Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4’-methylene diphenyl diisocyanate exposure

Background: Occupational exposure to the most widely used diisocyanate, 4,4’-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA.

Objective: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA.

Materials and methods: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580?±?1497?μg/m³ MDI-aerosol for 60?minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY? miRs qPCR Profiling Service was used to profile circulate miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan^® miR assays.

Results: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens.

Discussion and conclusion: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations.     

Tumor suppressive microRNA-485-5p targets PRRX1 in human skin melanoma cells,regulating epithelial–mesenchymal transition and apoptosis

Melanoma is one of the most aggressive skin cancers. Existing evidence has reported the aberrant expression of microRNAs (miRNAs) in melanoma, but their putative targets and underlying downstream effects remain to be further understood. Herein, we explored the suppressive role of miR-485-5p in melanoma progression. Initial bioinformatics analyses showed that the PRRX1 gene was differentially expressed in melanoma, while miR-485-5p was predicted to be a potential regulatory miRNA binding to PRRX1 mRNA. We confirmed that PRRX1 was upregulated, while miR-485-5p was downregulated in human melanoma samples compared with adjacent normal skin tissues. We then showed that PRRX1 was a target gene of miR-485-5p by dual-luciferase reporter gene assay. Moreover, a reduction in the expression of PRRX1 and downregulation of important proteins of the transforming growth factor-beta (TGFβ) signaling pathway was observed after miR-485-5p overexpression. Furthermore, miR-485-5p overexpression or PRRX1 knockdown suppressed epithelial–mesenchymal transition, cell viability, migration, and invasion, and promoted cell apoptosis in melanoma cells. Our study demonstrates the tumor-suppressive functions of miR-485-5p in the development of human melanoma, providing a potential target for therapy.     

hsa-miR-342-3p生物信息学分析

通过生物信息学方法预测hsa-miR-342-3p靶基因及其功能机制。检索Pub Med有关hsa-miR-342-3p的研究报道并进行功能分析;检索miRBase获取hsa-miR-342-3p序列;通过Target Scan,Pictar和PITA数据库预测靶基因并取交集,对其进行组织和疾病特异性表达谱分析、功能富集分析(GO enrichment analysis)、信号转导通路富集分析(Pathway enrichment analysis)和蛋白质相互作用网络分析(PPI analysis)。结果发现:hsa-miR-342-3p序列在多物种间具有高度保守性;hsa-miR-342-3p在肾脏组织和急性淋巴细胞性白血病、乳腺癌疾病中表达水平较高(RPM≥1 000);预测得到14个hsa-miR-342-3p靶基因;靶基因分子功能分别富集于转化生长因子活性、DNA结合和蛋白激酶激活等(P0.05);hsa-miR-342-3p靶基因GO生物学过程主要集中于大分子代谢抑制,肺部组织发育、呼吸系统发育及管状组织发育建成(P0.05);细胞信号通路主要富集于TGF-Beta信号通路、细胞因子、受体作用信号通路及前列腺疾病信号通路(P0.01)。hsa-miR-342-3p在体内分布广泛,预测的靶向TGF-Beta信号通路可能在疾病发生中发挥重要调控作用,是具有潜在研究价值的生物学靶标。     

p8-应激调节的多功能核蛋白

1997年,Iovanna等在研究急性胰腺损伤的分子变化中首先克隆得到了p8基因.人的p8分子是一个由82个氨基酸组成的小分子核蛋白,分子内含有一个保守的核定位序列,蛋白质二级结构与HMG-I/Y蛋白十分相似,该分子在包括胰腺、肝脏、肾脏在内的诸多脏器受到损伤刺激后,能够在短时间内迅速、高水平地上调,因此被认为是一个应激分子(stress associated protein).随后的研究显示该基因具有复杂的表达调节模式,在个体发育、肿瘤形成、组织损伤、心肌肥大以及糖尿病性肾病中都发挥重要作用,并且其功能表现在不同组织、细胞中不尽相同.     

