHV-8 DNA. Analysis for differential miRNA expression revealed a significant upregulation of miR-193a-5p in HHV-8 latently infected patients (\u003cstrong\u003e\u003cem\u003etable 5\u003c/em\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5: \u003c/strong\u003eDifferentially expressed circulating miRNAs in KS negative patients testing positive or negative for HHV-8 DNA\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003emiRNA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e\u003cstrong\u003eFold change (log10)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eUp/Down regulation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eAdjusted p-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003ehsa-miR-193a-5p*\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e\u003cstrong\u003e6.565646\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003e\u003cstrong\u003eUp\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003e2.13E-06\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.00026667\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-10a-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e2.786356\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e1.25E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05386547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-99a-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e3.72719\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e1.42E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05386547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-143-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e4.498237\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e1.72E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.05386547\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-375-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e2.964285\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e2.64E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.06611451\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-483-3p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e4.872215\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e6.32E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.1217676\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-942-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e-8.418699\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e6.82E-03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.1217676\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-122-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e2.75821\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e2.59E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.37944146\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-10b-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e3.901264\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e2.73E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.37944146\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003ehsa-miR-100-5p\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e3.984967\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"161\"\u003e\n\u003cp\u003en.s\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e3.10E-02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.38552523\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\" width=\"623\"\u003e\n\u003cp\u003e*Indicates significantly dysregulated miRNA, n.s- Not significant\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential miRNA expression and duration from first HIV Diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants were stratified in to four (4) groups from when they were diagnosed with HIV (i.e., 0-2 years, 2-5 years, 5-10 years, and above 10 years). Consequently, differential expression of circulating miRNAs was compared between these different groups. Analysis revealed significant differences in miRNA expression between patients diagnosed HIV positive 0-2 years and 2-5 years ago (\u003cstrong\u003e\u003cem\u003etable 6\u003c/em\u003e\u003c/strong\u003e). Between these two groups, 4 circulating miRNAs were down regulated, whereas 20 circulating miRNAs where up regulated (volcano plot in \u003cstrong\u003e\u003cem\u003efigure 5\u003c/em\u003e\u003c/strong\u003e). There were no significant differences in the circulating miRNA expression when the remaining categories were compared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6: \u003c/strong\u003eNumber of differentially upregulated or downregulated miRNAs at different time points from index HIV diagnosis\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\" width=\"629\"\u003e\n\u003cp\u003e\u003cstrong\u003eTime since HIV Diagnosis (Years)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003eExpression\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e\u003cstrong\u003e0-2 Vs 2-5\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003eExpression\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003e0-2 Vs 2-5\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003eExpression\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003e0-2 Vs 