GENETIC AND FUNCTIONAL DISSECTION OF THE MIR-17-92 CLUSTER OF MIRNAS

Background: The miR-17∼92 cluster was among the first cluster of miRNAs to be linked to cancer. It is directly activated by the members of the MYC family of transcription factors and acts as a bona fide oncogene in several types of cancer, including lymphomas. miR-17∼92 is essential for mammalian development. Knockout mice die in utero or at birth with cardiovascular defects and impaired B-cell maturation. Aims: Although miR-17∼92 is important for tumorigenesis and development both the molecular mechanisms and the prominent miRNAs acting in different contexts are still unknown. The main aim of our study was to identify the role played by different members of the miR-17∼92 cluster in B-cell lymphomas and vascular development. Materials and Methods: A mouse model of B-cell lymphomas and a conditional miR-17~92 KO mouse strain were employed to investigate the role of miR-17~92 in Myc-induced cancers. A GFP-based in vitro assay allowed us to rapidly verify the oncogenic potential of each miRNA family and to determine how mutations in the seed/non seed sequence affect miRNA function. Gene-expression profiling, computational prediction and an shRNA-mediated validation screening were used to identify miR-19 targets. miR-17∼92 involvement in vascular development was studied by means of Real time, western blot, ELISA and the CD-31 (PECAM) vascular staining. Results and Conclusions : Our results suggest that the expression of two members of the cluster, miR-19a and miR-19b, is necessary and largely sufficient to recapitulate the oncogenic potential of the full miR-17∼92 cluster in Myc-driven B cell lymphomas. A systematic mutational analysis of miR-19b showed that single point mutation in the seed sequence impaired miR-19b anti-apoptotic activity. Finally, a chimeric miRNA (miR-19b seed/miR-20 non-seed) acted as effectively as miR-19b, suggesting that the specific sequence of non-seed region of miR-19 provides little additional substrate specificity. We identified the tumor suppressor PTEN as the critical miR-19b target and the principal mediator of its pro-survival activity. We also showed that miR- 17~92–null embryos have upregulation of VEGF, although it is unclear at this time whether miR-17~92 suppresses VEGF expression directly or indirectly

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season

Stercorary aseptic peritonitis due to diastatic caecal perforation: computed tomography findings.

Caecal perforation is a complication secondary to colon obstruction. It may present with insidious clinical features and may be associated with chronic constipation. The event may become severe due to the peritonitic development. We present a case of caecal perforation associated with sub-occlusive carcinoma of the left colon and hypotonic colitis caused by chronic lavative abuse, demonstrated with Computed Tomography

The mitochondrial translation machinery as a therapeutic target in Myc-driven lymphomas

The oncogenic transcription factor Myc is required for the progression and maintenance of diverse tumors. This has led to the concept that Myc itself, Myc-activated gene products, or associated biological processes might constitute prime targets for cancer therapy. Here, we present an in vivo reverse-genetic screen targeting a set of 241 Myc-activated mRNAs in mouse B-cell lymphomas, unraveling a critical role for the mitochondrial ribosomal protein (MRP) Ptcd3 in tumor maintenance. Other MRP-coding genes were also up regulated in Myc-induced lymphoma, pointing to a coordinate activation of the mitochondrial translation machinery. Inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired respiratory activity and tumor cell survival in vitro, and significantly extended lifespan in lymphoma-bearing mice. We have thus identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-overexpressing tumors

Intact p53-Dependent Responses in miR-34–Deficient Mice

<div><p>MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34–deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation <em>in vitro</em>, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34–deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc–initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.</p> </div

Response to p53 activation in miR-34^TKO/TKO mouse embryonic fibroblasts (MEFs).