24p3-24p3受体系统：一种新的铁转运途径

小鼠24p3是脂质运载蛋白lipocalin家族的成员之一,可在白介素3(interleukin-3,IL-3)缺乏时诱导细胞发生凋亡,并参与细胞的铁转运过程.最近,Devireddy等又成功克隆到了24p3的细胞表面受体(24p3 receptor,24p3R),进一步确定了24p3-24p3R这一新的铁转运途径.该途径的发现不仅增加了对铁转运理论新的认识,更重要的是,这一发现为铁代谢调控细胞凋亡理论的建立奠定了基础.     

miRNA-139-5p与肿瘤

转移和复发是恶性肿瘤的重要特征。肿瘤细胞的增殖、浸润、侵袭能力与肿瘤的复发和转移息息相关。非编码小RNA通过转录后水平参与肿瘤的发生和发展过程。其中,miR-139-5p成为研究热点之一。在许多肿瘤中都发现miR-139-5p的异常表达,它参与了肿瘤细胞的增殖、分化、转移和浸润,调节肿瘤细胞对化疗药物敏感性,并与肿瘤患者的预后相关。因此,miR-139-5p可能成为肿瘤治疗的一个新靶点。本文对miR-139-5p与肿瘤的关系作一综述。系统地了解它在恶性肿瘤进程中的作用,为广大研究者及临床工作者提供新思路新方法。     

miR-305-3p和miR-71-5p通过调控谷胱甘肽代谢途径参与斜纹夜蛾应对植物次生物质

昆虫和植物在长期进化过程中形成相互作用的关系。其中植物次生物质是植物防御昆虫的主要机制,而昆虫则以解毒酶来应对。为了发现可用于害虫防治的miRNA,通过对农业害虫斜纹夜蛾Spodoptera litura进食芥菜Brassica juncea后中肠的测序分析,获得了一系列差异表达的miRNA。通过生物信息学分析,发现斜纹夜蛾miR-305-3p靶向了谷胱甘肽代谢中的谷氨酸半胱氨酸连接酶的催化亚基(Slgclc),这是一个谷胱甘肽从头合成的限速酶;而miR-71-5p靶向调控gclc和解毒酶表达的转录因子SlNrf2。用芥菜的次生物质吲哚-3-甲醇处理斜纹夜蛾Spli-221细胞株后,实时荧光定量PCR证明Slgclc和SlNrf2表达上调,miR-305-3p和miR-71-5p表达下调。分别在斜纹夜蛾Spli-221细胞中超表达miR-305-3p及miR-71-5p mimics,Slgclc或SlNrf2转录水平下调,并且miR-71-5p mimic处理抑制了SlNrf2下游基因Slgclc和解毒酶谷胱甘肽S-转移酶(GSTs)的表达。结果表明:miR-305-3p和miR-71-5p响应植物次生物质而分别促进Slgclc和SlNrf2表达。然而双荧光素酶实验显示miR-305-3p并不与Slgclc直接结合,miR-71-5p也不与SlNrf2直接结合,推测miR-305-3p和miR-71-5p可能间接调控Slgclc和SlNrf2的表达。研究结果表明,斜纹夜蛾miR-305-3p和miR-71-5p通过调控解毒酶GSTs表达及其底物谷胱甘肽的生成,而参与昆虫抵抗植物次生物质。     

14-3-3τ Regulates Ubiquitin-Independent Proteasomal Degradation of p21, a Novel Mechanism of p21 Downregulation in Breast Cancer

