目的:建立用于甲状腺结节分子分型的二代靶向测序方案.方法:基于生物素化探针靶向捕获和高通量测序技术,对 112 个甲状腺结节相关基因进行靶向测序,建立用于甲状腺结节分子分型与分化水平的二代靶向测序方案,甲状腺结节Panel(ThyNod Panel,甲结Panel).基因包括甲状腺良性结节相关基因、甲状腺恶性肿瘤相关基因、甲状腺发育和功能基因、细胞来源标志基因等,检测目的基因碱基替换、插入缺失、拷贝数变异和融合等突变类型以及基因表达水平.使用甲结Panel对良恶性甲状腺结节进行分子分型.结果:成功构建并应用甲结Panel,完成856 例良恶性甲状腺结节检测.其中 676 例甲状腺结节检出意义明确的突变,占比 79.0%.627 例甲状腺结节病理诊断明确,良性结节占比 17.6%.甲状腺恶性肿瘤病理类型包括经典性乳头状癌、滤泡亚型乳头状癌、髓样癌等.最常见的突变检出基因为BRAF(n=426),其次是RET(n=68),RET/PTC 融合(n=68),DICER1(n=35),还有端粒酶逆转录酶(telomerase reverse transcriptase,TERT)(n=35)、HRAS(n=24)、NRAS(n=23)、神经营养酪氨酸受体激酶(neurotrophin receptor kinase,NTRK3)融合基因(n=19)、真核翻译起始因子 1A,X染色体(eukaryotic translation initi-ation factor 1A,X-chromosomal,EIF1AX)基因(n=11)等.对检出突变统计结节恶性率,BRAF V600E、RET融合变异等结节恶性比例高于 90.0%,而RAS-类变异恶性率仅为 18.9%.结论:甲结Panel可有效进行甲状腺结节的分子分型.

目的 探讨 miRNA-146 是否通过调控 Toll 样受体 4/核因子-κB(TLR4/NF-κB)信号通路促进滋养层细胞HTR-8/SVneo增殖和迁移,以期为不明原因复发性流产(uRSA)的发病机制研究和防治提供参考.方法(1)细胞研究:将转染后的胎盘滋养层细胞 HTR-8/SVneo 分成 miRNA-146 抑制剂组、抑制剂对照组、miRNA-146 模拟物组、模拟物对照组,检测各组 HTR-8/SVneo细胞生长的活力、增殖、凋亡、迁移情况,通过实时荧光定量PCR(qRT-PCR)和 Western blot检测各组TLR4、NF-κB、肿瘤坏死因子(TNF-α)和白介素-6(IL-6)的mRNA和蛋白相对表达水平.(2)动物研究:建立 Dicer酶敲除的 uRSA小鼠模型,将正常妊娠小鼠作为对照组,通过 qRT-PCR检测胎盘组织的 miRNA-146 mRNA表达情况,采用免疫组织化学法检测胎盘组织的 TLR4、NF-κB、TNF-α、IL-6 蛋白表达情况.结果(1)细胞研究:与模拟物对照组比较,miRNA-146 模拟物组 HTR-8/SVneo细胞克隆形成率、增殖能力及迁移能力显著增加(P<0.05),细胞凋亡率显著降低(P<0.05);与抑制剂对照组和miRNA-146 模拟物组比较,miRNA-146 抑制剂组 HTR-8/SVneo 细胞克隆形成率、增殖能力及迁移能力显著降低(P<0.05),而细胞凋亡率显著增加(P<0.05).与模拟物对照组比较,miRNA-146 模拟物组的 TLR4、NF-κB、TNF-α、IL-6 蛋白和mRNA相对表达量显著降低(P<0.05);与抑制剂对照组和miRNA-146 模拟物组比较,miRNA-146抑制剂组的TLR4、NF-κB、TNF-α、IL-6 蛋白和mRNA相对表达量显著增加(P<0.05).不同浓度(10～80 μmol/L)miRNA-146对 HTR-8/SVneo细胞生长活力的增长均有显著影响(P<0.05),并呈浓度依赖性.(2)动物研究:与对照组比较,uRSA组小鼠胎盘组织中miRNA-146 mRNA的相对表达量显著降低[(0.32±0.19)vs.(0.96±0.24),t=12.473,P<0.05],而TLR4、NF-κB、TNF-α、IL-6 的蛋白表达有增高趋势.结论 上调 miRNA-146 的表达具有促进胎盘滋养层细胞的增殖和迁移作用,其机制可能与负性调控TLR4/NF-κB通路相关.miRNA-146 有望成为防治 uRSA的潜在靶标.

