De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of β-glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.
Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.
Long noncoding RNAs (lncRNAs) are a class of functional non-coding transcripts that are longer than 200 nt and regulate gene expression via diverse mechanisms in eukaryotes. In fact, they have emerged as critical epigenetic and transcriptional regulators of autophagy in mammals in response to various stressors. Autophagy not only plays a crucial role in maintaining cellular homeostasis, but it is also essential to immunity, targets intracellular pathogens for degradation, modulates inflammation, and participates in adaptive immune responses. However, the expression profile of lncRNA and its role in regulating autophagy in macrophages have been poorly defined. Here, we used transcriptomic and bioinformatics to analysis LncRNA expression profile during autophagy and functional studies to evaluate the function of the metastasis-associated lung adenocarcinoma transcript-1 ( Malat1 ) lncRNA in macrophages. A total of 1112 putative lncRNAs (240 novel lncRNAs) were identified, including 831 large intergenic, 129 intronic, and 152 anti-sense lncRNA, of which 59 differentially expressed transcripts exhibited a greater than 1.5-fold change under different conditions. The interaction of Malat1 lncRNA with microRNA ( mir ) -23-3p and lysosomal-associated membrane protein 1 ( Lamp1 ) was found, Malat1 releases inhibition of Lamp1 expression in macrophages through competitive adsorption of mir-23-3p . The results of this study provide a better understanding of lncRNA function in macrophages and a basis for further investigation into the roles and mechanisms of ncRNA in immunology, particularly the functions of Malat1 and mir-23-3p in the pathogenesis of macrophages.
Colorectal cancer (CRC) is one of the most malignant cancers with high morbidity and mortality. MicroRNAs (miRNAs) are small non-coding RNAs that affect biological processes by binding to mRNAs and regulating their expression, and epigenetic alterations including miRNA dysregulation are significantly involved in CRC development. Determining the effect of the miRNA-mRNA network on CRC could be helpful for developing novel therapeutic targets and prognostic biomarkers, and even improving survival. In this study, microarray assays were used to screen differentially expressed miRNAs (DE miRNAs) and mRNAs (DE mRNAs) in CRC and the adjacent normal tissues. Among the detected genes, 42 miRNAs and 142 mRNAs were significantly upregulated in CRC, while 23 miRNAs and 279 mRNAs were significantly downregulated. Through overlapping of predicted targets of DE miRNAs and anti-expressed DE mRNAs, networks of DE miRNAs and DE mRNAs in CRC were established. Additionally, the formation of a protein-protein interaction network of DE mRNAs possibly targeted by DE miRNAs, functional annotation and pathway analysis, stable subnetwork mining, and determination of hub genes provided the probable mechanism used by DE miRNAs and DE mRNAs to regulate CRC growth. Finally, validation of expression and prognostic potential of hub genes provided further support for the results above and indicated that CCL-28, GPR15, PNOC, NUSAP1, and their interacted miRNAs may be a potential signature for prognosis of CRC patients. In sum, we successfully established miRNA-mRNA regulatory networks based on microarray results targeting CRC, and these findings may elucidate the mechanisms used for CRC growth and identify miRNA-related signatures for prognosis and treatment of CRC.
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