14-3-3 proteins regulate many cellular functions, including proliferation. However, the detailed mechanisms by which they control the cell cycle remain to be fully elucidated. We report that one of the 14-3-3 isoforms, 14-3-3τ, is required for the G₁/S transition through its role in ubiquitin-independent proteasomal degradation of p21. 14-3-3τ binds to p21, MDM2, and the C8 subunit of the 20S proteasome in G₁ phase and facilitates proteasomal targeting of p21. This function of 14-3-3τ may be deregulated in cancer. The overexpression of 14-3-3τ is frequently found in primary human breast cancer and correlates with lower levels of p21 and shorter patient survival. Tenascin-C, an extracellular matrix protein involved in tumor initiation and progression and a known 14-3-3τ inducer, decreases p21 and abrogates adriamycin-induced G₁/S arrest. It has been known that p21 is required for a proper tamoxifen response in breast cancer. We show that the overexpression of 14-3-3τ inhibits tamoxifen-induced p21 induction and growth arrest in MCF7 cells. Together, the findings of our studies strongly suggest a novel oncogenic role of 14-3-3τ by downregulating p21 in breast cancer. Therefore, 14-3-3τ may be a potential therapeutic target in breast cancer.14-3-3 proteins are a family of about 30-kDa dimeric well-conserved α-helical phosphoserine/threonine binding proteins. They contain seven mammalian isoforms (β, ɛ, γ, η, σ, τ, ζ) and are able to bind to multiple protein ligands. The 14-3-3 binding proteins are very diverse; therefore, 14-3-3 is involved in many different cellular processes, including mitogenesis, DNA damage checkpoint, cell cycle control, and apoptosis (12). Most 14-3-3 ligands require phosphorylation to bind to 14-3-3; and their consensus motifs are R(S/Ar)XpSXP (mode 1), RX(Ar/S)XpSXP (mode 2) (12, 46), and (pS/pT)X_1-2-COOH (mode 3) (13). However, this consensus is not absolutely required, since a few 14-3-3 binding ligands have sequences significantly different from the sequences of these motifs or do not even require phosphorylation for binding (12, 46).In general, 14-3-3 proteins play a role in promoting survival and repressing apoptosis (33). However, each isoform may have unique functions in certain physiological contexts. For example, 14-3-3τ binds to ATM-phosphorylated E2F1 during DNA damage and promotes E2F1 stability, leading to the induction of E2F1 proapoptotic target genes such as p73, Apaf1, and caspases (44). Like other 14-3-3 isoforms, however, there appears to be a role for 14-3-3τ in cell survival as well. The deletion of 14-3-3τ in mice leads to embryonic lethality, probably due to developmental arrest (25). Examination of 14-3-3τ^+/− mice reveals a role for 14-3-3τ in cardiomyocyte survival (25). This is probably due to its activity that antagonizes ASK1 and sequesters BAD and FOXO family members. However, whether and how 14-3-3τ is involved in cell cycle progression remain poorly understood.In the study described here, we investigated the role of 14-3-3τ in cell cycle control and uncovered its involvement in the regulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). p21 is a p53 target gene and a major regulator that mediates p53-dependent G₁ arrest and senescence. The turnover of the p21 protein is under very tight control. p21 can be degraded through both ubiquitin-dependent and ubiquitin-independent mechanisms. In the ubiquitin-dependent pathway, Skp2 and CRL4(Cdt2) are responsible for p21 degradation in S phase (1, 5, 23, 30, 48), whereas APC/C^Cdc20 controls the degradation of p21 in prometaphase (3). There are also data demonstrating that Skp2 is not required for basal p21 ubiquitylation and degradation (8). p21 can also be directly targeted to the proteasome for degradation without ubiquitylation (38). This process is mediated by an interaction between p21 and the C8 α subunit of the 20S proteasome (40) and can be promoted by MDM2 and MDMX (20, 21, 49). The MDM2/MDMX-regulated degradation of p21 occurs at the G₁ and early S phases (21). The stability of the p21 protein is also regulated by heat shock proteins. An Hsp90 binding protein, WISp39, recruits Hsp90 to p21 and protects p21 from degradation during DNA damage (19). Therefore, it appears that several different regulators control the stability of the p21 protein, probably depending on the phases of the cell cycle and cellular contexts.Given the evidence of both ubiquitin-dependent and -independent degradation of p21, Pagano and colleagues proposed that both mechanisms of degradation of p21 in different protein complexes may occur in cells (3). It has been shown that free p21, but not cyclin E/cdk2-bound p21, can be degraded by the proteasome in vitro without ubiquitylation (4, 26, 27). Thus, when p21 is complexed with cdk2, it may be degraded by the ubiquitin-dependent pathway, while the ubiquitin-independent mechanism may target free p21 for degradation. It has been shown that this process involves the REGγ proteasome complex (7, 26), which forms the 11S lid and activates the 20S catalytic core proteasome. However, the regulatory mechanisms for the ubiquitin-independent degradation of p21 remain unclear.In the present study, we identify 14-3-3τ as the protein that regulates the ubiquitin-independent proteasomal degradation of p21 in G₁ phase. We demonstrate a direct role for 14-3-3τ in the 20S-mediated p21 degradation via facilitation of an interaction between p21, MDM2, and C8 in vitro. This new role of 14-3-3τ might have an important clinical implication. The extracellular matrix tenascin-C induces 14-3-3τ and degrades p21 through the induction of 14-3-3τ and ameliorates adriamycin-induced cell cycle arrest. 14-3-3τ is often overexpressed in breast cancer, and its overexpression is associated with the downregulation of p21 and shorter patient survival. Through the downregulation of p21, 14-3-3τ overexpression also leads to tamoxifen resistance in MCF7 breast cancer cells.     