<p>(A) MiR-34a and miR-34c expression in serially-passaged wild-type MEFs, as measured by qPCR. Error bars indicate 1 standard deviation (SD). (B) Cumulative population doublings of wild-type, miR-34^TKO/TKO and p53^−/− MEFs. Error bars indicate 1 SD. (C) Growth curves of wild-type and miR-34^TKO/TKO MEFs. Error bars indicate 1 SD. (D) Immunoblots of p53, p21 and Mdm2 in wild-type (W) and miR-34^TKO/TKO (K) MEFs treated with 0.2 µg/ml doxorubicin for the indicated time. (E) Expression of selected p53 targets in total RNA from doxorubicin-treated MEFs. Cells were treated with 0.2 µg/ml doxorubicin for 12 hours (Dox) or left untreated (U). Expression of the indicated genes was determined by qPCR. Error bars represent 1 SD. (F) Immunoblots showing p53 activation in three wild-type and three miR-34^TKO/TKO MEF lines. Cells were left untreated or treated with 0.2 µg/ml doxorubicin for 12 hours. (G) Time course of miR-34a and miR-34c expression in wild-type and p53^−/− cells treated with 0.2 µg/ml doxorubicin. MicroRNA expression was determined by qPCR. Error bars indicate 1 SD. (H, I) Cell cycle distribution of wild-type and miR-34^TKO/TKO MEFs. Asynchronously growing MEFs of the indicated genotype were treated with increasing doses of doxorubicin for 16 hours (H), or with 0.2 µg/ml doxorubicin for increasing time (I). Error bars indicate 1 SD. (J) Upper panel: cell cycle distribution of wild-type, miR-34^TKO/TKO, and p53^−/− MEFs after 72 hours in starvation medium (gray histogram). Starved cells were released in complete medium containing colcemid and mock-treated (light blue histogram) or exposed to 20 Gy irradiation (red histogram). Cells were analyzed by 7-AAD staining at the indicated time after release in complete medium. Lower panel: percentages of irradiated and untreated cells in G1 and G2-M phases after 24 hours in complete medium. Experiments were performed on three independent wild-type and three independent miR-34^TKO/TKO MEF lines. (K) Immunoblot detection of predicted miR-34 targets on three independent wild-type and three independent miR-34^TKO/TKO MEF lines.</p

Oncogene-induced transformation in miR-34^TKO/TKO fibroblasts and mice.

<p>(A) Representative focus formation assays of wild-type, miR-34^TKO/TKO, and p53^−/− MEFs. MEFS were infected with retroviruses expressing K-Ras^V12 alone or K-Ras^V12 and E1A. The results are representatitve of two independent experiments performed on a total of four wild-type and four miR-34^TKO/TKO MEF lines. (B) Bar plot showing the number of transformed foci. Error bars are 1 SD. (C) Survival curves of Eμ-Myc;miR-34^+/+ and Eμ-Myc;miR-34^TKO/TKO mice. P-value was calculated using the log-rank (Mantel-Cox) test. (D) Histopathology and cleaved caspase-3 (CC3) immunohistochemistry of representative lymphomas obtained from Eμ-Myc;miR-34^+/+ and Eμ-Myc;miR-34^TKO/TKO mice. (E) Bar plot showing the number of CC3-positive cells per low magnification field. Five Eμ-Myc;miR-34^+/+ tumors and and four Eμ-Myc;miR-34^TKO/TKO tumors were analyzed. Error bars indicate 1 SD.</p

p53-dependent apoptosis in thymocytes and <i>in vivo</i>.

<p>(A) Percentages of viable wild-type, miR-34^TKO/TKO, and p53^−/− thymocytes 16 hours after treatment with increasing doses of irradiation (0, 2, 4, 6, 8, and 10 Gy). Error bars represent 1 SD. (B) Percentages of viable wild-type, miR-34^TKO/TKO, and p53^−/− thymocytes 4, 8, and 24 hours after irradiation (5 Gy). Error bars correspond to 1 SD. (<b>C</b>) Expression levels of p53 transcriptional targets in the thymi and spleens of untreated (U) and irradiated (IR, 10 Gy) wild-type, miR-34^TKO/TKO and p53^−/− mice by qPCR. (D, E) Representative cleaved caspase-3 immunohistochemistry of the thymus (D) and the small intestine (E) of untreated and irradiated (10 Gy) wild-type, miR-34^TKO/TKO and p53^−/− mice (n = 3 mice per group). Brown staining indicates cleaved caspase-3 (CC3). (F,G) Quantification of apoptosis in the thymus (F) and in the intestine (G) of control and irradiated animals. In panel F the relative staining intensity averaged over three microscopic fields per sample is plotted. In panel G, the average number of CC3-positive cells per crypt is plotted. At least 25 randomly selected crypts per sample were counted. Error bars correspond to 1 SD. P values were calculated using the two-tailed Student's t-test.</p