Table 2_Free circulating versus extracellular vesicle-associated microRNA expression in canine T-cell lymphoma.xlsx

IntroductionCanine lymphoma (cL) is one of the most frequent cancers in dogs. The T-cell lymphoma (TcL) is not the most common phenotype but presents an aggressive behavior. MicroRNAs (miRNAs), are small, single-stranded, non-coding RNA molecules which can circulate freely in blood or be associated with extracellular vesicles (EVs). The dysregulation of certain miRNAs has been identified in numerous types of human cancers and they have been largely investigated as possible tumors biomarkers in human medicine, while research in veterinary oncology is still scarce. The aim of this study was to compare the expression patterns of free circulating and EV-associated miRNAs in dogs with T-cell lymhoma (TcL) and healthy dogs.
MethodsEight dogs with TcL were selected as the lymphoma group (LG) and eight dogs were included as controls (Ctrl). Plasma samples were collected at the time of the diagnosis and EVs isolated with ultracentrifugation. miRNAs were extracted from both the circulating EVs and the plasma supernatant, obtaining EV-associated and free-miRNAs. Quantitative real-time PCR was performed to analyze the expression of 88 target miRNAs.
ResultsTen and seven differentially expressed miRNAs between LG and Ctrl were detected in EV-associated and free-miRNAs, respectively. Among EV-associated and free-miRNAs, only has-miR-222-3p was overexpressed in both conditions.
DiscussionAll the differentially expressed miRNAs detected in this study, have been already described as dysregulated in other human or canine cancers. The EV-associated miRNAs, which appear to be more stable and better conserved than free-miRNAs, could be investigated in further larger studies to better assess their use as possible biomarkers for TcL.

引言
犬淋巴瘤（canine lymphoma, cL）是犬类最常见的恶性肿瘤之一。T细胞淋巴瘤（T-cell lymphoma, TcL）并非最常见的亚型，却具有侵袭性生物学行为。微小RNA（microRNAs, miRNAs）是一类小型单链非编码RNA分子，可在血液中自由循环，或与细胞外囊泡（extracellular vesicles, EVs）结合。已有研究证实，多种人类癌症中存在部分miRNAs的表达失调，此类分子在人类医学中已被广泛作为潜在的肿瘤生物标志物开展研究，但在兽医肿瘤学领域的相关研究仍较为匮乏。本研究旨在对比T细胞淋巴瘤（TcL）患犬与健康犬的循环游离miRNAs及与EVs结合的miRNAs的表达谱。

材料与方法
本研究选取8只TcL患犬作为淋巴瘤组（lymphoma group, LG），另纳入8只健康犬作为对照组（control group, Ctrl）。于确诊时采集血浆样本，采用超速离心法分离细胞外囊泡。从循环EVs及血浆上清液中分别提取miRNAs，得到与EVs结合的miRNAs及游离miRNAs。采用实时荧光定量PCR技术对88个目标miRNAs的表达水平进行检测。

结果
在LG与Ctrl组的对比中，分别于与EVs结合的miRNAs及游离miRNAs中检测到10个和7个差异表达miRNAs。在两类miRNAs中，仅has-miR-222-3p在两组中均呈现高表达。

讨论
本研究中检测到的所有差异表达miRNAs，均已在其他人类或犬类癌症中被报道存在表达失调现象。与游离miRNAs相比，结合EVs的miRNAs稳定性更强、保守性更优，后续可开展更大样本量的研究，以进一步评估其作为TcL潜在生物标志物的应用价值。