miR-505-3p对GT1-7细胞株基因表达谱的影响

目的了解微小RNA 505-3p(miR-505-3p)对下丘脑GnRH神经元GT1-7稳转细胞株表达谱的影响。方法用慢病毒转染GT1-7细胞获得稳定表达miR-505-3p的细胞株,感染实验分为3组:空白对照组(未经处理的GT1-7细胞),阴性对照组(感染pLenti6.3-nc空病毒),实验组(感染pLenti6.3-miR-505-3p病毒)。采用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)技术检测稳定细胞株中miR-505-3p的表达丰度,并进一步利用芯片检测对照组和实验组细胞的表达谱结合生物信息学方法探寻差异表达基因。采用GO分析和Pathway分析差异表达基因,解析miR-505-3p的功能。结果在稳定表达miR-505-3p的GT1-7细胞中,165个基因的表达量变化在2倍以上,这些基因主要参与细胞粘附、GTP分解代谢、神经系统发育等生物过程。结论 miR-505-3p可能通过影响下丘脑GnRH神经元中相应靶基因的表达调控细胞粘附、GTP分解代谢、神经系统发育等生物过程,和间隙连接、轴突导向和胃酸分泌等信号通路。     

miR-9-5p靶向Ambra1促进舌癌细胞增殖

miRNAs在肿瘤中异常表达,且与肿瘤的发生发展密切相关。目前发现,miR-9-5p在肿瘤中可能发挥原癌或抑癌效应,功能尚未完全阐述清楚。本文拟探讨miR-9-5p在舌癌中的作用。前期研究中收集10例舌癌组织及配对的癌旁组织,实时荧光定量PCR技术检测后发现,miR-9-5p在舌癌组织中的表达量显著高于癌旁组织,且其在舌癌细胞中的表达量也明显高于正常舌上皮细胞。此外,在舌癌细胞Tca8113中过表达miR-9-5p显著增加细胞的增殖能力。生物信息学预测及双荧光素酶报告基因实验证实,miR-9-5p可直接结合在自噬/苄氯素1调节因子1（activating molecule in beclin1-regulated autophagy, Ambra1）的 3′-UTR区域,靶向抑制Ambra1表达。Western印迹结果证实过表达miR-9-5p降低Ambra1的表达,反之亦然。Ambra1在舌癌细胞中的表达量显著低于正常舌上皮细胞。BrdU实验证实在舌癌细胞SCC-25中过表达Ambra1可显著抑制其增殖能力;相反,使用siRNA技术沉默Ambra1能够显著促进Tca8113细胞的增殖。在干预miR-9-5p的细胞中同时干预Ambra1的表达,结果发现Ambra1可显著逆转miR-9-5p对舌癌细胞增殖的促进作用。总之,miR-9-5p在舌癌中可能发挥原癌基因样作用,通过直接靶向抑制Ambra1表达进而促进舌癌细胞发生增殖。     