2-5\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cstrong\u003eExpression\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003e0-2 Vs 2-5\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eDown\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eNot significant\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eNot significant\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eNot significant\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eNot significant\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e187\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"73\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eSerum miRNAs have been proposed as potential biomarkers of normal physiology and disease, and serum microRNA signatures are currently used as diagnostic and prognostic markers of disease in prostate cancer, renal cell carcinoma, and lung cancer. In the current study, we profiled the unique miRNAs found in serum of patients with epidemic KS, by comparing circulating miRNAs in patients with epidemic KS to those of a comparative group of patients with no KS. Additionally, we analyzed the associations between the level of expression of dysregulated miRNAs and participant clinical data. We found a unique miRNA expression signature in 49 miRNAs when comparing serum collected from patients with epidemic KS to those with no epidemic KS. Moreover, three participants from the comparative group who tested positive for HHV-8 DNA (latent KS infection) differentially upregulated circulating levels of miR-193a compared to their counterparts (HIV-positive, HHV-8 DNA negative). Further analysis of serum of epidemic KS patients revealed a miRNA signature according to KS tumor status (1 downregulated miRNA) and time since first HIV diagnosis (1 upregulated and 4 down regulated miRNAs). However, no differential expression was found when comparing chemotherapeutic cycles, nature of KS lesions and duration since the first KS diagnosis in the epidemic KS patient group.\u003c/p\u003e\n\u003cp\u003emiRNAs are prime candidates for use as non-invasive biomarkers in molecular diagnostics of disease (Blondal et al., 2013). The utility of miRNA\u0026rsquo;s in diagnostics is derived from their specificity for a particular type of tissue or cell, their abundance, as well as their remarkable stability in body tissues and fluids such as serum, urine, saliva, milk and cerebrospinal fluid (Dave et al., 2019; Gustafson et al., 2016). Even though the precise roles of circulating miRNA are still largely unknown, they have been found to be stable and survive conditions such as extreme variations in pH, boiling, multiple freeze thaw cycles, and extended storage, as well as degradation from RNAse enzymes. (Blondal et al., 2013; Wang, Peng, Wang, Qin, \u0026amp; Xue, 2018). In contrast to miRNAs, common RNA species like messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) are degraded within several seconds after being placed in a nuclease rich extracellular environment (Chen et al., 2008; Turchinovich, Samatov, Tonevitsky, \u0026amp; Burwinkel, 2013). In certain cancers, such as non-small cell lung cancer, differential expression of circulating miRNAs has been observed at different stages of disease, thereby contributing to diagnosis, treatment and prognosis (Wang et al., 2018; Yu, Guan, Cuk, Zhang, \u0026amp; Brenner, 2019). In the current study, we determined the differential expression profile of circulating miRNA by comparing the serum miRNA expression profiles of patients with Epidemic KS to that of a comparative group of HIV-positive patients with no KS. We found a unique miRNA expression signature in 49 miRNAs in patients with epidemic KS compared to those with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs down regulated. The top 10 significantly upregulated miRNAs were miR-4446-3p, miR-411-5p, miR-1304-3p, miR-6819-3p, miR-654-5p, miR-1908-5p, miR-431-5p, miR-223-5p, miR487a-5p, and miR-379-5p. The significantly downregulated miRNAs were miR-451a, miR-4732-3p, miR-93-5p, miR-486-5p, miR-185-5p, miR-375-3p, miR-25-3p, and miR-7-5p. These findings imply that the above patterns of differential expression of circulating miRNAs could be used to diagnose epidemic KS in HIV infected individuals.\u003c/p\u003e\n\u003cp\u003eThe life cycle of HHV-8 consists of latent and lytic replication phases. During the latent phase of the infection, only a limited number of HHV-8 genes are expressed in a bid to promote persistent infection, evade host immune responses, and induce HHV-8-related malignancies, such as KS (Ye, Lei, \u0026amp; Gao, 2011). In vitro studies conducted in cell lines derived from HHV-8 associated tumors have implicated the HHV-8 viral miRNA, miR-k12-11 as a key player in reprograming na\u0026iuml;ve B-cells towards supporting long-term latency. In the current study, three out of the 29 KS negative, HIV positive subjects in the comparison group tested positive for HHV-8 DNA, pointing to possible latent HHV-8 infection. Differential profiling of circulating miRNA in the latently infected individuals in our study revealed a significant upregulation in the expression of miR-193a-5p compared to HIV positive, HHV-8 PCR negative participants. This could imply that circulating levels of miR-193a-5p could be used as a none-invasive biomarker of latent epidemic KS infection. Indeed miR-193a-5p has been reported to be upregulated in primary lymphatic endothelial cells with in 72hrs of HHV-8 infection (Lagos et al., 2010), further strengthening the fact that they could be used as early disease biomarkers in KS. miR-193a-5p has also been reported to be upregulated in other cancers, such as prostate (Yang et al., 2017) and hepatocellular carcinoma (Ji et al., 2009), as well as during hypoxic conditions (Viollet et al., 2017).