Mechanism underlying the effect of Liujunzi decoction on advanced-stage non-small cell lung cancer in patients after first-line chemotherapy.

OBJECTIVE: To further clarify the anticancer mechanisms of Liujunzi decoction and provide possible targets for the treatment of advanced-stage nonsmall cell lung cancer (NSCLC) by re-analyzing differential gene expression profile of peripheral blood mononuclear cells (PBMCs) from Liujunzi decoctiontreated NSCLC patients receiving first-line chemotherapy. METHODS: The PBMC gene expression microarray data set GSE61926 was retrieved from a high throughput gene expression database. Differentially expressed genes (DEGs) were screened by paired sample t-test and the multiple ratio method. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed using the DAVID database. The protein-protein interaction (PPI) network was constructed using interaction gene library retrieval tools and Cytoscape software. RESULTS: A total of 162 DEGs were identified, with 67 upregulated genes and 95 downregulated genes. The functional distribution of Gene Oncology (GO) genes showed that DEGs were mostly concentrated in extracellular regions, calcium ion binding, and transcriptase activity. KEGG pathway analysis showed that cytokine-cytokine receptor interactions were significantly enriched. PPI network analysis screened out the top 10 central protein-coding genes with the highest nodal degree: IL2, PIWIL4, DICER1, PIWIL2, SAA1, XCL1, IL22RA1, ARHGAP11A, DCP1A, and GDNF. Among them, the central protein-coding gene with the highest node degree was IL2. In addition, the central protein-coding genes with high node degrees and high molecular complex detection (MCODE) scores were PIWIL4, DICER1, PIWIL2, and DCP1A, all of which are related to tumor development. CONCLUSIONS: One signaling pathway and 10 central protein-coding genes related to anticancer mechanisms were screened by re-analysis of GSE61926 data. IL2, PIWIL4, DICER1, PIWIL2, and DCP1A may have important roles in the mechanism of Liujunzi decoction treatment against NSCLC. Our results suggest that the anticancer mechanism of Liujunzi decoction may be related to gene silencing by RNA and the biological processes of piwi-interacting RNA and other small RNAs.

RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant.

BACKGROUND: In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. METHODS: We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. RESULTS: Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). CONCLUSION: Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

Dietary sulfur amino acid restriction upregulates DICER to confer beneficial effects.

OBJECTIVE: Dietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation. METHODS: We performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets. RESULTS: We found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin. We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner. CONCLUSIONS: We propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.

Chemical enhancers of posttranscriptional gene silencing in Arabidopsis.

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.

MicroRNAs are involved in the hypothalamic leptin sensitivity.

The central nervous system monitors modifications in metabolic parameters or hormone levels (leptin) and elicits adaptive responses such as food intake and glucose homeostasis regulation. Particularly, within the hypothalamus, pro-opiomelanocortin (POMC) neurons are crucial regulators of energy balance. Consistent with a pivotal role of the melanocortin system in the control of energy homeostasis, disruption of the Pomc gene causes hyperphagia and obesity. Pomc gene expression is tightly controlled by different mechanisms. Interestingly, recent studies pointed to a key role for micro ribonucleic acid (miRNAs) in the regulation of gene expression. However, the role of miRNAs in the leptin sensitivity in hypothalamic melanocortin system has never been assessed. We developed a transgenic mouse model (PDKO) with a partial deletion of the miRNA processing enzyme DICER specifically in POMC neurons. PDKO mice exhibited a normal body weight but a decrease of food intake. Interestingly, PDKO mice had decreased metabolic rate by reduction of VO2 consumption and CO2 production which could explain that PDKO mice have normal weight while eating less. Interestingly, we observed an increase of leptin sensitivity in the POMC neurons of PDKO mice which could explain the decrease of food intake in this model. We also observed an increase in the expression of genes involved in the function of brown adipose tissue that is in polysynaptic contact with the POMC neurons. In summary, these results support the hypothesis that Dicer-derived miRNAs may be involved in the effect of leptin on POMC neurons activity.

Dicer-like and RNA-dependent RNA polymerase gene family identification and annotation in the cultivated Solanum tuberosum and its wild relative S. commersonii.

MAIN CONCLUSION: We provide advances in DCL and RDR gene diversity in Solanaceae. We also shed light on DCL and RDR gene expression in response to cold stress. DICER-like (DCL) and RNA-dependent RNA polymerase (RDR) genes form the core components to trigger small non-coding RNA (ncRNA) production. In spite of this, little is known about the two gene families in non-model plant species. As their genome sequences are now available, the cultivated potato (Solanum tuberosum) and its cold-tolerant wild relative Solanum commersonii offer a valuable opportunity to advance our understanding of the above genes. To determine the extent of diversification and evolution of DCLs and RDRs in these species, we performed a comparative analysis. Seven DCLs were identified in the two species, whereas seven and six RDR genes were found in S. tuberosum and S. commersonii, respectively. Based on phylogenetic analysis with DCLs and RDRs from several species, we provide evidence for an increase in their number in both potato species. We also disclosed that tandem duplications played a major role in the evolution of these gene families in Solanaceae. DCL and RDR expression was investigated in different tissues and under cold and virus stresses, with divergent profiles of the tandem duplicated genes being found in different tissues. DCL paralogs showed a contrasting expression in S. tuberosum and S. commersonii following cold stress and virus infection. By contrast, no change in RDR transcript activity was detected following both stresses. Overall, this study provides the first comparative genomic analysis of the core components of the RNAi machinery in Solanaceae and offers a scaffold for future functional analysis of these gene families.

