Genome-wide interrogation of transfer RNA-derived small RNAs in a mouse model of traumatic brain injury

Transfer RNA(t RNA)-derived small RNAs(ts RNAs) are a recently established family of regulatory small non-coding RNAs that modulate diverse biological processes. Growing evidence indicates that ts RNAs are involved in neurological disorders and play a role in the pathogenesis of neurodegenerative disease. However, whether ts RNAs are involved in traumatic brain injury-induced secondary injury remains poorly understood. In this study, a mouse controlled cortical impact model of traumatic brain injury was established, and integrated ts RNA and messenger RNA(m RNA) transcriptome sequencing were used. The results revealed that 103 ts RNAs were differentially expressed in the mouse model of traumatic brain injury at 72 hours, of which 56 ts RNAs were upregulated and 47 ts RNAs were downregulated. Based on micro RNA-like seed matching and Pearson correlation analysis, 57 differentially expressed ts RNA-m RNA interaction pairs were identified, including 29 ts RNAs and 26 m RNAs. Moreover, Gene Ontology annotation of target genes revealed that the significantly enriched terms were primarily associated with inflammation and synaptic function. Collectively, our findings suggest that ts RNAs may be associated with traumatic brain injury-induced secondary brain injury, and are thus a potential therapeutic target for traumatic brain injury. The study was approved by the Beijing Neurosurgical Institute Animal Care and Use Committee(approval No. 20190411) on April 11, 2019.

Transfer RNA-derived small RNA serves as potential non-invasive diagnostic marker and a novel therapeutic target for acute pancreatitis

Transfer RNA(tRNA)-derived fragments,a new type of tRNA-derived small RNA(tsRNA),can be cleaved from tRNA by enzymes to regulate target gene expression at the transcriptional and translational levels.tsRNAs are not only degradation fragments but also have biological functions,including those in immune inflammation,metabolic disorders,and cell death.tsRNA dysregulation is closely associated with multiple diseases,including various cancers and acute pancreatitis(AP).AP is a common gastrointestinal disease,and its incidence increases annually.AP development is associated with tsRNAs,which regulate cell injury and induce inflammation,especially pyroptosis and ferroptosis.Notably,serum tRF36 has the potential to serve as a non-invasive diagnostic biomarker and leads to pancreatic acinar cell ferroptosis causing inflammation to promote AP.We show the characteristics of tsRNAs and their diagnostic value and function in AP,and discuss the potential opportunities and challenges of using tsRNAs in clinical applications and research.

Genome-wide analysis of hippocampal transfer RNA-derived small RNAs identifies new potential therapeutic targets of Bushen Tiansui formula against Alzheimer’s disease

Objective:Bushen Tiansui formula(BSTSF),a traditional Chinese medicine prescription,has been widely used to treat Alzheimer’s disease(AD).However,the mechanisms underlying its effects remain largely unknown.In this study,a rat AD model was used to study the effects of BSTSF on cognitive performance and expression of transfer RNA-derived small RNAs(tsRNAs)in the hippocampus,to determine whether treatment of AD with BSTSF could regulate the expression of tsRNAs,a novel small non-coding RNA.Methods:To generate a validated AD model,oligomeric amyloid-β_(1-42)(Aβ_(1-42))was injected intracerebroventricularly into rats.The Morris water maze(MWM)test was used to evaluate rat cognitive performance,and tsRNA-sequencing was conducted to examine tsRNA expression in the rat hippocampus.Potential targets were validated by quantitative real-time polymerase chain reaction(qRT-PCR).Bioinformatic analyses were conducted to investigate the biological function of candidate tsRNAs.Results:The learning and memory deficits of Aβ_(1-42)-induced AD rats,assessed by MWM tests,were clearly ameliorated by BSTSF treatment.A total of 387 tsRNAs were detected in the rat hippocampus.Among them,13 were significantly dysregulated in AD rats compared with sham control rats,while 57 were markedly altered by BSTSF treatment,relative to untreated AD rats(fold change>2 and P<0.05).Moreover,six BSTSF treatment-related tsRNAs were identified and validated by qRT-PCR.Bioinformatic analyses indicated that the six treatment-related tsRNAs had potential therapeutic roles,via multiple signaling pathways and Gene Ontology biological functions,including cyclic adenosine monophosphate and retrograde endocannabinoid signaling.Conclusion:This study identified a previously uncharacterized mechanism underlying the effects of BSTSF in alleviating the learning and memory deficits in Aβ_(1-42)-induced AD rats,demonstrating that tsRNAs are potential therapeutic targets of BSTSF in the treatment of AD.

肺腺癌相关tsRNA的筛选和验证

目的通过分析tsRNA在肺腺癌中的差异表达情况及其表达水平与患者预后的关系,进一步筛选并验证肺腺癌相关tsRNA,以了解其在肺腺癌发生和进展中的相关机制。方法基于计算医学中心数据库筛选出在肺腺癌组织和正常组织中差异表达的tsRNA;基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析tsRNA表达水平对肺腺癌患者预后的影响;基于TRFtarget2.0和tRFTar数据库预测靶基因;基于DAVID、KOBA KEGG在线网站进行基因本体论(Gene Ontology,GO)富集分析和京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析;基于阿拉巴马大学伯明翰分校癌症数据分析门户(the University of Alabama at Birmingham CANcer data analysis Portal,UALCAN)分析靶基因在肺腺癌组织和正常组织中的表达水平。采用增殖实验、迁移实验、侵袭实验验证tRF-19-69M8LOJX在肺腺癌细胞中的生物学功能。结果与正常组织相比,tRF-19-69M8LOJX在肺腺癌组织中表达上调(log2FC=4.28,FDR<0.05)。高表达水平的tRF-19-69M8LOJX预示着更短的无进展生存期(HR=1.565,95%CI=1.142~2.145,P=0.005);过表达tRF-19-69M8LOJX促进A549细胞的增殖、迁移(P<0.001)和侵袭(P=0.009);COL1A1(P=0.002)和VCAN(P=0.022)在tRF-19-69M8LOJX过表达细胞模型中显著上调。结论tRF-19-69M8LOJX在肺腺癌组织的表达水平上调,与患者不良预后密切相关,可能在肺腺癌的发生发展中起着重要作用。

Identification of tRNA-derived Fragments and Their Potential Roles in Atherosclerosis

Objective Atherosclerosis(AS),a chronic inflammatory disease,is the basis of cardiovascular disease(CVD).Although the treatment has been greatly improved,AS still imposes a large burden on human health and the medical system,and we still need to further study its pathogenesis.As a novel biomolecule,transfer RNA-derived fragments(tRFs)play a key role in the progression of various disease.However,whether tRFs contribute to atherosclerosis pathogenesis remains unexplored.Methods With deep sequencing technology,the change of tRFs expression profiles in patients with AS compared to healthy control group was identified.The accuracy of the sequencing data was validated using RT qPCR.Subsequently,we predicted the potential target genes of tRFs by online miRNA target prediction algorithms.The potential functions of tRFs were evaluated with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses.Results There were 13 tRFs differentially expressed between patients with AS and healthy controls,of which 2 were up-regulated and 11 were down-regulated.Validation by RT-qPCR analysis confirmed the sequencing results,and tRF-Gly-GCC-009 was highly up-regulated in the AS group based on the results of sequencing which was confirmed by RT-qPCR analysis.Furthermore,GO enrichment and KEGG pathway analyses indicated that 10 signaling pathways were related to tRF-Gly-GCC-009.These pathways might be physiopathological fundamentals of AS,mainly involving in Apelin signaling,Notch signaling and calcium signaling.Conclusion The results of our study provide important novel insight into the underlying pathogenesis and demonstrate that tRFs might be potential biomarkers and therapeutic targets for AS in the future.

The biogenesis and biological roles of tRNA-derived short RNAs

In recent years, next-generation sequencing (NGS) technologies targeting the microRNA (miRNA)transcriptome revealed the existence of tRNA-derived short RNAs: tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs). These small RNAs represent a novel type of small non-coding RNAs (sncRNAs), which are heterogeneous in size, nucleotide composition and biogenesis, and have been suggested to be involved in translation, cell proliferation, priming of viral reverse transcriptases, regulation of gene expression, modulation of the DNA damage response, tumor suppression and neurological disorders. Herein, we review the mechanism of their biogenesis and discuss in detail the regulatory roles they play in cell physiology. We also point out that the biological function of tRNA-derived short RNAs will be understood better as research moves forward, and that this knowledge will find its way into clinical application in the near future.

基于TCGA数据筛选乳腺癌预后相关的tsRNA标志物

目的筛选乳腺癌中差异表达的转运RNA衍生小RNA(tsRNA),构建与乳腺癌患者预后相关的预测模型。方法收集癌症基因组图谱(TCGA)数据库中的乳腺癌患者的tsRNA表达谱以及临床相关数据,利用最小绝对值收敛和选择算子算法(LASSO)筛选出关键tsRNA,构建风险评分模型和列线图预测模型。结果从乳腺癌差异表达的106条tsRNA中识别了11条非零系数的tsRNA,据此构建了评估乳腺癌预后的风险评分模型和列线图预测模型,经测试集和校准曲线证实两模型均具有良好的预测能力。进一步预测上述11条tsRNA的靶基因,发现其富集了8条KEGG通路,包括多条与肿瘤的进展相关的通路;经相关性分析发现,NR2C2和ZNFX1与tRF-21-YBOZZ7ND的表达水平呈负相关。结论成功构建了风险评分模型和列线图预测模型,有望为乳腺癌的预后评估提供新的方法。

tsRNA在急性心肌梗死早期诊断中的价值

目的 探讨转运RNA衍生的小RNA(transfer RNA-derived small RNA,tsRNA)在急性心肌梗死(AMI)早期诊断中的价值及其作为AMI诊断标志物的潜能。方法 将年龄及体质量匹配的雄性C57小鼠(8~10周龄)随机分为MI组及对照组(Sham组),每组4只。两组均于建模24 h后提取左室心肌组织RNA,经去修饰预处理,连接人工化接头后扩增,筛选包含15~40个核苷酸的小RNA扩增产物,构建文库并测序,将测序结果与GtRNAdb数据库成熟的t RNA和tRNA前体序列比对,获得在AMI前后心肌中差异表达的tsRNA谱。采用生物信息学分析同一密码子对应的tRNA在AMI前后剪切方式的改变。依据AMI前后tsRNA表达谱差异,获得在AMI后特异性高丰度表达的tsRNA,并在AMI小鼠心肌和血浆中验证,探讨tsRNA作为AMI诊断标志物的潜能。结果 tsRNA表达谱在组内有较好的重复性,组间差异较大。发生AMI后,Asn-GTT、Glu-TTC、Gly-ACC、Gly-GCC、His-GTG、Ile-AAT、Ile-GAT、Pro-TGG、Ser-AGA和Trp-CCA在心肌中剪切方式发生改变,生成有明显差异的tsRNA表达谱。与Sham组相比,MI组有268个tsRNA表达上调,1 228个tsRNA表达下调,且有64个特异性tsRNA。AMI建模第1天,小鼠tRF-Gly-CCC-2-31、tiRNA-Val-CAC-1-32、tiRNA-ValAAC-2-32、tiRNA-Glu-TTC-2-32和tiRNA-Lys-TTT-1-34在心肌中特异性表达,其中tiRNA-Val-AAC-2-32和tiRNA-LysTTT-1-34在AMI小鼠血浆中特异性高丰度表达,并随AMI持续时间动态变化。结论 tsRNA表达谱在AMI前后差异显著,其中在小鼠心肌和血浆中特异性表达的tiRNA-Val-AAC-2-32和tiRNA-Lys-TTT-1-34有AMI诊断潜能,可能在AMI修复过程中发挥重要作用。

