Versican 3′-untranslated region (3′UTR) promotes dermal wound repair and fibroblast migration by regulating miRNA activity

Versican is an extracellular chondroitin sulfate proteoglycan which functions as a structural molecule but can also regulate a variety of cellular activities. This study was designed to explore the roles of versican in the process of dermal wound repair. To elevate levels of versican, we ectopically expressed the versican 3′-untranslated region (3′UTR) as a competitive endogenous RNA to modulate expression of versican. We demonstrated that wounds closed faster in transgenic mice expressing the versican 3′UTR, as compared to those in wildtype mice. We stably expressed versican 3′UTR in NIH3T3 fibroblasts and found that the 3′UTR-transfected cells showed increased migratory capacity relative to vector-transfected cells. Interestingly, we found that the 3′UTRs of versican and β-catenin shared common microRNAs (miRNAs) including miR-185, miR-203*, miR-690, miR-680, and miR-434-3p. Luciferase assays showed that all of these miRNAs could target the 3′UTRs of both versican and β-catenin, when the luciferase constructs contained fragments harboring the miRNA binding sites. As a consequence, expression of both versican and β-catenin was up-regulated, which was confirmed in vitro and in vivo. Transfection with small interfering RNAs (siRNAs) targeting the versican 3′UTR abolished the 3′UTR's effects on cell migration and invasion. Taken together, these results demonstrate that versican plays important roles in wound repair and that versican messenger RNAs (mRNAs) could compete with endogenous RNAs for regulating miRNA functions.     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

NOVA1 acts as an oncogene in melanoma via regulating FOXO3a expression

Increasing studies have suggested that dysregulation of RNA‐binding proteins (RBPs) contributes to cancer progression. Neuro‐oncological ventral antigen 1 (NOVA1) is a novel RBP and plays an important role in tumour development. However, the expression and role of NOVA1 in melanoma remain unknown. In this study, we indicated that NOVA1 expression was up‐regulated in melanoma samples and cell lines. Moreover, we demonstrated that knockdown of NOVA1 suppressed melanoma cell proliferation, migration and invasion in both A375 and A875 cell lines. In addition, we showed that suppressed expression of NOVA1 enhanced forkhead box O3a (FOXO3a) expression while inhibited AKT expression in melanoma cell. Furthermore, we demonstrated that inhibited expression of FoxO3A rescued NOVA1‐mediated cell proliferation, migration and invasion in melanoma cell line A375. These results suggested that NOVA1 acted as an oncogene in the development of melanoma partly through regulating FoxO3A expression.     

Tumor-suppressive activity of 1,25-dihydroxyvitamin D3 against kidney cancer cells via up-regulation of FOXO3

1,25-Dihydroxyvitamin D3 has been known to have the tumor-suppressive activity in various kinds of tumors. However, the exact effect and working mechanism of 1,25-dihydroxyvitamin D3 on the tumor-suppressive activity in human kidney cancer cells remains poorly understood. 1,25-Dihydroxyvitamin D3 has cytotoxicity to ACHN cells and inhibited ACHN cell proliferation compared to the vehicle control. 1,25-Dihydroxyvitamin D3 increased the expression of the cleaved PARP1, active Caspase3, Bax, and Bim but decreased the expression of Bcl2 in ACHN cells. Moreover, 1,25-dihydroxyvitamin D3 down-regulated the phosphorylated Akt and Erk which might lead to apoptosis through activation of FOXO3 in ACHN cells. Transfection of siRNA against FOXO3 attenuated the pro-apoptotic BimEL expression in ACHN cells treated with 1,25-dihydroxyvitamin D3. These results suggest that FOXO3 is involved in the apoptosis induced by 1,25-dihydroxyvitamin D3.     

SIRT1 regulates tyrosine hydroxylase expression and differentiation of neuroblastoma cells via FOXO3a

To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.     

Cardamonin induces G2/M arrest and apoptosis via activation of the JNK–FOXO3a pathway in breast cancer cells

Cardamonin (CD), a naturally occurring chalcone isolated from large black cardamom, was previously reported to suppress the proliferation of breast cancer cells. However, its precise molecular anti‐tumor mechanisms have not been well elucidated. In this study, we found that CD markedly inhibited the proliferation of MDA‐MB 231 and MCF‐7 breast cancer cells through the induction of G2/M arrest and apoptosis. Reactive oxygen species (ROS) plays a pivotal role in the inhibition of CD‐induced cell proliferation. Treatment with N‐acetyl‐cysteine (NAC), an ROS scavenger, blocked CD‐induced G2/M arrest and apoptosis in this study. Quenching of ROS by overexpression of catalase also blocked CD‐induced cell cycle arrest and apoptosis. We showed that CD enhanced the expression and nuclear translocation of Forkhead box O3 (FOXO3a) via upstream c‐Jun N‐terminal kinase, inducing the expression of FOXO3a and its target genes, including p21, p27, and Bim. This process led to the reduction of cyclin D1 and enhancement of activated caspase‐3 expression. The addition of NAC markedly reversed these effects, knockdown of FOXO3a using small interfering RNA also decreased CD‐induced G2/M arrest and apoptosis. In vivo, CD efficiently suppressed the growth of MDA‐MB 231 breast cancer xenograft tumors. Taken together, our data provide a molecular mechanistic rationale for CD‐induced cell cycle arrest and apoptosis in breast cancer cells.     

miR-204 functions as a tumor suppressor by regulating SIX1 in NSCLC

The involvement of miR-204 in lung cancer development is unclear. In our study, we analyzed the expression of miR-204 in tumor- and adjacent-tissue samples from 141 patients with non-small cell lung cancer (NSCLC). MiR-204 expression was decreased in tumor samples compared with non-cancerous tissue-derived controls. Moreover, miR-204 expression negatively correlated with homeobox protein SIX1 expression, tumor size and metastasis. MiR-204 silencing in miR-204-positive NSCLC cell lines promoted cell invasion and proliferation. Concomitantly, MiR-204 overexpression resulted in reduced cell proliferation and invasion, upregulated E-cadherin and downregulated N-cadherin and Vimentin expression. SIX1 was identified as a potential target of miR-204, and SIX1 silencing partially compromised the invasive and proliferative capacity of miR-204-deficient cells. Thus, miR-204 may be involved in the NSCLC development.     

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood.In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination.In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.     

Fine mapping of interactions between eEF1α protein and 3′UTR of metallothionein-1 mRNA

The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3′untranslated region (3′UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1α) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1α, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3′UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3′UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3′UTR is critical for binding of eEF1α and histidine-tRNA ligase, and that binding of eEF1α is facilitated through domains I and III.     

Human Tudor staphylococcal nuclease (Tudor-SN) protein modulates the kinetics of AGTR1-3′UTR granule formation

Human Tudor staphylococcal nuclease (Tudor-SN) interacts with the G3BP protein and is recruited into stress granules (SGs), the main type of discrete RNA-containing cytoplasmic foci structure that is formed under stress conditions. Here, we further demonstrate that Tudor-SN binds and co-localizes with AGTR1-3′UTR (3′-untranslated region of angiotensin II receptor, type 1 mRNA) into SG. Tudor-SN plays an important role in the assembly of AGTR1-3′UTR granules. Moreover, endogenous Tudor-SN knockdown can decrease the recovery kinetics of AGTR1-3′UTR granules. Collectively, our data indicate that Tudor-SN modulates the kinetics of AGTR1-3′UTR granule formation, which provides an additional biological role of Tudor-SN in RNA metabolism during stress.     

GSK‐3β inhibition protects the rat heart from the lipopolysaccharide‐induced inflammation injury via suppressing FOXO3A activity

Sepsis‐induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK‐3β is involved in the modulation of sepsis. However, the signalling details of GSK‐3β regulation in endotoxin lipopolysaccharide (LPS)‐induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK‐3β phosphorylation at its active site (Y216) and up‐regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK‐3β inhibitors and further reversed through β‐catenin knock‐down. This pharmacological inhibition of GSK‐3β attenuated the LPS‐induced cell injury via mediating β‐catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK‐3β suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS‐induced model was also blocked by inhibition of GSK‐3β, which curbed both ERK and NF‐κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP‐activated protein kinase (AMPK). Our results demonstrate that GSK‐3β inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.     

MicroRNA‐539 functions as a tumour suppressor in prostate cancer via the TGF‐β/Smad4 signalling pathway by down‐regulating DLX1

Prostate cancer (PCa) is the second leading cause of cancer‐related death in males, primarily due to its metastatic potential. The present study aims to identify the expression of microRNA‐539 (miR‐539) in PCa and further investigate its functional relevance in PCa progression both in vitro and in vivo. Initially, microarray analysis was conducted to obtain the differentially expressed gene candidates and the regulatory miRNAs, after which the possible interaction between the two was determined. Next, ectopic expression and knock‐down of the levels of miR‐539 were performed in PCa cells to identify the functional role of miR‐539 in PCa pathogenesis, followed by the measurement of E‐cadherin, vimentin, Smad4, c‐Myc, Snail1 and SLUG expression, as well as proliferation, migration and invasion of PCa cells. Finally, tumour growth was evaluated in nude mice through in vivo experiments. The results found that miR‐539 was down‐regulated and DLX1 was up‐regulated in PCa tissues and cells. miR‐539 was also found to target and negatively regulate DLX1 expression, which resulted in the inhibition of the TGF‐β/Smad4 signalling pathway. Moreover, the up‐regulation of miR‐539 or DLX1 gene silencing led to the inhibition of PCa cell proliferation, migration, invasion, EMT and tumour growth, accompanied by increased E‐cadherin expression and decreased expression of vimentin, Smad4, c‐Myc, Snail1 and SLUG. In conclusion, the overexpression of miR‐539‐mediated DLX1 inhibition could potentially impede EMT, proliferation, migration and invasion of PCa cells through the blockade of the TGF‐β/Smad4 signalling pathway, highlighting a potential miR‐539/DLX1/TGF‐β/Smad4 regulatory axis in the treatment of PCa.     

Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

PPARγ activation induces autophagy in breast cancer cells

It has been previously shown that PPARγ ligands induce apoptotic cell death in a variety of cancer cells. Given the evidence that these ligands have a receptor-independent function, we further examined the specific role of PPARγ activation in this biological process. Surprisingly, we failed to demonstrate that MDA-MB-231 breast cancer cells undergo apoptosis when treated with sub-saturation doses of troglitazone and rosiglitazone, which are synthetic PPARγ ligands. Acridine orange (AO) staining showed acidic vesicular formation within ligand-treated cells, indicative of autophagic activity. This was confirmed by autophagosome formation as indicated by redistribution of LC3, an autophagy-specific protein, and the appearance of double-membrane autophagic vacuoles by electron microscopy following exposure to ligand. To determine the mechanism by which PPARγ induces autophagy, we transduced primary mammary epithelial cells with a constitutively active mutant of PPARγ and screened gene expression associated with PPARγ activation by genome-wide array analysis. HIF1α and BNIP3 were among 42 genes up-regulated by active PPARγ. Activation of PPARγ induced HIF1α and BNIP3 protein and mRNA abundance. HIF1α knockdown by shRNA abolished the autophagosome formation induced by PPARγ activation. In summary, our data shows a specific induction of autophagy by PPARγ activation in breast cancer cells providing an understanding of distinct roles of PPARγ in tumorigenesis.