HMGA1-pseudogene expression is induced in human pituitary tumors

Numerous studies have established that High Mobility Group A (HMGA) proteins play a pivotal role on the onset of human pituitary tumors. They are overexpressed in pituitary tumors, and, consistently, transgenic mice overexpressing either the Hmga1 or the Hmga2 gene develop pituitary tumors. In contrast with HMGA2, HMGA1 overexpression is not related to any rearrangement or amplification of the HMGA1 locus in these tumors. We have recently identified 2 HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, acting as competitive endogenous RNA decoys for HMGA1 and other cancer related genes. Here, we show that HMGA1 pseudogene expression significantly correlates with HMGA1 mRNA levels in growth hormone and nonfunctioning pituitary adenomas likely inhibiting the repression of HMGA1 through microRNAs action. According to our functional studies, these HMGA1 pseudogenes enhance the proliferation and migration of the mouse pituitary tumor cell line, at least in part, through their upregulation. Our results point out that the overexpression of HMGA1P6 and HMGA1P7 could contribute to increase HMGA1 levels in human pituitary tumors, and then to pituitary tumorigenesis.     

Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.     

miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.     

The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.     

Let-7 Prevents Early Cancer Progression by Suppressing Expression of the Embryonic Gene HMGA2

The microRNA let-7 regulates late embryonic development by suppressing expression of a number of genes such as c-myc and RAS as well as the embryonic gene high mobility group, A2 (HMGA2). We now demonstrate that HMGA2 is more efficiently targeted by let-7 than RAS. Its expression inversely correlates with the expression of let-7 in the NCI60 cells lines, and the expression of RAS does not change when amounts of let-7 that efficiently silence expression of HMGA2 are introduced into tumor cells. We did not find a difference in the expression of HMGA2 between primary ovarian cancer samples and matching metastases, suggesting that the expression of HMGA2 represents an early event during cancer progression. The late repression of HMGA2 by let-7 during embryonic development, and the early reexpression of HMGA2 during cancer development, is in line with the hypothesis that cancer development represents a case of reverse embryogenesis.     

Up-regulation of KISS1 as a novel target of Let-7i in melanoma serves as a potential suppressor of migration and proliferation in vitro

Melanoma is a kind of skin cancer that is begun by the alteration of melanocytes. miRNAs are small non-coding RNA molecules that regulate a variety of biological processes. KISS1, the metastasis-suppressor gene, encodes kisspeptins which inhibits migration and proliferation of cancers. This study was aimed to determine the role of Let-7i and KISS1 in melanoma cell migration and proliferation. At first, the expression of Let-7i and KISS1 was determined in patients with melanoma. In the in vitro part of the study, Let-7i mimics were transfected and the impact of its restoration on target gene expression, proliferation, migration and apoptosis of SK-MEL-3 melanoma cell line was assessed by real-time PCR and Western blotting, MTT assay, wound-healing assay and flow cytometry, respectively. Besides, KISS1 inhibitor siRNA alone and along with Let-7i was transfected to determine their probable correlation. The results revealed that either Let-7i or KISS1 were down-regulated in patients with melanoma. The results obtained from the in vitro part of the study revealed that restoration of Let-7i reduced the expression of metastasis- and proliferation-related target genes. Moreover, it was revealed that up-regulation of Let-7i attenuated migration and proliferation capability of SK-MEL-3 cells. Besides, it was demonstrated that Let-7i restoration induced apoptosis in melanoma cells. More importantly, the KISS1 inhibitor caused a prominent cell migration and proliferation, attenuated by Let-7i re-expression. To sum up, the present study revealed the impressive role of Let-7i restoration along with its correlation with KISS1 on melanoma carcinogenicity which may be applicable in future in vivo studies.     

有翅和无翅豌豆蚜中翅型分化信号通路相关微小RNA及其靶基因的表达差异

【目的】前期研究发现麦长管蚜Sitobion avenae孤雌蚜有翅和无翅个体中存在很多差异表达的微小RNA(microRNA, miRNA),本研究旨在进一步明确这些miRNA在豌豆蚜Acyrthosiphon pisum中发挥作用的发育阶段,探索miRNA调控孤雌蚜翅两型性分化的机制。【方法】选择在麦长管蚜有翅蚜和无翅蚜中显著差异表达,且靶基因为蜕皮激素、胰岛素信号通路及翅型发育关键基因的5个miRNA(Let-7,miR-92a, miR-92b, miR-92a-1-p5和miR-277),利用qPCR检测这些miRNA及其靶标基因在豌豆蚜3-4龄若蚜和成虫有翅和无翅个体中的表达谱;同时利用双荧光素酶活性检测法对上述miRNA的靶基因进行验证。【结果】表达谱分析发现,这5个miRNA在豌豆蚜成虫中表达量均高于其在若蚜中的表达量,而其预测的靶基因在4龄若蚜中的表达量均高于其在成虫中的表达量,表明miRNA对其靶基因的调控作用可能集中在成虫阶段。分析豌豆蚜有翅和无翅个体中5个miRNA的表达情况发现,在成虫有翅个体中5个miRNA的表达量均高于无翅个体中的,其中miR-277表达差异最显著,成虫有翅个体中的表达量是无翅个体中表达量的7.5倍;其次为Let-7,表达差异达3倍。而Let-7在3龄有翅若蚜和无翅若蚜中表达差异最显著,有翅个体中的表达量是无翅个体中的37.8倍;其次为miR-277,表达差异达7.6倍。比较5个miRNA与其靶基因在豌豆蚜3-4龄若蚜及成虫有翅和无翅个体中的表达发现,miRNA Let-7和miR-92b的表达趋势分别与其靶基因abrupt和Foxo的基本相反。荧光素酶活性检测结果显示,Let-7的真实靶标为abrupt,共转染Let-7模拟物后与对照相比,荧光素酶活性下降53%,达极显著水平。其他miRNA与靶标基因的互作不显著。【结论】首次发现miRNA对豌豆蚜孤雌蚜翅型分化相关基因的调控可能发生在成虫阶段。Let-7可能通过调控abrupt基因参与孤雌蚜翅型分化。该研究为进一步探索miRNA参与孤雌蚜翅两型性分化的机制奠定了基础。     

Target Repression Induced by Endogenous microRNAs: Large Differences,Small Effects

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.     

Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production

IL-23R gene variants have been identified as risk factors for two major inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, but how they contribute to disease is poorly understood. In this study, we show that the rs10889677 variant in the 3'-untranslated region of the IL-23R gene displays enhanced levels of both mRNA and protein production of IL-23R. This can be attributed to a loss of binding capacity for the microRNAs (miRNAs) Let-7e and Let-7f by the variant allele. Indeed, inhibition and overexpression of these miRNAs influenced the expression of the wild type but not the variant allele. Our data clearly demonstrate a role for miRNA-mediated dysregulation of IL-23R signaling, correlated with a single nucleotide polymorphism in the IL-23R strongly associated with IBD susceptibility. This implies that this mutation, in combination with other genetic risk factors, can lead to disease through sustained IL-23R signaling, contributing to the chronicity of IBD.