DataSheet_1_mRNA-miRNA networks identify metabolic pathways associated to the anti-tumorigenic effect of thyroid hormone on preneoplastic nodules and hepatocellular carcinoma.zip

BackgroundThyroid hormones (THs) inhibit hepatocellular carcinoma (HCC) through different mechanisms. However, whether microRNAs play a role in the antitumorigenic effect of THs remains unknown.MethodsBy next generation sequencing (NGS) we performed a comprehensive comparative miRNomic and transcriptomic analysis of rat hepatic preneoplastic lesions exposed or not to a short-term treatment with triiodothyronine (T3). The expression of the most deregulated miRs was also investigated in rat HCCs, and in human hepatoma cell lines, treated or not with T3.ResultsAmong miRs down-regulated in preneoplastic nodules following T3, co-expression networks revealed those targeting thyroid hormone receptor-β (Thrβ) and deiodinase1, and Oxidative Phosphorylation. On the other hand, miRs targeting members of the Nrf2 Oxidative Pathway, Glycolysis, Pentose Phosphate Pathway and Proline biosynthesis – all involved in the metabolic reprogramming displayed by preneoplastic lesions– were up-regulated. Notably, while the expression of most miRs deregulated in preneoplastic lesions was not altered in HCC or in hepatoma cells, miR-182, a miR known to target Dio1 and mitochondrial complexes, was down-deregulated by T3 treatment at all stages of hepatocarcinogenesis and in hepatocarcinoma cell lines. In support to the possible critical role of miR-182 in hepatocarcinogenesis, exogenous expression of this miR significantly impaired the inhibitory effect of T3 on the clonogenic growth capacity of human HCC cells.ConclusionsThis work identified several miRNAs, so far never associated to T3. In addition, the precise definition of the miRNA-mRNA networks elicited by T3 treatment gained in this study may provide a better understanding of the key regulatory events underlying the inhibitory effect of T3 on HCC development. In this context, T3-induced down-regulation of miR-182 appears as a promising tool.</p

(A) TSA breast cancer cells were subcutaneously injected in WT and Sema4D KO mice

Tumor volume was evaluated at the indicated days. As shown, tumor burden was dramatically less in Sema4D KO versus WT mice. Bars indicate mean tumor volume ± SEM. *, P < 0.0001 ( = 10 mice per group). (B and C) Macro- and micrometastases present in the lungs of mice bearing tumors of comparable size were evaluated as indicated in Materials and methods and scored. Error bars indicate mean ± SD. *, P < 0.0001 ( = 10 lungs). (D) Representative pictures of lungs and lung slices for WT and Sema4D KO mice. As shown, the number of metastases was significantly lower in Sema4D KO mice. Statistical differences between groups were determined by using the two-tailed Student's test. Bars: (whole mount) 0.5 cm; (micrograph) 0.2 mm.<p><b>Copyright information:</b></p><p>Taken from "Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1673-1685.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442644.</p><p></p

(A and B) Immunofluorescence analysis of tumors grown in WT or Sema4D KO animals

In A, staining with CD45 and F4/80 (specific for macrophages) reveals that most of the leukocytes in the tumor stroma are macrophages (∼90%; not depicted). In B, staining with F4/80 and Sema4D antibody reveals that TAMs express Sema4D. Bars, 50 μm. (C) Western blot analysis of total cell lysates of TAMs derived from WT or Sema4D KO mice. The blot was probed with anti-Sema4D antibodies and, as a loading control, with anti–β-actin. (D) PCR analysis performed on RNAs obtained from peritoneal macrophages of control mice (left) or of mice injected i.p. with CFA. As shown, only activated macrophage express Sema4D. (E) Transwell motility assays. The ability of supernatants of cultures of TAMs, purified from tumors grown either in WT or KO mice, to promote EC (HUVEC) migration was evaluated. Supernatants of WT TAMs efficiently promoted EC migration, whereas supernatants of KO TAMs were significantly less effective. Moreover, the chemoattractive activity of the WT supernatant was significantly decreased in the presence of Sema4D or plexin B1 blocking antibodies, and of the c-Met inhibitor PHA-665752. Chemoattractive positive controls included Sema4D (4D) and HGF, whereas anti-VSV was used as a control antibody. Bars represent the mean ± SD (experiments were performed twice in triplicate). Statistical differences between groups were determined by using the two-tailed Student's test. *, P = 0.0003; **, P = 0.0002; ***, P = 0.0013; and #, P = 0.0001.<p><b>Copyright information:</b></p><p>Taken from "Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1673-1685.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442644.</p><p></p

(A) Matrigel plugs containing either WT or KO TAMs were subcutaneously implanted in KO mice

After 14 d, mice received dextran-FITC and were killed, and plugs were microscopically analyzed. As shown, plugs containing WT TAMs were well vascularized and showed well-formed and branched vessels, whereas plugs containing KO TAMs displayed small and poorly organized vessels. Addition of blocking plexin B1 antibodies, as well as of the c-Met inhibitor PHA-665752, resulted in the lack of an organized vessel tree. Plugs containing PBS were used as a negative control, whereas plugs containing known angiogenic factors (HGF and b-FGF) were used as positive controls. ( = 3 plugs per group). Bar, 2 mm. (B) TSA tumor cells were injected in KO mice alone or together with TAMs obtained either from WT or KO animals. TSA cells alone were also injected in WT mice as a control. Coinjection of TSA cells and WT TAMs resulted in the formation of significantly larger tumor masses in comparison to those originated by TSA plus KO TAMs. Bars represent the mean ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages"</p><p></p><p>The Journal of Experimental Medicine 2008;205(7):1673-1685.</p><p>Published online 7 Jul 2008</p><p>PMCID:PMC2442644.</p><p></p