Overexpression of miR‐483‐5p/3p cooperate to inhibit mouse liver fibrosis by suppressing the TGF‐β stimulated HSCs in transgenic mice

The transition from liver fibrosis to hepatocellular carcinoma (HCC) has been suggested to be a continuous and developmental pathological process. MicroRNAs (miRNAs) are recently discovered molecules that regulate the expression of genes involved in liver disease. Many reports demonstrate that miR‐483‐5p and miR‐483‐3p, which originate from miR‐483, are up‐regulated in HCC, and their oncogenic targets have been identified. However, recent studies have suggested that miR‐483‐5p/3p is partially down‐regulated in HCC samples and is down‐regulated in rat liver fibrosis. Therefore, the aberrant expression and function of miR‐483 in liver fibrosis remains elusive. In this study, we demonstrate that overexpression of miR‐483 in vivo inhibits mouse liver fibrosis induced by CCl₄. We demonstrate that miR‐483‐5p/3p acts together to target two pro‐fibrosis factors, platelet‐derived growth factor‐β and tissue inhibitor of metalloproteinase 2, which suppress the activation of hepatic stellate cells (HSC) LX‐2. Our work identifies the pathway that regulates liver fibrosis by inhibiting the activation of HSCs.     

Computational identification of conserved microRNAs and their putative targets in the Hypericum perforatum L. flower transcriptome

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John’s wort (Hypericum perforatum L., 2n = 4x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

Identification and expression analysis of miRNAs in germination and seedling growth of Tibetan hulless barley

Germination and seedling growth are crucial for plant development and agricultural production. While, the regulatory mechanisms during these processes in Tibetan hulless barley (Hordeum vulgare L. var. nudum) are not well understood. Given the regulatory roles of microRNAs (miRNAs) in crop plants and the irreplaceability of barley in the highland area of China, we herein presented a genome-wide survey of miRNAs to reveal a potential regulatory network in the early developmental stages of two Tibetan hulless barleys, from which a total of 156 miRNAs was identified including 35 known and 121 novel ones. Six of the identified novel miRNAs were further experimentally validated. According to the evolutionary analysis, miR156, miR166, miR168, and miR171 were conserved across Tibetan hulless barleys and eight other seed plants. Expression profiles of ten known miRNAs showed that they were involved in phytohormone signaling, carbohydrate and lipid metabolism, as well as juvenile-adult transition during barley development. Moreover, a total of 1280 genes targeted by 101 miRNAs were predicted from both barley libraries. Three genes (PLN03212, MATE eukaryotic, and GRAS) were validated via the RNA ligase-mediated 5′-rapid amplification of cDNA ends (RLM-5' RACE) to be the targets of hvu-miR159a, hvu-miR166a, and hvu-miR171-3p, respectively. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of putative targets, the most abundant pathways were related to “metabolism”.These results revealed that miRNA-target pairs participating in the regulation of multigene expression and the embryonic development of Tibetan hulless barleys were controlled by complex mechanisms involving the concordant expression of different miRNAs and feedback loops among miRNAs as well as their targets. The study provides insight into the regulatory network of barley miRNAs for better understanding of miRNA functions during germination and seedling growth.     

Exploring the evolutionary differences of SBP-box genes targeted by miR156 and miR529 in plants

The combinatorial control of one target by multiple miRNAs brings big challenges to elucidate its precise evolutionary mechanism. Squamosa promoter binding protein-like (SBP) gene family exhibits the different regulatory patterns, in which some members are only regulated by miR156 and others by miR156 and miR529. Here, we explored the different evolutionary patterns and rates between miR156 targets and miR529 ones in three species (moss, rice, and maize). Our work found that the miR529 targets were members of miR156 target dataset, indicative of cooperative control. Further phylogenetic analyses as well as gene structure features demonstrated that miR529 targets derived from a monophyletic branch of miR156 targets which evolved into two independent branches duo to the ancient gene duplication. Moreover, inspection of evolutionary rate parameters (d_N/d_S, d_N and d_S) for miR156 targets and miR529 ones revealed they were under different selection strength. MiR529 targets were more constraint by strong purifying selection and evolved conservatively with a slow rate. By contrast, miR156 targets evolved more rapidly and experienced more relaxed purifying selection, which may contribute to their functional diversification. Our results will enhance the understanding of different evolutionary fates of SBP-box genes regulated by the different numbers of miRNA families before functional studies.     