Viruses can infect host plants to cause severe diseases and substantial agricultural loss,while plants have evolved RNA interference (RNAi) strategy to defend against viral infection.Despite enormous efforts,only a few host proteins in RNAi pathway were shown to mediate antiviral defense,including RNA-dependent RNA polymerase 1 (RDR1),RDR6,DICER-LIKE 2 (DCL2) and DCL4.In this study,we carried out a genetic screen for antiviral factors of RNAi pathway in Arabidopsis rdr6 background via inoculation with a 2b-deficient Cucumber Mosaic Virus (CMV-△2b).We identified a mutant susceptible to CMV-△2b,referred to as (en)hancer (of) (r)dr6 (enor) 3-1 rdr6,and found that ENOR3 encodes a functionally unknown protein with high homology to the mammalian Non Imprinted in Prader-Willi/Angelman (NIPA) magnesium transporters.ENOR3 inhibits accumulation of CMV-△2b and acts additively with RDR1,RDR6,DCL2 and DCL4 in antiviral defense.These results uncover that ENOR3 is a key component in antiviral RNAi pathway,and provide new insights into antiviral immunity.

RNA silencing (RNAi) is a nucleotide sequence-specific process that results in blockage of gene expression (Baulcombe,2004,2005;Gunter Meister,2004;Vaucheret,2006;Chinnusamy and Zhu,2009;Heo and Kim,2009;Matzke et al.,2009;Simon and Meyers,2011).In plants,post-transcriptional gene silencing (PTGS) occurs in the cytoplasm that is induced by small RNAs (sRNAs),which are the products of double-stranded RNAs (dsRNAs)processed by Dicer-like ribonucleases and achieve specificity through base pairing with targeted RNA sequences (Carmell and Hannon,2004;Gunter Meister,2004;Gasciolli et al.,2005).In an alternative pathway occurring in the nucleus,sRNAs corresponding to promoter sequences direct the silencing machinery to block the transcription of homologous promoters (transcriptional gene silencing,TGS).This process requires 24-nt sRNAs for de novo DNA methylation,a process known as RNA-directed DNA methylation (RdDM) (James P.Jackson and Jacobsen,2002;Matzke and Birchler,2005;Matzke et al.,2007;Zilberman et al.,2007;Wierzbicki et al.,2008;Zilberman,2008).Histone modifications also play an important role in the establishment and maintenanceof DNA methylation (Zuzana Jasencakova,2003;Ooi et al.,2007;Cedar and Bergman,2009;Law and Jacobsen,2010).In plants,it has been shown that the transcription of exogenous transgene transcribing inverted-repeat (exo-IR) sequences produces dsRNAs,triggering exo-IR PTGS that is negatively autoregulated through methylation spreading/transitive silencing.This transitive silencing reinforces the self-silencing of exo-IR and leads to reduced exo-IR PTGS and exo-IR-derived sRNA production.exo-IR-derived sRNAs function as mobile signals to trigger sRNA-mediated non-cell autonomous silencing of an endogenous homologous target gene (endo-gene) (Dong et al.,2011).It remains unknown whether histone modifications play a role in the exo-IR-triggered endo-gene silencing.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor impairment and progressive loss of dopamine (DA) neurons.At present,the acute application of neurotoxic drugs such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are commonly used to simulate the pathology of PD;however,it is difficult to induce the progressive pathogenesis of PD with these models.In this study,we employed DAT promoter-mediated Cre transgenic mice to establish tamoxifen-inducible Dicer conditional knockout (cKO) mice in an effort to mimic the progressive loss of DA neurons and the development of PD-like behavioral phenotypes.The results showed that Dicer cKO mice exhibited progressive loss of DA neurons in the substantia nigra (SN) following tamoxifen administration.Significant DA loss was observed 6 weeks after tamoxifen administration;accordingly,progressive motor function impairment was also observed.We also found that a significant neuroinflammatory response,as evidenced by microglial proliferation,another hallmark of PD pathogenesis,accompanied the loss of DA neurons.The acute application of levo-DOPA (L-DOPA) relieved the PD-like motor impairments in Dicer cKO mice to exert its antiparkinsonian action,indicating that the model can be used to evaluate the antiparkinsonian efficacy of PD drugs.To further elucidate the potential application of this novel PD animal model for PD drug development,we employed the powerful neuroprotective agent dihydromyricetin (DHM) (10 mg/kg) and the selective sigma-1 receptor agonist PRE-084 (1 mg/kg),both of which were previously shown to produce antiparkinsonian effects.The results indicated that the chronic administration of either DHM or PRE-084 attenuated the Dicer cKO-induced loss of DA neurons and motor impairments,although the two drugs acted through different mechanisms.These data indicate that the Dicer cKO mouse model may be a useful model for investigating the pathological development of PD and intervention-mediated changes.In conclusion,this transgenic mouse model appears to simulate the progressive pathogenesis of PD and may be a potentially useful model for PD drug discovery.

