AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea.
Up to now, miRBase database (20.0, ) contains 24, 521 miRNA loci from 206 species and 30, 424 mature miRNA products. However, there is still no report of miRNAs from S. miltiorrhiza. Identification of the tissue-specific miRNAs and their target genes will help understand their role in a variety of metabolic pathways. Therefore, the goal of this study is to identify tissue-specific miRNAs and their potential targets in S. miltiorrhiza, and provide valuable information for future studies of the miRNA-mediated biosynthesis of tanshinones.
Mitogen-activated protein kinase (MAPK) and Akt signaling pathways play key roles in the anticancer effects of melatonin.
AIMS: The present study investigated whether MAPK and Akt signaling pathways are involved in melatonin's antiproliferative actions on the human MG-63 osteosarcoma cells.
METHODS/RESULTS: Western blot analysis confirmed that melatonin significantly inhibited phosphorylation of ERK1/2 but not p38, JNK, or Akt.
Co-immunoprecipitation further confirmed that there was an interaction between p-ERK1/2 and cyclin D1, CDK4, cyclin B1, or CDK1, which was blunted in the presence of melatonin or PD98059.
CONCLUSION: These findings suggest that melatonin's antiproliferative action is mediated by inhibition of the ERK1/2 signaling pathway rather than the p38, JNK, or Akt pathways.
Identification of the targets of these identified miRNAs will help understand their role in a variety of metabolic pathways. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified, and 29 were homologous with the genes that have already been found in other plants (). Fortunately, one target of miR5072, acetyl-CoA C-acetyltransferase, was identified in S. miltiorrhiza, and related to the biosynthesis of terpenoid compounds and tanshinones in plant (). Tanshinones are abietane-type norditerpenoid quinones identified in root of S. miltiorrhiza, which mainly including tanshinone I, tanshinone IIA, dihydrotanshinone I and cryptotanshinone, and showing diverse pharmacological activities, such as antibacterial, antioxidant, antiinflammatory, cytotoxic, neuroprotective, cardioprotective, antiplatelet, and antitumor effects , . In plant, diterpenoids are generated from geranylgeranyl diphosphate, which is synthesized from isopentenyl diphosphate and its allylic isomer dimethylallyldiphosphate via two different pathways, the mevalonic acid (MVA) pathway in the cytosol and the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway in cellular plastids –. Generally, the main MVA derived isoprenoid end-products are certain sesquiterpenes, sterols and the side chain of mitochondrial ubiquinones, whereas monoterpenes, certain sesquiterpenes and photosynthesis-related isoprenoids, are derived from the DXP pathway . The two pathways are not separated absolutely. In some extents, there are some forms of crosstalk between them. The isoprenoids generated from MVA can get into the plastid carried by membrane and form monoterpene or diterpene . In the MVA pathway, two units of acetyl-CoA are first catalyzed to acetoacetyl-CoA by acetyl-CoA C-acetyltransferase, and then catalyzed to mevalonate by 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) . Considering the important role of acetyl-CoA C-acetyltransferase in the initial reaction of MVA pathway, miR5072 must have involved in regulating the biosynthesis of tanshinones in S. miltiorrhiza. In addition, acetyl-CoA C-acetyltransferase was also involved in the biosynthesis of a variety of products –, indicating the other crucial role of miR5072 in S. miltiorrhiza.
Analyzing the temporal and spatial expression patterns of miRNAs would provide useful information about their molecular functions. In plants, more and more evidence showed that miRNAs have differential expression in specific developmental stages and tissues . For example, miR159 mainly expressed in the leaf of potato, and were considered to have crucial function in leaf development . MiR164 mainly expressed in roots of several plant species, and showed essential role in plant root development through their NAC transcription factor targets-mediated downstream pathways , . In addition, recent studies also showed that miR166 mainly expressed in barley roots, miR171 mainly expressed in opium poppy roots, miR397 mainly expressed in opium poppy leaves, and miR156 and miR408 mainly expressed in barley leaves , , which indicated their important roles in plant specific tissues growth and development . In the present study, based on the deep sequencing counts, many miRNAs showed tissue-specific expression in S. miltiorrhiza. Among them, 62, 95, 19 and 71 miRNAs only express in root, stem, leaf and flower, respectively (). In addition, miR156 and miR167 were highly abundant in flower and leaf, miR164 were highly abundant in flower and root, and miR166 were highly abundant in all tissues, especially in flower (), which were quite different from the patterns found in other plants , . These suggested that besides some common mechanism sharing with different plant species, there were species-specific miRNA regulatory mechanisms in S. miltiorrhiza.
Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.">
The release of cytochrome from mitochondriainto the cytosol is one of the major apoptosis pathways (). To elucidate whether cytochrome release in TSIIA-induced apoptosis, we determined thesubcellular localization of cytochrome byimmunofluorescent labeling. As shown in , the staining pattern becamediffuse in most cells treated with 20 μM TSIIA for 48 h, consistentwith a translocation of cytochrome into the cytosol,whereas, cytochrome displayed a dotted pattern inuntreated cells, consistent with its location within themitochondria. The condensation and fragmentation of the chromatinof the nucleus was also observed by Hoechst 33258 in these cellswith diffuse cytochrome staining.
The change of mitochondrial morphology anddecreasing of MMP are associated with mitochondrial damage linkedto apoptosis (). Thus, we nextevaluated the effect of TSIIA on mitochondria. Cell shrinkage andcytoplasm vacuolization were observed in TSIIA-treated A549 cells(). As one of threeresults shown in ,after treatment with DMSO (0.1%) or TSIIA (20 μM), the JC-1red/green fuorescent ratio in A549 cells was 5.83±0.862 and1.14±0.156, respectively, suggesting that TSIIA induces a markeddecrease of MMP in A549 cells (p). As previous studies showedthat TSIIA significantly induced cell apoptosis on a panel of humantumor cell lines, we thought that TSIIA could induce NSCLC A549cell apoptosis through the mitochondria pathway.
Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.
Furthermore, the inhibition of USP39 expression decreased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, indicating that ERK signaling pathways might be involved in the regulation of melanoma cell proliferation by USP39.
Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy- d -xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity.