miR-217 inhibits invasion of hepatocellular carcinoma cells through direct suppression of E2F3

The poor prognosis of hepatocellular carcinoma (HCC) is mainly due to the development of invasion and metastasis. Recent data strongly suggests the important role of miRNAs in cancer progression, including invasion and metastasis. Here, we found miR-217 expression was much lower in highly invasive MHCC-97H HCC cells and metastatic HCC tissues. Restored miR-217 expression with miR-217 mimics inhibited invasion of MHCC-97H cells. Inversely, miR-217 inhibition enhanced the invasive ability of Huh7 and MHCC-97L cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated E2F3 was a novel direct target of miR-217. Moreover, E2F3 protein level was positively associated with HCC metastasis and functional analysis confirmed the positive role of E2F3 in HCC cell invasion. Our findings suggest miR-217 function as a potential tumor suppressor in HCC progression and miR-217-E2F3 axis may be a novel candidate for developing rational therapeutic strategies.     

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-β signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1-155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-β-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA(12)LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-β by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3'-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-β with possible implications in several human diseases where homeostasis of TGF-β might be altered.     

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells

MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.     

miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.     

lncRNA NEAT1 facilitates melanoma cell proliferation,migration, and invasion via regulating miR-495-3p and E2F3

Melanoma contributes a lot to skin cancer-related deaths. lncRNAs are implicated in various diseases, including melanoma. lncRNA NEAT1 is frequently dysregulated and can play important roles in multiple cancers. Nevertheless, little has been studied about the function of NEAT1 in melanoma progression. In our present research, we displayed NEAT1 was overexpressed in melanoma cells. A series of functional assays showed that overexpression of NEAT1 promoted the proliferation, migration, and invasion of melanoma cells. By contrast, NEAT1 knockdown obviously restrained melanoma cell progression. Mechanistically, it was revealed that NEAT1 could directly bind with miR-495-3p, which led to a negative effect on miR-495-3p levels. In addition, miR-495-3p was significantly decreased in melanoma cells. Furthermore, E2F3 was postulated as the target of miR-495-3p and overexpression of this miR could suppress the levels of E2F3. Meanwhile, it was exhibited that melanoma cell proliferation, migration, and invasion induced by E2F3 silence was abrogated by miR-495-3p. Moreover, an in vivo xenograft nude mice model was established using A375 cells and it was indicated that NEAT1 promoted melanoma progression in vivo via regulating the miR-495-3p/E2F3 axis. In conclusion, we suggest that NEAT1 exerts an oncogenic effect on melanoma development via inhibition of miR-495-3p and induction of E2F3. NEAT1 might serve as a crucial prognostic biomarker of melanoma.