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition.Here,we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation.Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs)and miRNA processing core proteins,such as Dicer-like 1,SERRATE,and HYPONASTIC LEAVES 1,whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels.However,the levels of most miRNAs do not notably increase in response to light.To reconcile this inconsistency,we demonstrated that an unknown suppressor decreases miRNA-processing activity and lightinduced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings.Taken together,these data suggest a novel mechanism,miRNA-biogenetic inconsistency,which accounts for the intricacy of miRNA biogenesis during de-etiolation.This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

甘蔗黑穗病菌(Sporisorium scitamineum)是典型的二型态真菌,由其引起的甘蔗黑穗病是一个全球性的病害,对甘蔗产量和含糖量造成巨大损失.RNA干扰是一种保守的小RNA介导的转录后基因沉默机制.目前甘蔗黑穗病菌的RNA干扰系统尚未明确.本研究用蛋白保守结构域对甘蔗黑穗病菌基因组进行搜索,发现了一个RNA干扰组件DICER-LIKE (SsDcl)蛋白.序列分析显示此蛋白具有剪切双链RNA所必需的RNaseⅢ结构域和双链RNA识别区域.为了研究ssdcl基因的功能,本研究以甘蔗黑穗病菌单倍体JG36为出发菌株,构建了ssdcl基因的缺失突变株.与野生型相比,Δssdcl突变体的生长速度较慢,与野生型JG35配合后形成的二倍体菌丝更为细小,且分支较多,表明ssdcl基因的缺失可能影响了黑穗病菌菌丝的生长发育过程.另外,Northern blot和qRT-PCR的结果显示,ssdcl基因缺失突变体中milR-4的表达量下调,提示ssdcl基因在甘蔗黑穗病菌milRNAs形成过程中起重要作用.

MicroRNAs (miRNAs) are a class of endogenous small RNAs that regulate target gene expression at the post-transcriptional level.miRNAs play an important role in almost all physiological activities including the processes of embryonic stem cell (ESC) pluripotency maintenance and differentiation, as well as induced pluripotent stem cell (iPSC) reprogramming.In this study, we identified eight novel miRNAs by mining the deep sequencing dataset from mouse ESCs and three kinds of reprogrammed pluripotent cells.Most of them are conserved at least in Murinae animals.Seven of them are derived from gene introns.We further showed that miR-novel-41 is a mirtron that derives from the intron of Man2c1, a protein-coding gene.Mirtrons are a kind of special non-canonical miRNAs encoded in introns.Their precursors are not processed by Drosha but generated by intron splicing.Intron splicing is required for the maturation of mirtrons and allows pre-miRNA-like hairpins to form [1].Both ends of the precursor of a canonical mirtron are precisely generated by the splicing reaction.Some spliced pre-miRNA-like hairpins have a single-stranded tail on either the 5'or 3'end, named as 5'-or 3'-tailed mirtrons, which will be digested by exonuclease to leave a pre-miRNA for Dicer processing [2,3].The potential functions of these novel miRNAs were also preliminarily explored by target genes prediction.

Plants resist viral infection through multiple mechanisms.These include physical barriers,activation of NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT PROTEINS(NLRs),autophagy,biosyn-thesis of defense-related phytohormones,and RNA interference(RNAi).RNAi suppresses the expression of viral RNA and is conserved across several eukaryotic kingdoms.During infection,RNA-DEPENDENT RNA POLYMERASES(RDRs)amplify viral RNA fragments into double-stranded RNAs(dsRNAs).DICER-LIKE ENDORIBONUCLEASES(DCLs)then cleave these dsRNAs into small interfering RNAs(siRNAs),which associate with RNASE H-LIKE ARGONAUTES(AGOs)and form RNA-INDUCED SILENCING COMPLEXES(RISCs).siRNAs guide RISCs to cleave homologous viral RNA fragments and repress viral replication(Fang and Qi,2016).While RNAi has been extensively studied over the past few decades,the initial cues that prime the expression of RNAi-related genes during viral infection remain unclear.A recent study by Liu and colleagues sheds new light on the regulation of RNAi during the early stage of infection(Wang et al.,2021).

植物Dicer-like蛋白家族属于核糖核酸酶Ⅲ (RNase Ⅲ),主要在植物体内产生miRNA与siRNA等非编码RNA,在植物的生长发育、器官发生、昼夜节律、生物和非生物胁迫反应以及对病毒和转座子的防御中都起重要作用.本文综述了Dicer-like蛋白的结构、功能、种类、定位,以及在生物与非生物逆境研究中的进展,以期为Dicer-like家族在植物中的进一步的研究与利用提供理论参考.