高通量转录组测序技术在植物雄性不育研究中的应用

植物雄性不育是指植物雄蕊发育受阻不能产生正常有功能花粉的现象。植物雄性不育不仅是生殖生理研究的宝贵材料,也是植物杂种优势利用的重要工具。由于高通量转录组测序技术几乎可以检测细胞内所有mRNA及非编码RNA的信息,已被广泛应用于生命科学研究的各项领域。在植物雄性不育相关研究中,高通量转录组测序技术在不同物种、不同败育类型中的应用已有报道,这为研究者在转录组水平综合了解植物雄性不育的分子机制及代谢网络提供了帮助。本文从测序文库构建策略、差异表达基因、非编码RNA的功能特征等方面综述了高通量转录组测序在植物雄性不育机理方面的研究进展,并探讨了转录组测序技术在花粉败育机制解析及育性相关基因定位中的应用价值,以期为植物雄性不育的相关研究提供参考。     

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.     

MicroRNA expression and its implications for diagnosis and therapy of tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is the most common type of oral squamous cell carcinomas and is well known for its high rate of lymph nodal metastasis. Despite the identification of many molecular mechanisms in TSCC, the number of deaths associated with TSCC increased during the past 5 years. MicroRNAs (miRNAs) are a family of small non‐coding RNA molecules, which regulate gene expression by either translational inhibition or mRNA degradation. miRNAs have been proven to be key regulators of various biological and pathological processes including cell proliferation, development and tumourigenesis. Increasing evidence has demonstrated that the deregulated miRNAs are implicated in the diagnosis and treatment of TSCC. In this review, we summarized the expressions and roles of miRNAs in TSCC and comment on the potential roles of miRNAs in diagnosis, prognosis and treatment of this malignancy.     

LUCAT1 as an oncogene in tongue squamous cell carcinoma by targeting miR-375 expression

Emerging studies suggested that lncRNAs play a crucial molecular role in cancer development and progression. LncRNA LUCAT1 has been proved as oncogenic molecular in lung cancer, glioma, osteosarcoma, renal carcinoma and oesophageal squamous cell carcinoma. However, its roles and function mechanisms in tongue squamous cell carcinoma (TSCC) are still unknown. We showed that the expression of LUCAT1 was up-regulated in the TSCC cells and tissues and the higher LUCAT1 expression was associated with the poor overall survival (OS). Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cycle and migration. In addition, we demonstrated that miR-375 overexpression inhibited the luciferase activity of LUCAT1 wild-type and knockdown LUCAT1 promoted the miR-375 expression in TSCC cell. Furthermore, we indicated that miR-375 expression was down-regulated in the TSCC cell lines and tissues and the lower expression of miR-375 was associated with poor OS. The expression of miR-375 was inversely correlated with LUCAT1 expression in the TSCC tissues. Knockdown LUCAT1 promoted TSCC cell proliferation, cell cycle and migration partly through regulating miR-375 expression. In summary, this study suggested the tumorigenic effect of lncRNA LUCAT1 in TSCC cells by targeting miR-375 expression.     

Identification,characterization, and functional investigation of circular RNAs in subventricular zone of adult rat brain

Adult neural stem cells (NSCs) are able to self-renew and generate new neural cells. Identifying regulators of NSCs is significant for the development of NSC-based therapies for neurodegenerative diseases and brain injuries. Recently, circular RNAs (circRNAs) have been characterized in various cell lines and brain tissues, and found to participate in multiple biological processes. However, the expression pattern of circRNAs in adult NSCs is still unknown. Here, the subventricular zone (SVZ) of the lateral ventricle was isolated as the niche of NSCs in adult rat brain for RNA sequencing and the characteristics of circRNAs profiling in both SVZ and cerebral cortex were also investigated. As a result, 29 049 and 31 975 circRNAs were identified in SVZ and cortex, respectively. Among them, 41 were SVZ-specific and 48 were cortex-specific. 467 circRNAs were also found to express predominately in SVZ, while the cortex had other 423 circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the SVZ-specific circRNAs have close relationship with the regulation of NSC expansion and NSC-niche interaction, while the other differentially expressed circRNAs might be involved in neural cellular construction and nerve system function. Furthermore, the interactions between circRNAs and microRNAs were also explored, and the result showed that one SVZ-specific circRNA was capable to competitively bind miR-138-5p as a potential derepressive regulator in NSCs proliferation. Hence, our work has laid the foundations to decipher regulation mechanisms of circRNAs in adult NSCs and to develop circRNAs as novel biomarkers for adult NSCs.     