Post-Translational Regulation of the Dicing Activities of Arabidopsis DICER-LIKE 3 and 4 by Inorganic Phosphate and the Redox State.

In Arabidopsis thaliana, small interfering RNAs (siRNAs) generated by two Dicer isoforms, DCL3 and DCL4, function in distinct epigenetic processes, i.e. RNA-directed DNA methylation and post-transcriptional gene silencing, respectively. Plants often respond to their environment by producing a distinct set of small RNAs; however, the mechanism for controlling the production of different siRNAs from the same dsRNA substrate remains unclear. We established a simple biochemical method to visualize the dsRNA-cleaving activities of DCL3 and DCL4 in cell-free extracts prepared from Arabidopsis seedlings. Here, we demonstrate that different nutrient statuses of a host plant affect the post-translational regulation of the dicing activity of DCL3 and DCL4. Phosphate deficiency inhibited DCL3, and the activity of DCL3 was directly activated by inorganic phosphate. Sulfur deficiency inhibited DCL4 but not DCL3, and the activity of DCL4 was recovered by supplementation of the cell-free extracts with reductants containing a thiol group. Immunopurified DCL4 was activated by recombinant Arabidopsis thioredoxin-h1 with dithiothreitol. Therefore, DCL4 is subject to redox regulation. These results demonstrate that post-translational regulation of DCL activities fine-tunes the balance between branches of the gene silencing pathway according to the growth environment.

Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Comparative analysis of the Dicer-like gene family reveals loss of miR162 target site in SmDCL1 from Salvia miltiorrhiza.

DCL1, the core component for miRNA biogenesis, is itself regulated by miR162 in Arabidopsis. MiRNA-mediated feedback regulation of AtDCL1 is important to maintain the proper level of DCL1 transcripts. However, it is unknown whether the miRNA-mediated regulation of DCL1 is conserved among plants. We analyzed the SmDCL gene family in Salvia miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies, using a comprehensive approach integrating genome-wide prediction, molecular cloning, gene expression profiling, and posttranscriptional regulation analysis. A total of five SmDCLs were identified. Comparative analysis of SmDCLs and AtDCLs showed an apparent enlargement of SmDCL introns in S. miltiorrhiza. The absence of miR162 in S. miltiorrhiza and the loss of miR162 target site in SmDCL1 were unexpectedly found. Further analysis showed that the miR162 target site was not present in DCL1 from ancient plants and was gained during plant evolution. The gained miR162 target site might be lost in a few modern plants through nucleotide mutations. Our results provide evidence for the gain and loss of miR162 and its target sites in Dicer-like genes during evolution. The data is useful for understanding the evolution of miRNA-mediated feedback regulation of DCLs in plants.

Hypothalamic miR-103 protects from hyperphagic obesity in mice.

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

Docosahexaenoic acid modulates the enterocyte Caco-2 cell expression of microRNAs involved in lipid metabolism.

Consumption of the long-chain ω-3 (n-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) is associated with a reduced risk of cardiovascular disease and greater chemoprevention. However, the mechanisms underlying the biologic effects of DHA remain unknown. It is well known that microRNAs (miRNAs) are versatile regulators of gene expression. Therefore, we aimed to determine if the beneficial effects of DHA may be modulated in part through miRNAs. Loss of dicer 1 ribonuclease type III (DICER) in enterocyte Caco-2 cells supplemented with DHA suggested that several lipid metabolism genes are modulated by miRNAs. Analysis of miRNAs predicted to target these genes revealed several miRNA candidates that are differentially modulated by fatty acids. Among the miRNAs modulated by DHA were miR-192 and miR-30c. Overexpression of either miR-192 or miR-30c in enterocyte and hepatocyte cells suggested an effect on the expression of genes related to lipid metabolism, some of which were confirmed by endogenous inhibition of these miRNAs. Our results show in enterocytes that DHA exerts its biologic effect in part by regulating genes involved in lipid metabolism and cancer. Moreover, this response is mediated through miRNA activity. We validate novel targets of miR-30c and miR-192 related to lipid metabolism and cancer including nuclear receptor corepressor 2, isocitrate dehydrogenase 1, DICER, caveolin 1, ATP-binding cassette subfamily G (white) member 4, retinoic acid receptor ß, and others. We also present evidence that in enterocytes DHA modulates the expression of regulatory factor X6 through these miRNAs. Alteration of miRNA levels by dietary components in support of their pharmacologic modulation might be valuable in adjunct therapy for dyslipidemia and other related diseases.