\u003c/p\u003e\n\u003cp\u003emiRNAs are actively secreted via \"exosomes\". Exosomes are small vesicles released from cells, into which miRNAs are specifically sorted and accumulated. Exosomes help to protect circulating miRNA from degradation by RNases in the extracellular environment (Sohel, 2016). Host-encoded miRNAs have previously been found in exosomes released from KSHV-infected lymphoma cell lines (Hoshina et al., 2016). Indeed, some of the dysregulated miRNA in patients with epidemic KS in the current study have been reported to be expressed in KS endothelial tumor cells. Specifically, miR-146a-5p, miR-199a-3p, and miR-126-3p that we found to be upregulated in serum collected from patients with epidemic KS in our study, were also upregulated in tumor specimens collected from patients with both classical and epidemic KS in earlier studies (Punj et al., 2010; Wu et al., 2015), implying that they originated in the KS tumor lesions. Regarding the roles of these miRNA in KS pathophysiology, KSHV infection activates the transcription factor nuclear factor kappa B (NF\u0026kappa;B), which is believed to up-regulate the expression of miR-146. miR-146a then down-regulates the chemokine ligand CXCR4, which promotes the release of KSHV-infected endothelial cells into the circulation (Hussein \u0026amp; Akula, 2017). miR-126a is believed to promote growth of KS by inducing the expression of Vascular Endothelial Growth Factor -A (VEGF-A), whereas miR-199a promotes KS development by inducing the proliferation and survival of endothelial cells (Shatseva, Lee, Deng, \u0026amp; Yang, 2011).\u003c/p\u003e\n\u003cp\u003eDuring human immunodeficiency virus type 1 (HIV-1) infection, host miRNA profiles are altered either as a host response against the virus or as a mechanism for the virus to facilitate viral replication and infection, or to maintain latency (Su et al., 2018). Moreover, it has been shown that HIV-infected subjects have unique circulating miRNA profiles compared to HIV-uninfected persons. However, what is currently unknown is whether this pattern of differential expression stays constant during the lifecycle of HIV infection. In the current study, we compared the differential miRNA expression patterns at different time points from index HIV diagnosis up to a period of more than 10 years after index HIV diagnosis. We found an upregulation of 20 differentially expressed miRNAs, as well as down regulation of four miRNAs in patients diagnosed with in 0-2 years of the study compared to 2-5 years from index HIV diagnosis at the time of the study. The 10 most significantly upregulated miRNA were miR-1307-3p, miR-151a-5p, miR-151b-5p, miR490-5p, miR-222-3p, miR-6819-3p, miR-487a-5p, miR-154-5p, miR-431-5p, and miR-224-5p, whereas miR-451a, miR-486-5p, miR-150-5p, and miR-4732-3p were significantly downregulated. From amongst the upregulated miRNAs, miR-151 has previously been reported as an early biomarker of HIV-1 infection in a panel of circulating miRNAs that distinguished HIV-1 infected individuals from health HIV-negative controls (Qi et al., 2017). Conversely, downregulation of circulating miR-150 as described in the current study was previously reported to predict HIV/AIDS disease progression and therapy. miR-150 is a key regulator of immune cell differentiation and activation and is expressed in monocytes, as well as in mature and resting B and T lymphocytes (Munshi, Panda, Holla, Rewari, \u0026amp; Jameel, 2014). Additionally, differential co-expression of miR-150 in plasma has been used to identify cognitive impairment in HIV-infected patients (Kadri et al., 2016). These results add to the existing findings that HIV infection alters the differential expression of circulating miRNAs.\u003c/p\u003e\n\u003cp\u003eUse of highly active antiretroviral therapy (HAART) reduces the incidence of epidemic KS in HIV positive, KS negative individuals. In individuals with epidemic KS, use of HAART alone can lead to resolution of KS (Stebbing, Portsmouth, \u0026amp; Gazzard, 2003). In addition to HAART, patients with epidemic KS at the Uganda Cancer Institute are treated with chemotherapy consisting of a combination of bleomycin and vincristine for varying treatment cycles, depending on the patients\u0026rsquo; response. Although miRNAs have previously been used to predict response to chemotherapy in pancreatic ductal adenocarcinoma, colorectal cancer, and osteosarcoma (Review by (Wang et al., 2018)), we did not find any differential circulating miRNA expression when we compared epidemic KS patients who had received only three or less cycles of chemotherapy, compared to epidemic KS patients who had received more than 3 cycles of chemotherapy. Taken together, these results imply that circulating miRNAs are poor predictors of chemo-response or chemo-resistance in epidemic KS.