Screening and bioinformatics analysis of mRNA,long non-coding RNA and circular RNA expression profiles in mucoepidermoid carcinoma of salivary gland

Mucoepidermoid carcinoma (MEC) of salivary gland is a disease characterized by high rate of diatant metastasis, and associated with poor outcomes. However, the molecular mechanisms underlying the MEC remain poorly understand. Here, we simultaneously detected, for the first time, the expression profiles of mRNAs, lncRNAs, and circRNAs in four pairs of MEC and matched non-carcinoma tissues by microarrays. A total of 3612?mRNA, 3091 lncRNAs, and 284 circRNAs were altered during the pathogenesis of MEC. The functions of these differentially expressed RNAs were predicted by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were conducted to uncovered the hidden ceRNA mechanisms. Moreover, NONHSAT154433.1 that associated with ADAM12 and hsa_circ_0012342 were further screened and confirmed using qRT-PCR analysis. In conclusion, this study provides a systematic perspective on the potential function of non-coding RNAs (ncRNAs) in the molecular mechanisms of MEC. Among these, NONHSAT154433.1 and hsa_circ_0012342 might be served as potential prognostic biomarkers and therapeutic target of MEC.     

Large-scale investigation of zoonotic viruses in the era of high-throughput sequencing

Zoonotic diseases considerably impact public health and socioeconomics. RNA viruses reportedly caused approximately 94% of zoonotic diseases documented from 1990 to 2010, emphasizing the importance of investigating RNA viruses in animals. Furthermore, it has been estimated that hundreds of thousands of animal viruses capable of infecting humans are yet to be discovered, warning against the inadequacy of our understanding of viral diversity. High-throughput sequencing (HTS) has enabled the identification of viral infections with relatively little bias. Viral searches using both symptomatic and asymptomatic animal samples by HTS have revealed hidden viral infections. This review introduces the history of viral searches using HTS, current analytical limitations, and future potentials. We primarily summarize recent research on large-scale investigations on viral infections reusing HTS data from public databases. Furthermore, considering the accumulation of uncultivated viruses, we discuss current studies and challenges for connecting viral sequences to their phenotypes using various approaches: performing data analysis, developing predictive modeling, or implementing high-throughput platforms of virological experiments. We believe that this article provides a future direction in large-scale investigations of potential zoonotic viruses using the HTS technology.     

Reconstruction and analysis of the aberrant lncRNA‐miRNA‐mRNA network based on competitive endogenous RNA in CESC

A growing body of studies has demonstrated that long non‐coding RNA (lncRNA) are regarded as the primary section of the ceRNA network. This is thought to be the case owing to its regulation of protein‐coding gene expression by functioning as miRNA sponges. However, functional roles and regulatory mechanisms of lncRNA‐mediated ceRNA in cervical squamous cell carcinoma (CESC), as well as their use for potential prediction of CESC prognosis, remains unknown. The aberrant expression profiles of mRNA, lncRNA, and miRNA of 306 cervical squamous cancer tissues and three adjacent cervical tissues were obtained from the TCGA database. A lncRNA‐mRNA‐miRNA ceRNA network in CESC was constructed. Meanwhile, Gene Ontology (GO) and KEGG pathway analysis were performed using Cytoscape plug‐in BinGo and DAVID database. We identified a total of 493 lncRNA, 70 miRNA, and 1921 mRNA as differentially expressed profiles. An aberrant lncRNA‐mRNA‐miRNA ceRNA network was constructed in CESC, it was composed of 50 DElncRNA, 18 DEmiRNA, and 81 DEmRNA. According to the overall survival analysis, 3 out of 50 lncRNA, 10 out of 81 mRNA, and 1 out of 18 miRNA functioned as prognostic biomarkers for patients with CESC (P value < 0.05). We extracted the sub‐network in the ceRNA network and found that two novel lncRNA were recognized as key genes. These included lncRNA MEG3 and lncRNA ADAMTS9‐AS2. The present study provides a new insight into a better understanding of the lncRNA‐related ceRNA network in CESC, and the novel recognized ceRNA network will help us to improve our understanding of lncRNA‐mediated ceRNA regulatory mechanisms in the pathogenesis of CESC.     