hsa-miR-192-3p的靶基因预测及功能分析

通过生物信息学方法预测hsa-miR-192-3p的靶基因及其靶基因的可能功能。首先通过miRbase在线数据库对hsamiR-192-3p的碱基序列及序列在各物种间的保守性进行分析,再通过miRGator v3. 0在线数据库查看hsa-miR-192-3p在各个组织器官中的表达丰富度情况;其次,应用Target Scan和miRanda在线数据库预测hsa-miR-192-3p的靶基因;最后,将预测得到的两个数据库的靶基因交集用DAVID在线数据库进行功能富集分析和信号转导通路富集分析。结果表明:hsa-miR-192-3p在人、家鼠、猕猴等生物中存在高度保守性; hsa-miR-192-3p在胃肠道、肾脏、肝胆系统、干细胞、鼻、脾、胸腺中表达丰富度较高;通过两个靶基因预测软件预测的靶基因取交集后共有190个;功能富集分析发现hsa-miR-192-3p靶基因富集在细胞质、细胞核、质膜、高尔基体等15个细胞组件(p0. 05),参与蛋白结合、GTP酶活性、锌离子跨膜转运蛋白活性等7个分子功能(p0. 05),涉及金属离子运输、RNA聚合酶II启动子的转录阳性调控、基因表达调节、钙离子跨膜运输、胚胎发育等18个生物过程(p0. 05);预测靶基因集合显著富集于癌症通路与催乳素信号通路中(p0. 05)。得出结论:hsa-miR-192-3p预测的靶基因集合富集于多个生物过程,与肿瘤密切相关,生物信息预测为今后的研究奠定了一定的理论基础,为后续实验验证提供了研究方向。     

慢性牙周炎患者血清miR-205-5p、miR-28-5p的表达与诊断价值研究

摘要 目的：探讨慢性牙周炎（CP）患者血清微小核糖核酸（miRNA）-205-5p、miR-28-5p表达与牙周指标、炎症反应的关系及诊断价值。方法：选取2021年1月～2022年12月我院口腔科收治的102例CP患者为CP组,根据病情严重程度分为轻度组31例、中度组37例、重度组34例,另选取同期我院65名体检健康者为对照组。采用实时荧光定量聚合酶链式反应（PCR）检测血清miR-205-5p、miR-28-5p表达,酶联免疫吸附法检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）水平,检查各组牙周指标[菌斑指数（PLI）、牙龈出血指数（BI）、附着丧失（AL）、探诊深度（PD）]。CP患者血清miR-205-5p、miR-28-5p表达与牙周指标和炎症指标的相关性采用Pearson相关性分析。受试者工作特征（ROC）曲线分析血清miR-205-5p、miR-28-5p表达诊断CP的价值。结果：与对照组比较,CP组血清miR-205-5p、miR-28-5p表达降低,PLI、BI、AL、PD和IL-1β、IL-6、TNF-α水平升高（P＜0.05）。轻度组、中度组、重度组血清miR-205-5p、miR-28-5p表达依次降低,PLI、BI、AL、PD和IL-1β、IL-6、TNF-α水平依次升高（P＜0.05）。Pearson相关性分析显示,CP患者血清miR-205-5p、miR-28-5p表达与PLI、BI、AL、PD、IL-1β、IL-6、TNF-α呈负相关（P＜0.05）。ROC曲线分析显示,血清miR-205-5p联合miR-28-5p诊断CP的曲线下面积分别为0.885大于miR-205-5p、miR-28-5p单独检测的0.792、0.790。结论：CP患者血清miR-205-5p、miR-28-5p低表达,与牙周指标、炎症反应密切相关,血清miR-205-5p、miR-28-5p的表达情况可能成为CP辅助诊断指标,且miR-205-5p联合miR-28-5p诊断CP的价值较高。     

p53家族新成员：p51

p53是一种广谱的肿瘤抑制基因,其新家族成员p51具有同p53相似的DNA结合特笥和相似的功能,同样可以转录激活p53基因的内源性靶分子,如细胞周期抑制基因p21、导致细胞凋亡和生长受抑。本文阐述了它的研究进展。     

