海马中特异性敲除Dicer1蛋白的小鼠的构建和表型

目的：在小鼠海马中特异性敲除RNA酶Ⅲ酶（RNAase Ⅲenzyme）Dicer1,并初步观察Dicer1在小鼠海马的生长发育中起到的基本作用。方法：用在海马中特异性表达Cre酶的Frizzled9-CreERTM小鼠和Dicer1（flox/flox）小鼠交配,得到Dicer1（flox/＋）;Frizzled9-CreERTM小鼠,并再次与Dicer1（flox/flox）交配,得到Dicer1（flox/flox）;Frizzled9-CreERTM小鼠,用他莫昔芬（TM）诱导Cre酶的表达,使Dicer1在海马中特异性敲除,观察小鼠的表型。结果：TM诱导的Dicer1全敲的小鼠出生率低,大部分在出生后40天左右死亡,海马中CA3区域变薄,猜测与海马中缺乏Dicer1酶有关。结论：Dicer1是miRNA产生过程中的重要因素,在海马中敲除Dicer1后影响小鼠发育,小鼠易猝死。     

Molecular characterization and gene functional analysis of Dicer‐2 gene from Nilaparvata lugens (Hemiptera: Geometroidea)

Abstract Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer‐2 (Dcr‐2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full‐length complementary DNA (cDNA) of N. lugens Dcr‐2 (NlDcr‐2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr‐2 gene. The complete Dcr‐2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr‐2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr‐2 was significantly reduced by NlDcr‐2 double‐stranded RNA (dsRNA) (dsDcr‐2). Fifty‐five per cent decrease of NlDcr‐2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion.     

Silencing of Dicer1 temporally separates pro- and anti-apoptotic signaling and confers susceptibility to chemotherapy in p53 mutated cells

miRNAs are critically implicated in the initiation process of and progression through cancerogenesis. The mechanisms, however, by which miRNAs interfere with the signalosomes of human cancer cells, are still obscure. We utilized the p53-mutated human keratinocyte cell line HACAT to investigate the biological significance and extent to which miRNAs regulate proliferation, cell growth, and apoptosis in transformed phenotypes. Silencing of the miRNA-processing enzyme Dicer1 resulted in cell cycle arrest at the G₁/S border, along with restoration of CDK inhibitor p21^CIPexpression. Employing a cell cycle-wide phospho-proteomic approach, we detected neglectable changes in abundance and schedule of overall and cell cycle periodic protein expression despite cell cycle arrest of Dicer1-depleted cells. Instead, we found substantially delayed post-translational modifications of some, but not all, signaling nodes. Phospho-site-specific analyses revealed that pro-apoptotic information elicited by Myc, β-catenin, and other mitotic pathways early in G₁ are absorbed and balanced by anti-apoptotic signaling from AKT and NFκB in Dicer1-competent cells. The absence of regulatory miRNAs, however, led to a substantial delay of anti-apoptotic signaling, leaving pro-apoptotic stress unbalanced in Dicer1-deprived cells. We here show that this temporal separation of pro- and anti-apoptotic signaling induced by inhibition of Dicer1 is synergistic and synthetic lethal to low-dose 5-FU chemotherapy in p53-mutated HACAT cells. The findings reported here contribute to the understanding of the complex interactions of miRNAs with the signalosom of transformed phenotypes and may help to design novel strategies to fight cancer.     

Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

Micro RNAs（mi RNAs） are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 mi RNAs in brown（BAT） and white adipose tissue（WAT） during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six mi RNAs including let-7a, let-7b, mi R-107, mi R-150, mi R-222 and mi R-31 was significantly downregulated in WAT（P 〈 0.05）, which was 16%–54% of euthermic non-torpid control squirrels,whereas expression of three mi RNAs including mi R-143, mi R-200 a and mi R-519 d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more mi RNAs was downregulated in BAT during torpor. We detected reduced expression of 6 mi RNAs including mi R-103 a, mi R-107, mi R-125 b, mi R-21, mi R-221 and mi R-31（48%–70% of control）, while only expression of mi R-138 was significantly upregulated（2.91 ± 0.8-fold of the control, P 〈 0.05）. Interestingly,mi RNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas mi RNAs with altered expression in BAT during torpor were linked to mitochondrial b-oxidation. mi RPath target prediction analysis showed that mi RNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase（MAPK） signaling, while the mi RNAs upregulated in WAT were linked to transforming growth factor b（TGFb） signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-mi RNAs for the mi RNAs used in this study, suggesting no structure-influenced changes in pre-mi RNA processing efficiency in the squirrel. As well, the expression of mi RNA processingenzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of mi RNA expression in adipose tissues may be linked     

microRNA-215异常激活抑制Dicer1促进肝癌细胞迁移和转化

microRNA异常表达促进癌症的发生发展.本研究通过microRNA表达谱分析2个肝癌细胞和2个正常细胞microRNA的表达,寻找与肝癌相关的microRNA,发现microRNA-215在肝癌细胞中高表达,q RT-PCR验证microRNA-215在肝癌细胞呈显著高表达.进一步研究发现,microRNA-215直接靶向Dicer1基因的3′UTR并抑制Dicer1蛋白表达,Dicer1是microRNA加工成熟过程中必需的蛋白.过表达microRNA-215抑制Dicer1从而促进肝癌细胞迁移和转化,而抑制microRNA-215表达起相反作用.Dicer1抑制后,许多抑癌microRNA表达被抑制,从而促进迁移和转化.相对于癌旁组织,Dicer1在肝癌组织呈明显低表达.本研究揭示,microRNA-215异常活化并抑制Dicer1表达与肝癌发展相关.     

Reduction of protein kinase C α (PKC‐α) promote apoptosis via down‐regulation of Dicer in bladder cancer

In clinic, we examined the expression of protein kinase C (PKC)‐α and Dicer in the samples of bladder cancer patients, and found that the two proteins have a line correlation. Our study confirmed this correlation existing by clearing the decreasing expression of Dicer after the PKC‐α knockdown. Treatment of bladder cancer cell lines (T24, 5637) with the PKC‐α or Dicer knockdown and the PKC inhibitors (Calphostin C and Gö 6976) can promote the apoptosis. Inhibition of PKC can increase the activities of caspase‐3 and PARP, however, decrease the expression of Dicer. And knockdown of the PKC‐α or Dicer can also activate the caspase‐3 or the PARP. Considering the reduction of PKC‐α can induce the Dicer down‐regulation, we make the conclusion that the reduction of PKC‐α can promote the apoptosis via the down‐regulation of Dicer in bladder cancer.