\u003c/p\u003e\n\u003cp\u003eOur results are encouraging as they advance the potential of circulating miRNAs as biomarkers for diagnosis of epidemic Kaposi\u0026rsquo;s sarcoma. As it is with all observational studies, causality cannot be inferred from this study. Thus, further work is needed, using a larger, controlled prospective study to fully validate the differential circulating miRNA expression patterns observed in the current study. Moreover, the differential fold expression results for circulating miRNAs in the current study have to be validated by real-time polymerase chain reaction (RT-PCR). Additionally, the methodology adopted for the current study does not provide insights into the causes of the differential expression of the circulating miRNAs observed, which could have resulted from factors other than epidemic KS.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have potential roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection. Additionally, duration from index HIV diagnosis also significantly contributed to alterations in the differential expression of circulating miRNAs in all study subjects.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e3\u0026rsquo;UTR three prime untranslated region\u003c/p\u003e\n\u003cp\u003eAIDS Acquired Immune Deficiency Syndrome\u003c/p\u003e\n\u003cp\u003eART Anti-retroviral therapy\u003c/p\u003e\n\u003cp\u003ecART Combined Antiretroviral Therapy\u003c/p\u003e\n\u003cp\u003ecDNA Complementary DNA\u003c/p\u003e\n\u003cp\u003eDNA deoxyribonucleic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEBV Epstein Bar Virus\u003c/p\u003e\n\u003cp\u003eELISA Enzyme-linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eFC Fold change\u003c/p\u003e\n\u003cp\u003eFDR False discovery rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHAART Highly Active Antiretroviral Therapy\u003c/p\u003e\n\u003cp\u003eHHV-8 Human Herpes Virus 8\u003c/p\u003e\n\u003cp\u003eHIV Human Immune Virus\u003c/p\u003e\n\u003cp\u003eINSTI Integrase Strand Transfer Inhibitor\u003c/p\u003e\n\u003cp\u003eISS clinic Immune suppression syndrome clinic\u003c/p\u003e\n\u003cp\u003eKS Kaposi\u0026rsquo;s Sarcoma\u003c/p\u003e\n\u003cp\u003eKSHV Kaposi's Sarcoma-associated Herpesvirus\u003c/p\u003e\n\u003cp\u003elog CPM log2 counts per million\u003c/p\u003e\n\u003cp\u003emiRNA microRNA\u003c/p\u003e\n\u003cp\u003emRNA messenger RNA\u003c/p\u003e\n\u003cp\u003eNF\u0026kappa;B nuclear factor kappa B\u003c/p\u003e\n\u003cp\u003eNNRTI Non-nucleoside Reverse Transcriptase Inhibitors\u003c/p\u003e\n\u003cp\u003eNRTIs Nucleoside Reverse Transcriptase Inhibitors\u003c/p\u003e\n\u003cp\u003eORF Open Reading Frame\u003c/p\u003e\n\u003cp\u003ePAGE polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003ePCR Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003ePI Protease Inhibitors\u003c/p\u003e\n\u003cp\u003eRIN RNA Integrity Number\u003c/p\u003e\n\u003cp\u003eRNA ribonucleic acid\u003c/p\u003e\n\u003cp\u003erRNA ribosomal RNA\u003c/p\u003e\n\u003cp\u003eRT primers Reverse Transcription primers\u003c/p\u003e\n\u003cp\u003eSBS-REC School of Biomedical Sciences Research Ethics Committee\u003c/p\u003e\n\u003cp\u003etRNA transfer RNA\u003c/p\u003e\n\u003cp\u003eUCI Uganda Cancer Institute\u003c/p\u003e\n\u003cp\u003eUMIs Unique Molecular Identifiers ()\u003c/p\u003e\n\u003cp\u003eUNCST Uganda National Council for Science and Technology\u003c/p\u003e\n\u003cp\u003eVEGF-A Vascular Endothelial Growth Factor -A\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried out in compliance with research principles adopted from the Helsinki Declaration, and approval to conduct the study was obtained from the Institutional Review Board of the \u003cstrong\u003eMakerere University, College of Health Sciences, School of Biomedical Sciences Research and Ethics Committee (SBS-REC) (Protocol number: SBS-519\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. Written informed consent was obtained from all study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and material \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is part of a research fellowship supported by Grant Number D43TW010132 supported by Office of the Director, National Institutes of Health (OD), National Institute of Dental \u0026amp; Craniofacial Research (NIDCR), National Institute of Neurological Disorders and Stroke (NINDS), National Heart, Lung, And Blood Institute (NHLBI), Fogarty International Center (FIC), National Institute on Minority Health and Health Disparities (NIMHD), as well as the Microbiology and Immunology Training in HIV and related infection in Uganda (MITHU) fellowship under the Uganda Case Western Research collaboration. The funding agencies were not involved in the design, data collection, analysis, and interpretation of data; and in writing the manuscript. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the supporting offices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor\u0026rsquo;s contributions \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHM, HK, DN, DPK, and FB contributed to study conceptualization and design. HM, HK and FN conducted the laboratory analysis. CA conducted bioinformatics analysis on sequencing data for the study, and contributed to manuscript writing and revision. HM wrote the first draft of the manuscript. JK, AL, IK, JN, SLF, AMD, DN, DPK, FB, and FS contributed critical revisions and important intellectual content to the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors deeply appreciate the study subjects for agreeing and volunteering time to participate in this study. The authors also appreciate the contributions Mr. Edgar Kigozi and Mr. Ashaba Fred made to arrange for experimental procedures to be conducted for this study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAblashi, D. V., Chatlynne, L. G., Whitman, J. E., Jr., \u0026amp; Cesarman, E. (2002). Spectrum of Kaposi's sarcoma-associated herpesvirus, or human herpesvirus 8, diseases. \u003cem\u003eClin Microbiol Rev, 15\u003c/em\u003e(3), 439-464. doi:10.1128/cmr.15.3.439-464.2002\u003c/p\u003e\n\u003cp\u003eAhmed, F. E., Jeffries, C. D., Vos, P. W., Flake, G., Nuovo, G. J., Sinar, D. R., . . . Marcuard, S. P. (2009). 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Circulating MicroRNA Biomarkers for Lung Cancer Detection in East Asian Populations. \u003cem\u003eCancers (Basel), 11\u003c/em\u003e(3). doi:10.3390/cancers11030415\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microRNA, circulating microRNA, Kaposi’s Sarcoma, biomarker, blood","lastPublishedDoi":"10.21203/rs.3.rs-116856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-116856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003eThe Human herpesvirus 8 (HHV-8), causes Kaposi's sarcoma (KS). Kaposi sarcoma in HIV/AIDS patients is referred to as epidemic KS, and is the most common HIV-related malignancy worldwide. Lack of a diagnostic assay to detect latent and early stage disease has increased disease morbidity and mortality. Serum miRNAs have previously been used as potential biomarkers of normal physiology and disease. In the current study, we profiled the unique serum miRNAs in patients with epidemic KS to generate baseline data to aid in developing a miRNA-based non-invasive biomarker assay for Epidemic KS.\u0026nbsp;\u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eThis was a comparative cross-sectional study involving 27 patients with epidemic KS, and 27 HIV-positive adults with no prior diagnosis, or clinical manifestation of KS. DNA and RNA were isolated from blood and serum collected from study participants respectively. Nested PCR for circulating HHV-8 DNA was performed on the isolated DNA, whereas miRNA library preparation and sequencing for circulating miRNA was performed on the RNA samples. The miRge2 pipeline and EdgeR were used to analyze the sequencing data. \u003c/p\u003e\u003cp\u003eResults \u003c/p\u003e\u003cp\u003eFifteen out of the 27 epidemic KS positive subjects (55.6%) tested positive for HHV-8 DNA, whereas only 3 (11.1%) out of the 27 HIV positive, KS negative subjects tested positive for HHV-8 DNA. Additionally, we found a unique miRNA expression signature in 49 circulating miRNAs in epidemic KS subjects compared to subjects with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs down regulated. Subjects with latent KS infection had a differential upregulation of circulating miR-193a compared to HIV-positive, KS negative subjects for whom circulating HHV-8 DNA was not detected. Further analysis of serum from epidemic KS patients revealed a miRNA signature according to KS tumor status and time since first HIV diagnosis. \u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eThis study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have crucial roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection.\u003c/p\u003e","manuscriptTitle":"Unique Circulating microRNA Profiles in Epidemic Kaposi’s Sarcoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-03 15:58:21","doi":"10.21203/rs.3.rs-116856/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6b90d39-7f9c-4a11-96a5-2ff8ebd6c5da","owner":[],"postedDate":"December 3rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1312111,"name":"Cancer Biology"},{"id":1312112,"name":"Oncology"}],"tags":[],"updatedAt":"2022-04-10T01:55:18+00:00","versionOfRecord":{"articleIdentity":"rs-116856","link":"https://doi.org/10.1016/j.ncrna.2022.02.002","journal":{"identity":"non-coding-rna-research","isVorOnly":true,"title":"Non-coding RNA Research"},"publishedOn":"2022-04-01 01:55:18","publishedOnDateReadable":"April 1st, 2022"},"versionCreatedAt":"2020-12-03 15:58:21","video":"","vorDoi":"10.1016/j.ncrna.2022.02.002","vorDoiUrl":"https://doi.org/10.1016/j.ncrna.2022.02.002","workflowStages":[]},"version":"v1","identity":"rs-116856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-116856","identity":"rs-116856","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}