KSR2基因3'UTR双荧光素酶报告基因载体构建及其与hcmv-miR-UL70-3p和hcmv-miR-US4-3p靶向关系验证

人巨细胞病毒(Human cytomegalovirus,HCMV)的miR-UL70-3p和miR-US4-3p是HCMV microRNA(miRNA)家族中的一员,前期经生物信息学分析发现,Ras激酶抑制剂2(Kinase Suppressor 2 of Ras,KSR2)的3'-UTR区为miR-UL70-3p和miR-US4-3p的高度可疑结合靶点.为进一步鉴定miR-UL70-3p和miR-US4-3p与KSR2基因的靶标关系,本研究通过构建GV272-KSR2-3'UTR野生和突变型载体,进行双荧光素酶(Luciferase)报告基因实验,并测定其荧光值活性的方法判定miR-UL70-3p和miR-US4-3p是否能与KSR2基因结合.先将293T细胞复苏、扩增并进行质粒转染,转染分6组,每组重复做3孔,分组分别为①KSR2-3'UTR阴性载体与miRNA无关序列载体②KSR2-3'UTR阴性载体与miRNA有效序列载体③KSR2-3'UTR野生序列载体与miRNA无关序列载体④KSR2-3'UTR野生序列载体与miRNA有效序列载体⑤KSR2-3'UTR突变序列载体与miRNA无关序列载体⑥KSR2-3'UTR突变序列载体与miRNA有效序列载体.酶切及DNA测序结果显示GV272-KSR2-3'UTR双荧光素酶报告基因载体构建成功.Luciferase检测显示:同一Luciferase质粒转染的两个组中,将miRNA-NC组Luciferase相对表达量均一化为1,即将实验组①③⑤均一化为1后,比较实验组③和④组、⑤和⑥组、④和⑥组之间的Luciferase检测结果,显示实验组④和实验组③相比,有显著性降低(P＜0.05),实验组⑥和实验组⑤相比,实验组⑥和实验组④相比,差异具有统计学意义(P＜0.05),说明hcmv-miR-UL70-3p和hcmv-miR-US4-3p可与KSR2-3'UTR结合,抑制KSR2表达.     

p63和p63参与p53依赖的细胞凋亡过程

人们通常用经典的操作式学习方法来训练动物的行为 ,使动物学会根据外部信号 (如声音 )产生特定的行为反应 ,以获取奖赏 (如食物 )。而本文的作者用脑内植入微电极进行脑区刺激的方法教会动物如何学习 ,可以去除用来产生信号和奖赏的外部环境对实验的限制。这一动物模型使操作者能远距离地指挥动物的行为 ,很像控制智能机器人的方法。电刺激能否产生等同于信号或奖赏的效应 ,取决于它所刺激的脑区。作者在自由活动大鼠的躯体感觉皮层 (ＳＩ)左右两侧胡须代表区和内侧前脑束 (ＭＦＢ)内植入电极加以刺激 ,以产生信号和奖赏效应 ,据此准确地指…     