Transfer RNA-derived fragment tRF-23-Q99P9P9NDD promotes progression of gastric cancer by targeting ACADSB

Gastric cancer(GC)is one of the most common gastrointestinal tumors.As a newly discovered type of non-coding RNAs,transfer RNA(tRNA)-derived small RNAs(tsRNAs)play a dual biological role in cancer.Our previous studies have demonstrated the potential of tRF-23-Q99P9P9NDD as a diagnostic and prognostic biomarker for GC.In this work,we confirmed for the first time that tRF-23-Q99P9P9NDD can promote the proliferation,migration,and invasion of GC cells in vitro.The dual luciferase reporter gene assay confirmed that tRF-23-Q99P9P9NDD could bind to the 3'untranslated region(UTR)site of acyl-coenzyme A dehydrogenase short/branched chain(ACADSB).In addition,ACADSB could rescue the effect of tRF-23-Q99P9P9NDD on GC cells.Next,we used Gene Ontology(GO),the Kyoto Encyclopedia of Genes and Genomes(KEGG),and Gene Set Enrichment Analysis(GSEA)to find that downregulated ACADSB in GC may promote lipid accumulation by inhibiting fatty acid catabolism and ferroptosis.Finally,we verified the correlation between ACADSB and 12 ferroptosis genes at the transcriptional level,as well as the changes in reactive oxygen species(ROS)levels by flow cytometry.In summary,this study proposes that tRF-23-Q99P9P9NDD may affect GC lipid metabolism and ferroptosis by targeting ACADSB,thereby promoting GC progression.It provides a theoretical basis for the diagnostic and prognostic monitoring value of GC and opens upnew possibilities for treatment.

非病毒靶向载体递送小干扰RNA的研究进展

近年来由小干扰RNA(siRNA)所介导的RNA干扰(RNAi)技术发展迅速,如何高效、安全地将siRNA转运至靶组织是决定这一技术应用前景的关键问题。本文重点围绕构建siRNA的非病毒靶向载体递送系统的研究进展进行综述。

神经鞘内转染siRNA-p300对大鼠神经病理痛阈值的影响

目的:探讨神经鞘内转染p300si RNA(si RNA-p300)对大鼠神经病理痛阈值的影响。方法:成年雄性清洁级SD大鼠27只随机分为假手术组、对照组和实验组,每组9只。对照组和实验组应用坐骨神经压迫损伤建立神经病理性痛模型并鞘内置管,分别鞘内转染无关4μg si RNA和4μg si RNA-p300。假手术组单纯进行模拟手术并注射等量生理盐水。分别于转染前及注射后2、4、6、8、10、12和14 d分别检测3组大鼠痛阈分值,于注射后14 d处死大鼠,取腰段脊髓采用Western-blot法检测大鼠脊髓中p300表达水平。结果:与假手术组(21.22±1.56)比较,对照组(14.33±4.02)和实验组(13.66±2.30)大鼠疼痛阈值在转染后第2 d开始显著降低(P<0.05)。与对照组(8.98±2.89)比较,实验组(11.23±2.20)大鼠疼痛阈值在转染后第6天开始显著升高(P<0.05);假手术组、对照组和试验组p300相对表达量分别为0.11±0.03、0.67±0.18和0.22±0.04,与假手术组比较,对照组和实验组p300表达显著上调(P<0.05);与对照组比较,实验组p300表达明显下调(P<0.05)。结论:鞘内注射si RNA-p300可显著降低大鼠神经病理性疼痛,提高痛阈。