Mild hyperthermia enhances the expression and induces oscillations in the Dicer protein.

PURPOSE: To investigate whether mild heat stress at 39.5°C altered Dicer protein and miRNA expression patterns in several cell types. METHODS: Multiple human and mouse cell types were cultured during the course of 9 h at temperatures from 37°C to 39.5°C. Dicer mRNA levels and microRNAs were quantified by TaqMan RT-qPCR assays and Dicer protein by western blotting. RESULTS: Dicer protein was substantially elevated on western analysis in response to heat stress at 39.5°C in the absence of significant changes in Dicer mRNA by RT-qPCR. CONCLUSIONS: Heat-induced regulation of Dicer expression occurs primarily post- transcriptionally, and the expression levels of Dicer protein are increased and often oscillate in response to fever-range hyperthermia in multiple mouse and human cells. Our studies suggest a potential role for Dicer and microRNAs in the response to mild thermal stress. Additional studies on the mechanisms involved in the stress-induced oscillations of Dicer protein and microRNAs will be of interest.

Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.

In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.

Contributions of microRNA dysregulation to cisplatin resistance in adenocarcinoma cells.

Cisplatin resistance in cancer cells is due to a pleiotropic phenotype transition that allows cells to resist cell death. miRNAs have been shown to be reliable markers of phenotype, critical in cell differentiation, and dysregulated in cancer and other pathologies. Here we investigate the influence of miRNA on cisplatin resistance in KB adenocarcinoma cells. Silencing both DICER and TRBP2 in the miRNA biosynthesis pathway in KB-3-1 (sensitive parental), KB-CP.5 (cisplatin-resistant), and KB-CP20 (highly cisplatin-resistant) cells resulted in the reversal of cisplatin resistance, with no effect on cell viability in the absence of cisplatin. We found miR-181 expression differences in the cell lines using RT-PCR, with several members of the miR-181 family overexpressed in two KB cisplatin-resistant lines and in two cisplatin-resistant lung cancer lines, compared to their respective parental cells. Functional assays showed minimal effects of miR-181 on cisplatin resistance. We conclude that the miRNA biosynthesis pathway is critical for maintaining the cisplatin-resistant phenotype, but that it is difficult to determine the precise miRNAs involved in cisplatin resistance simply using expression profiles of individual miRNA species. Functional assays are needed to determine the influence of a specific miRNA and different members of the same miRNA family may have opposite effects.

Selective autophagy degrades DICER and AGO2 and regulates miRNA activity.

MicroRNAs (miRNAs) form a class of short RNAs (∼ 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum.

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.

Comparative analysis of the expressed genome of the infective juvenile entomopathogenic nematode, Heterorhabditis bacteriophora.

We report the first cDNA-sequencing project of the entomopathogenic nematode, Heterorhabditis bacteriophora. A total of 1246 expressed sequence tags (ESTs) were generated by random sequencing of clones from a cDNA library of the infective juvenile stage. The ESTs were annotated resulting in 1072 useful ESTs that were categorized into functional categories according to Kyoto Encyclopedia of Genes and Genomes. Approximately 459 of 1072 ESTs (43%) had significant similarities to annotated sequences in GenBank. Of these, 417 had significant similarities to the free-living nematode Caenorhanditis elegans proteins. Most ESTs (18%) belonged to the genetic information processing category followed by metabolism (15% ESTs) and environmental information processing (15%) pathways. Several interesting ESTs were found that may have roles in the infectivity and survival of infective juveniles. These included proteases, dauer pathway genes (akt-1, pdk-1 & daf-7) and aging and stress resistance genes such as superoxide dismutase (sod-4), heat shock genes (hsp-4 & hsp-6), and eat genes, and signaling proteins like G-protein coupled receptors, regulators of G-protein signaling (rgs), and serine/threonine kinases. Other interesting ESTs include systemic RNAi defective protein (sid-1), ribonuclease III family members (rnh-2 &rnc) and transposase gene (Tc3A). About 67% of the ESTs did not find matches in any of the searched databases suggesting potentially novel genes in this enomopathogenic nematode. Note: Sequences described in this paper have been deposited in Genbank under the accessions DN 152655-DN 152999, and DN 153000-DN 153726.

Biochemical specialization within Arabidopsis RNA silencing pathways.

In plants, the RNA silencing machinery responds to numerous inputs, including viral infection, microRNAs, and endogenous siRNAs that may act both in trans and in cis. Additionally, the full spectrum of silencing outcomes has been demonstrated in plants, ranging from mRNA degradation to repression at the level of protein synthesis to chromatin remodeling. Genetic studies in Arabidopsis have indicated that individual response pathways are functionally compartmentalized. However, to date, no biochemical systems have been available to investigate the roles of specific proteins within silencing pathways or the effects of selected mutations on the biochemical activity of those components. Here, we describe the generation of Arabidopsis extracts that reproduce many aspects of RNA silencing reactions in vitro. We find that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNA silencing pathways in Arabidopsis.