Let-7g induces granulosa cell apoptosis by targeting MAP3K1 in the porcine ovary

Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.     

Novel lncRNA lncRNA001074 participates in the low salinity–induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3′-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

棉花miR397 LAC4模块调控棉铃虫抗性研究

miRNA(microRNA)通过调控其靶标基因在植物的生长、发育和抗逆过程中扮演着重要的角色。该研究采用分子生物学和生物化学等方法,探讨棉花miR397 LAC4参与植株木质素生物合成和对棉铃虫抗性响应机制。结果发现：(1)棉花miR397(ghr miR397)在转录后调控漆酶基因(GhLAC4)的表达,GhLAC4属于蓝铜氧化酶家族,通过调控木质素合成,抵御棉铃虫入侵棉花。(2)GUS报告基因融合表达和酶活性测定表明,ghr miR397在转录后切割靶标基因GhLAC4抑制其表达。(3)利用VIGS(virus induced gene silencing)技术在棉花中沉默和过表达ghr miR397,棉铃虫抗性检测分析表明,沉默miR397表达会增加棉花对棉铃虫的抗性,但过表达ghr miR397则会降低棉花的抗性。(4)选择性和非选择性棉铃虫实验分析、组织化学染色和木质素含量测定表明,沉默GhLAC4表达会减少木质素的积累,增加棉花对棉铃虫的敏感性。研究表明,ghr miR397 GhLAC4模块共同微调棉花木质素合成来参与棉花抗虫性调控,同时也为棉花抗虫育种提供了新思路。     

miRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

一种新型细胞微阵列的制作和应用

为了高通量地检测大量培养细胞中基因原位表达, 发明了一种制作细胞微阵列的新方法,成功地制作含20种细胞系共100个供体细胞石蜡混合物点阵的细胞微阵列.免疫组化检测P53, P21, PTEN、P16基因在细胞微阵列中的蛋白质表达.原位杂交检测BRD7、NGX6 基因在细胞微阵列中mRNA原位表达.建立了P53、P21、PTEN、P16蛋白和BRD7、NGX6 mRNA 在不同培养细胞中的原位表达谱.细胞微阵列为基因功能研究提供一种新的高通量工具.细胞微阵列可广泛用于DNA、RNA和蛋白质水平上的基因原位表达研究.细胞微阵列还可用于筛选药物作用靶标的研究.     

Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3β

Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3′-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.     

Expression of hybrid <Emphasis Type="Italic">cry</Emphasis>3aM–<Emphasis Type="Italic">lic</Emphasis>BM2 genes in transgenic potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.     

A General Mechanism for Viral Resistance to Suicide Gene Expression

Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids. Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort. T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product. Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase. Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved. A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages. A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited intermediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes. Alterations in both RNA polymerase and a second gene are thus responsible for resistance. These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing. Received: 18 July 2000 / Accepted: 8 February 2001     

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

A practical vector for efficient knockdown of gene expression in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene. These authors contributed equally to this work.     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.