SmacsiRNA慢病毒载体的构建及其对Smac基因沉默效果的鉴定

背景研究证实半胱氨酸天冬氨酸蛋白水解酶激活剂（Smac）蛋白是肿瘤细胞的促凋亡蛋白，我们先前的研究表明，Smac蛋白在白内障患者LECs中表达量明显高于正常人，因此推测沉默LECs中Smac的表达可抑制LECs的过度凋亡，但如何成功将SmacsiRNA转染进入LECs是研究的关键。目的构建人SmacsiRNA慢病毒载体并对人LECs进行感染，研究慢病毒载体构建方法及其在人晶状体上皮细胞HLE—B3中的干扰效率，利用RNA干扰（RNAi）技术建立Smac低表达的HLE—B3株。方法根据我们前期的工作设计并合成针对Smac基因序列的siRNA序列，将合成的双链DNA以T4噬菌体DNA连接酶与GV118慢病毒载体相连接，转化DH5α感受态细胞，采用PCR筛选阳性克隆，经测序鉴定正确后，感染293T细胞，收集上清液并标定病毒滴度。用重组慢病毒感染HLE—B3细胞，荧光显微镜下观察转染后的阳性HLE—B3细胞数并计算病毒感染复数（MOI）。将常规培养的HLE—B3细胞分为阴性对照组、空质粒组和GV118-Smac—siRNA1组，阴性对照组为常规培养的细胞，空质粒组细胞仅转染不含慢病毒载体的siRNA质粒，GV118-Smac·siRNA1组细胞转染GV118-Smac—siRNA1质粒，采用实时荧光定量PCR检测各组细胞中SmacmRNA的相对表达量。结果构建的重组载体GV118-Smac—siRNAl转化DH5c感受态细胞后阳性克隆产物大小为340bp，空载的克隆产物为299bp，与预期结果相符，测序结果显示合成的Smac—siRNA1与预期目的序列一致。构建的GV118-Smac—siRNA1病毒滴度为3．0x10。TU／m1。感染HLE．B3细胞后荧光显微镜下可见MOI为100时，细胞毒性低且感染效率高，约为82％。阴性对照组、空载质粒组和GV118-Smac—siRNA1质粒组细胞中SmacmRNA相对表达量分别为（101．290±8．349）％、（92．330±6．320）％和（32．540±4．221）％，3个组间总体比较差异有统计学意义（F=32．871，P〈0．01），其中GV118-Smac—siRNA1质粒组细胞中SmacmRNA相对表达量较阴性对照组明显下降，差异有统计学意义（P=0．000），而空载质粒组细胞中SmacmRNA相对表达量略低于阴性对照组，但组间差异无统计学意义（P=0．535）。结论本研究中成功构建了siRNA慢病毒载体GV118-Smac—siRNA1，其转染HLE—B3效率高，该siRNA载体能够有效地抑制人LECs中Smac基因的表达。

tsRNA作为肿瘤诊断和预后标志物的研究进展

转运RNA(transfer RNA,tRNA)可结合相应的氨基酸,并将其运送到核糖体上,促进蛋白质的翻译。tRNA衍生的小RNA(transfer RNA-derived small RNA,tsRNA)是tRNA被切割而产生的。tsRNA具有重要的生物学功能,可发挥调节基因表达和调控蛋白质翻译等作用。近年来,研究揭示了tsRNA在癌症中的双重调控作用,特别是其在癌症患者体液中的显著差异性,强调了tsRNA作为一种潜在的肿瘤诊断和预后评估生物标志物的重要性。结直肠癌相关tsRNA中,5′tiRNA-His-GTG上调可促进肿瘤的发生和发展;由血管生成素切割产生的5′-tiRNA-Val上调,促进肿瘤转移和生长;tRF-20-MEJB5Y13上调,可促进结直肠癌细胞迁移和侵袭。胃癌相关tsRNA中,tRF-19-3L7L73JD上调可促进恶性肿瘤的进展,而tRF-24-V29K9UV3IU、tRF-5026a和tRF-Val上调可抑制肿瘤的增殖及进展。临床应用方面,血浆5-tRF-GlyGCC表达升高,诊断结直肠癌的曲线下面积达0.882,血浆tRF-5026a下降,诊断结直肠癌曲线下面积为0.883。胃癌患者血清tRF-27-FDXXE6XRK45、tRF-29-R9J8909NF5JP和tRF-23-Q99P9P9NDD的表达显著升高,诊断胃癌曲线下面积分别为0.805、0.889和0.783;三阴性乳腺癌血清中tDR-000620下降,与淋巴结转移和疾病复发相关。胃癌患者的血浆外泌体中,tRF-38、tRF-25和tRF-18表达升高,这些指标可用于诊断胃癌,且可能是术后预测因子;肝癌患者血浆外泌体的tRNA-ValTAC-3、tRNA-GlyTCC-5、tRNA-ValAAC-5和tRNA-GluCTC-5的表达水平明显增加,可能是新兴的标志物。本文综述了tsRNA的生成、分类及生物学功能,重点阐述tsRNA作为肿瘤标志物的研究进展以及其在不同肿瘤中发挥的作用。

EntransterTM纳米载体介导大鼠角膜CD25siRNA转染的效果及安全性评估

背景 基因转染是多种眼病基因治疗的有效方法,理想的非病毒载体是研究角膜基因治疗的关键因素,选择高转染率、基因高表达、低毒性的非病毒载体是成功实施基因治疗的前提. 目的 探讨EntransterTM、脂质体非病毒载体对正常SD大鼠角膜转染CD25 siRNA的转染率和安全性,筛选角膜基因转染的最佳载体.方法 应用随机数字表法将80只雄性SPF级成年SD大鼠随机分为EntransterTM-CD25 siRNA 组、脂质体-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组,每组20只,均以右眼作为实验眼.实验眼眼表麻醉后刮除角膜上皮,按照分组不同分别用EntransterTM-CD25 siRNA、脂质体-CD25 siRNA、单纯CD25siRNA和生理盐水各50μl点眼.于点眼后12h、24 h、3d和7d在裂隙灯显微镜下观察大鼠眼表组织反应,检查各组大鼠角膜表面绿色荧光个数.分别于上述时间点处死各组大鼠各4只并获取角膜组织,采用苏木精-伊红染色法行角膜组织病理学检查;采用罗丹明染色行荧光检测,评估各组大鼠角膜基因转染的转染率;采用TUNEL染色法检测实验眼角膜细胞的凋亡情况以评估各种转染载体的安全性;采用免疫荧光技术检测角膜组织中CD11b的表达. 结果 EntransterTM-CD25 siRNA组大鼠角膜表面的荧光染色数量及强度明显高于脂质体-CD25 siRNA组,单纯CD25 siRNA组大鼠角膜荧光染色出现早,但转染后24 h角膜荧光染色消失.角膜组织病理学检查显示,各组大鼠行基因转染后脂质体-CD25 siRNA组大鼠角膜上皮水肿和角膜炎性细胞浸润程度较EntransterTM-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组严重,角膜基质层和内皮层未发现异常,脂质体-CD25 siRNA组大鼠角膜炎性细胞数明显多于EntransterTM-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组,差异均有统计学意义（均P=0.00）.TUNEL检测发现,基因转染后12h和3d,脂质体-CD25siRNA组大鼠角膜细胞凋亡数明显多于EntransterTM-CD25 siRNA组、单纯CD25 siRNA组和生理盐水组,差异均有统计学意义（均P=0.00）.免疫荧光检测显示,CD11b荧光主要分布于角膜上皮层和角膜基质层,随着基因转染后时间的延长,脂质体-CD25 siRNA组大鼠角膜组织中CD11b的表达量逐渐增加,基因转染后24 h时荧光强度达峰,随后逐渐减弱,至转染后第7天仍可见弱荧光.EntransterTM-CD25 siRNA组、单纯CD25siRNA组和生理盐水组大鼠角膜组织中均未发现CD11b的荧光表达. 结论 EntransterTM纳米材料作为载体转染CD25 siRNA于正常SD大鼠角膜具有转染效率高、毒性低、不引起角膜免疫反应的特点,可作为角膜疾病基因治疗的合适载体.