MiR‐106b regulates the apoptosis and tumorigenesis of hepatocellular carcinoma via targeting Zinc finger and BTB domain‐containing protein 7A (Zbtb7a)

MicroRNAs play vital regulatory roles in various type of tumorigenesis. We aimed to explore the functional microRNAs that might play as therapeutic targets in hepatocellular carcinoma (HCC). In this study, our results revealed that microRNA‐106b was significantly increased in HCC tumor tissues. However, miR‐106b knockdown remarkably suppressed the growth and increased the apoptosis of Hub‐7 HCC cells. Biological analysis indicated that miR‐106b directly targeted toZinc finger and BTB domain‐containing protein 7A (Zbtb7a) to regulate the apoptosis of Hub‐7 cells. Extensively, Zbtb7a overexpression reversed Huh‐7 cell apoptosis and growth in vitro. Furthermore, in vivo studies confirmed that miR‐106b inhibition or Zbtb7a overexpression retarded the growth of Hub‐7 xenograft tumor in nude mice. In conclusion, we provide the evidence for the regulatory role of miR‐106b in HCC, which is causally linked to targeting of Zbtb7a. This study may provide miR‐106b as a potential therapeutic strategy for HCC.     

Profiling of MicroRNAs under Wound Treatment in Aquilaria sinensis to Identify Possible MicroRNAs Involved in Agarwood Formation

Agarwood, a kind of highly valued non-timber product across Asia, is formed only when its resource trees -- the endangered genus Aquilaria are wounded or infected by some microbes. To promote the efficiency of agarwood production and protect the wild resource of Aquilaria species, we urgently need to reveal the regulation mechanism of agarwood formation. MicroRNAs (miRNAs) are a group of gene expression regulators with overwhelming effects on a large spectrum of biological processes. However, their roles in agarwood formation remain unknown. This work aimed at identifying possible miRNAs involved in the wound induced agarwood formation. In this study, the high-throughput sequencing was adopted to identify miRNAs and monitor their expression under wound treatment in the stems of A. sinensis. The miR171, miR390, miR394, miR2111, and miR3954 families remained at the reduced level two days after the treatment. 131 homologous miRNAs in the 0.5 h library showed over three-fold variation of read number compared with the control library, of which 12 exhibiting strong expression alterations were further confirmed by real-time quantitative PCR. Target prediction and annotation of the miRNAs demonstrated that the binding, metabolic process, catalytic activity, and cellular process are the most common functions of the predicted targets of these newly identified miRNAs in A.sinensis. The cleaveage sites of three newly predicted targets were verified by 5''RACE.     

龙眼miR396分子进化特性及响应蓝光的表达分析

关于蓝光对龙眼胚性愈伤组织miRNA组学研究中发现大量响应蓝光的miRNAs,挑选关键且差异表达的miR396a-3p_2和miR396b-5p进行分子进化特性分析,以进一步明确它们在龙眼响应蓝光过程中的作用。该研究对前体和成熟体进化树、前体二级结构、启动子顺式作用元件、靶基因预测及其响应蓝光表达模式等进行分析。结果表明:(1)由5p臂上形成的miR396成熟体序列保守性较高,由3p臂上形成的miR396成熟体序列特异性较大;茎序列的保守性高于环序列,5p臂上保守性高于3p臂。(2)miR396a-3p_2和miR396b-5p的启动子主要包括光、激素、生物和非生物胁迫响应元件,可能通过这些顺式元件在龙眼响应蓝光中起调控作用;其响应蓝光的靶基因或蛋白主要包括生长素调控因子(GRF2)、LRR类受体丝氨酸(FLS2)、乙烯不敏感蛋白(EIN3)和DNA定向RNA聚合酶α亚基(rpoA)等。(3)实时荧光定量PCR结果表明,miR396a-3p_2和miR396b-5p在不同光质的表达模式存在差异;在不同光强的蓝光条件下,miR396b-5p与其靶基因的表达趋势基本呈负相关,而miR396a-3p_2与rpoA的表达趋势未呈负相关,可能存在其他miR396家族成员或miRNAs同时调控,从而实现靶基因转录水平的调控。     

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.