Circ_0000052/miR-382-3p axis induces PD-L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma

A better understanding of the mechanisms underlying PD-L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD-L1 in forty-five HNSCC archival samples was determined by qRT-PCR. The biological function associated with malignant behaviour was assessed by PD-L1 depletion, miR-382-3p re-expression and regulation of circ_0000052. The interactions of PD-L1-miRNA and miRNA-circRNA were determined by qRT-PCR, Western blot analysis, dual-luciferase reporter assays and RNA immunoprecipitation assays. PD-L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD-L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN-γ/JAK2/STAT1 signalling pathway can induce PD-L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD-L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR-382-3p and alleviates its repression of PD-L1. This leads to overexpression of PD-L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR-382-3p/PD-L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti-PD-L1 therapy may improve personalized disease management.     

Expression and clinical value of PD-L1 which is regulated by BRD4 in tongue squamous cell carcinoma

The programmed cell death-ligand-1 (PD-L1) and bromodomain protein 4 (BRD4) are frequently overexpressed in cancer and have even been shown to act synergistically. The aim of this study was to determine their potential oncogenic role .in tongue squamous cell carcinoma (TSCC). We detected significantly higher expression levels of both PD-L1 and BRD4 in TSCC tissues compared to normal tissues (P ≤ .05). In addition, the high levels of PD-L1 were significantly associated with increased tumor lymphatic metastasis (P ≤ .05), tumor staging (P ≤ .01), as well as BRD4 expression (P ≤ .05). Genetic and pharmacological inhibition of BRD4 in TSCC cells not only reduced their growth rate but also PD-L1 levels (P ≤ .05), while overexpression of BRD4 upregulated PD-L1. Bioinformatics analysis showed that c-MYC and CDK9 were interactive partners of both BRD4 and PD-L1. While c-MYC clearly modulated the expression of PD-L1, as well as reversed the inhibitory effects of JQ1, no obvious association was observed between CDK9 and PD-L1. We report a novel regulatory axis consisting of BRD4, PD-L1, and c-MYC that likely drives TSCC progression, and is a potential prognostic marker and/or therapeutic target for TSCC.     

CA9 transcriptional expression determines prognosis and tumour grade in tongue squamous cell carcinoma patients

CA9 is a member of the carbonic anhydrases’ family, that is often expressed in cancer cells under hypoxic condition. However, the role of CA9 in the molecular mechanisms of tongue squamous cell carcinoma (TSCC) pathogenesis remains unclear. CA9 expression was analysed using the TCGA database, and its influence on survival was performed using Kaplan-Meier, LASSO and COX regression analyses. The correlation between CA9 and immune infiltration was investigated by CIBERSORT and ESTIMATE. Moreover, the relationship between CA9 expression and downstream molecular regulation pathways was analysed by GSEA, GO and WGCNA. CA9 expression correlated with clinical prognosis and tumour grade in TSCC. Moreover, CA9 expression potentially contributes to the regulation of cancer cell differentiation and mediates tumour-associated genes and signalling pathways, including apoptosis, hypoxia, G2M checkpoint, PI3K/AKR/mTOR signalling and TGF-beta signalling pathways. However, the follicular helper T cells, regulatory T cells, immune and stromal scores showed no significance between high and low CA9 expression groups. These findings suggested that CA9 plays a critical role of TSCC prognosis and tumour grade. CA9 expression significantly correlated with the regulation of cell differentiation, various oncogenes and cancer-associated pathways.     

单细胞RNA测序技术在病毒研究中的应用

单细胞RNA测序(single-cell RNA sequencing, scRNA-seq)技术已经成为不同领域中研究细胞异质性的有效工具。在病毒研究领域中,利用该技术分析病毒和细胞的转录组,可以在单细胞水平上检测病毒感染的动态变化,了解病毒与细胞间复杂的相互作用。本文简述了scRNA-seq技术,着重介绍病毒感染宿主细胞后scRNA-seq研究的最新进展,同时也描述了细胞周期、基因表达、细胞状态等细胞异质性对病毒感染过程的影响,以及病毒变异对其本身感染过程的影响。此外,本文还分析了scRNA-seq在研究病毒–宿主互作动态变化方面具有的独特优势,及其在病毒研究领域中广阔的应用前景,为揭示病毒的感染与致病机制、抗病毒靶标的开发等提供参考。