miR-15a-5p和miR-16-5p在骨肉瘤组织中的表达分析

目的:使用microRNAs基因芯片及实时定量PCR法测定骨肉瘤组织中miR-15a-5p和miR-16-5p的相对表达含量,并与瘤旁组织对比,分析骨肉瘤细胞内miR-15a-5p和miR-16-5p的表达变化。方法:选取34例骨肉瘤组织蜡块样本,使用microRNAs基因芯片观察miR-15a-5p和miR-16-5p在骨肉瘤和瘤旁组织内的表达差异;实时定量PCR法测定骨肉瘤组织和瘤旁组织中miR-15a-5p和miR-16-5p的相对表达含量,并将两种结果对比分析。结果:microRNAs基因芯片结果显示,在骨肉瘤组织中,miR-15a-5p在肿瘤中的表达较瘤旁组织低1.79倍,miR-16-5p较瘤旁组织低1.62倍。实时定量PCR实验结果表明,miR-15a-5p和miR-16-5p表达较瘤旁组织降低,差异有统计学意义(P0.05)。经过统计学计算,miR-15a-5p在肿瘤中的表达较瘤旁组织低3.14倍,miR-16-5p较瘤旁组织低5.65倍。结论:在骨肉瘤中,miR-15a-5p和miR-16-5p表达含量降低,提示这两种microRNAs在骨肉瘤中可能做为抑癌因子存在。     

miR-9-5p靶向抑制Ambra1促进舌癌细胞增殖

miRNAs在肿瘤中异常表达,且与肿瘤的发生发展密切相关。目前发现,miR-9-5p在肿瘤中可能发挥原癌或抑癌效应,功能尚未完全阐述清楚。本文拟探讨miR-9-5p在舌癌中的作用。前期研究中收集10例舌癌组织及配对的癌旁组织,实时荧光定量PCR技术检测后发现,miR-9-5p在舌癌组织中的表达量显著高于癌旁组织,且其在舌癌细胞中的表达量也明显高于正常舌上皮细胞。此外,在舌癌细胞Tca8113中过表达miR-9-5p显著增加细胞的增殖能力。生物信息学预测及双荧光素酶报告基因实验证实,miR-9-5p可直接结合在自噬/苄氯素1调节因子1(activating molecule in beclin1-regulated autophagy,Ambra1)的3'-UTR区域,靶向抑制Ambra1表达。Western印迹结果证实过表达miR-9-5p降低Ambra1的表达,反之亦然。Ambra1在舌癌细胞中的表达量显著低于正常舌上皮细胞。Brd U实验证实在舌癌细胞SCC-25中过表达Ambra1可显著抑制其增殖能力;相反,使用siRNA技术沉默Ambra1能够显著促进Tca8113细胞的增殖。在干预miR-9-5p的细胞中同时干预Ambra1的表达,结果发现Ambra1可显著逆转miR-9-5p对舌癌细胞增殖的促进作用。总之,miR-9-5p在舌癌中可能发挥原癌基因样作用,通过直接靶向抑制Ambra1表达进而促进舌癌细胞发生增殖。     

MiR-186-5p对绵羊黑色素细胞黑色素生成的调控

黑色素合成是极其复杂的过程,其中涉及多种基因和miRNAs的参与。miR-186-5p在绵羊不同毛色皮肤中差异表达,说明其可能与毛色形成有关,生物信息学预测和绵羊不同毛色皮肤中miR-186-5p和Mitf的差异表达间接说明二者可能存在靶向关系,双荧光报告直接证实了二者的靶向关系。为了证实miR-186-5p在黑色素形成中的作用,本研究构建miR-186-5p真核表达载体,并转染绵羊黑素细胞。结果显示,miR-186-5p通过与Mitf mRNAs的3′UTR结合抑制Mitf的表达和翻译。MITF是Tyr基因家族的调节因子。实时荧光聚合酶链反应(quantitative real time polymerase chain reaction, qRT-PCR)和Western免疫印迹结果表明,过表达miR-186-5p引起的MITF下调抑制了TYR、TYRP1和TYRP2的表达。其中,mRNA表达水平分别下降34%、66%和52%;蛋白质表达水平分别下降32%、6%和46%。分光光度检测结果显示,黑色素含量下降59%倍。上述结果表明,miR-186-5p通过抑制靶基因Mitf的表达,抑制了绵羊黑素细胞内黑色素的生成。