STAT1特异性siRNA真核表达载体的构建及体外转染优化

目的:构建STAT1特异性siRNA真核表达载体,并转染人气道上皮细胞(16HBE)。方法:根据GeneBank数据库提供的STAT1基因两种变异体的共同核苷酸序列,按照Tuschl设计原则,选择设计双链siRNA,再转化为能表达其小发卡结构RNA(small hairpin RNA,shRNA)的DNA序列,并将其连接到带有卡那霉素抗性和增强绿色荧光蛋白的pGenesil-1.1真核表达载体上,将其转染至气道上皮(16HBE)细胞中,观察荧光表达,鉴定其转染效率。结果:通过酶切鉴定和序列测定,成功地构建了二条表达小干扰RNA的质粒及其阴性对照质粒,并成功转染到(16HBE)细胞株中,质粒/脂质体为1μg:2μl条件,能有效地转染细胞,转染效率达到50%以上。结论:成功构建siRNA真核表达载体并能成功体外转染,为下一步气道炎症性疾病的基因沉默研究奠定了良好的基础。

探讨外泌体介导的微小RNA在结直肠癌转移诊断中的价值

目的探讨外泌体介导的微小RNA(miRNA)在结直肠癌转移诊断中的价值。方法选取新疆医科大第四附属医院2017年6月至2019年6月收治的100例结直肠癌患者作为研究对象,根据是否发生转移分成转移组和非转移组,每组各50例。采集其血清外泌体样本进行检验,比较2组患者微小RNA的表达情况。结果转移组患者的结直肠癌转移、外泌体微小RNA的表达指标显著高于未转移组,差异有统计学意义(P<0.05),多数患者表现为miRNA中的23C、204、615、1229表达指标上调,125b、230、675、1252表达指标下调。结论在结直肠癌转移的诊断中,外泌体介导的微小RNA表达指标情况能够为临床判断提供参考,使临床医师能够尽快对转移情况进行分析,尽早采取控制措施,降低疾病风险。

精子tsRNA在父系遗传中的作用

tsRNA(tRNA-derived small RNA)是来源于成熟转运RNA(transfer RNA,tRNA)或tRNA前体的一种小非编码RNA(small non-coding RNA,sncRNA)。研究表明tsRNA是一种重要的RNA调控因子,有望作为疾病预防、诊断和治疗的生物标志物。不同tsRNA表达可能参与蛋白质合成、信使RNA(messenger RNA,mRNA)的翻译转录以及代谢性疾病等多种疾病的发生、发展,并且在精子成熟与代谢性疾病的代际遗传中也扮演了关键角色。阐述tsRNA的分类和生物学功能,尤其是在雄性生殖即精子成熟过程中的表达以及在父系相关代际遗传中的作用,对子代健康的保护具有重要意义。

tRNA来源的非编码小RNA在消化系统肿瘤中的研究进展

转移核糖核酸(tRNA)来源的非编码小RNA(tsRNAs)由成熟或前体tRNA在不同位点特异性剪切生成,广泛存在于原核生物和真核生物转录组,是一类新的基因表达调控分子,具有多种生物学功能。tsRNAs参与应激反应、蛋白质翻译、核糖体生物合成以及调节免疫反应等生理和病理过程,其表达失调与人类多种疾病的发生、发展关系密切。本文就tsRNAs在消化系统肿瘤中的研究进展作一综述。

低氧性大鼠肺动脉平滑肌细胞tsRNA的表达谱分析

目的观察低氧性大鼠肺动脉平滑肌细胞(rat pulmonary artery smooth muscle cells,RPASMCs)中1类新型非编码RNA-tRNA衍生的小RNA(tRNA-derived small RNA,tsRNA)表达情况。方法采用1%O2的低氧培养箱和常氧培养箱分别培养RPASMCs 24 h,通过tsRNA高通量测序技术和生物信息学检测并分析RPASMCs中tsRNA的表达差异。结果tsRNA高通量测序检测到3806个tsRNA,其中具有明确类型的有504个(上调的tsRNA有195个,下调的tsRNA有309个),差异表达的上调和下调的tsRNA分别为116个和156个,在进一步剔除掉片段长度<16 nt的tsRNA之后,与常氧培养比较,低氧处理的RPASMCs表达显著上调的tsRNA有59个(P<0.01),表达显著下调的tsRNA有9个(P<0.01)。结论低氧刺激RPASMC后,tsRNA表达存在明显差异,可能影响PASMC增殖、凋亡和迁移等功能,可为深入阐明低氧性肺动脉高压的发生机制提供新方法和新思路。

5′tRF-LysCTT对小鼠心肌细胞铁死亡的调控作用及其机制

目的探究tRNA衍生的小RNA(tsRNA)对小鼠心肌细胞铁死亡的调控作用及其机制。方法采用实时荧光定量PCR(RT-qPCR)检测8周龄C57BL/6J小鼠心脏、肝脏、脾脏、肾脏、大脑、肌肉中5′tRF-LysCTT的相对表达水平。将原代心肌细胞分为A~D组,A组细胞使用完全培养基培养60 h,B组先使用完全培养基培养24 h,再依次进行饥饿处理12 h,H/R处理24 h,C组和D组细胞分别转染antagomir-NC和antagomir-5′tRF-LysCTT,并于转染24 h后再依次进行饥饿(12 h)和H/R处理(12 h);将原代心肌细胞分为E~G组,E组细胞使用完全培养基培养24 h,F组细胞转染agomir-NC 24 h,G组心肌细胞转染agomir-5′tRF-LysCTT 24 h。采用RT-qPCR检测A~G组小鼠心肌细胞中5′tRF-LysCTT和Ptgs2、Gpx4、Slc7a11的相对表达水平,分别采用亚铁离子比色法测试盒、脂质活性氧(ROS)染色、CCK8试剂盒检测A~G组小鼠心肌细胞内亚铁离子相对水平、ROS水平及心肌细胞的存活率,采用普鲁士蓝染色试剂盒观察A~G组小鼠心肌细胞内铁离子沉积情况。结果RT-qPCR检测结果显示,与肝脏、脾脏、肾脏、大脑、肌肉中相比较,小鼠心脏中5′tRF-LysCTT表达水平最高(F=16.21,t=3.81~7.93,P<0.05)。D组与B组相比,心肌细胞内5′tRF-LysCTT和Ptgs2相对表达水平降低、Gpx4以及Slc7a11相对表达水平升高、亚铁离子相对水平和ROS水平降低,心肌细胞的细胞存活率升高(t=3.26~15.61,P<0.05),细胞内铁离子沉积明显增多,而C组与B组相比,细胞内的上述指标的水平差异无显著性(P>0.05)。G组与E组相比,心肌细胞内5′tRF-LysCTT和Ptgs2相对表达水平升高、Gpx4和Slc7a11相对表达水平降低、亚铁离子相对水平和ROS水平升高,心肌细胞的细胞存活率降低(t=3.57~91.84,P<0.05),细胞内铁离子沉积明显减少,而F组与E组相比,细胞内的上述指标的水平比较差异无显著性(P>0.05)。结论5′tRF-LysCTT对于小鼠心肌细胞铁死亡具有调控作用,敲低细胞内5′tRF-LysCTT可以抑制H/R诱导的心肌细胞铁死亡,而过表达5′tRF-LysCTT可促进心肌细胞铁死亡的发生。