Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization.

BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

miR-93-5p suppresses cellular senescence by directly targeting Bcl-w and p21.

Cellular senescence, a distinctive type of irreversible growth arrest, develops in response to various stimuli. Bcl-w, an oncogene and member of the Bcl-2 family, has been reported to promote tumorigenicity in various cancer cells. Here, we sought to explore the potential role of Bcl-w in premature senescence, which has received relatively little research attention. Our findings demonstrate that Bcl-w enhances the activity of senescence-associated ß-galactosidase (SA-ß-gal) and promotes histone H3 tri-methylation at lysine 9 (H3K9me3) and expressions of p53, Notch2, p21, and p16-hallmarks of the senescent phenotype-in human U251 glioblastoma and H460 lung carcinoma cells. It is also known that microRNAs (miRNAs) regulate processes related to tumor development, such as cell proliferation, differentiation, survival, metabolism, inflammation, invasion, angiogenesis, and senescence. In this context, we found that miR-93-5p inhibited premature cellular senescence by directly suppressing Bcl-w and p21 expressions. Collectively, these findings suggest that targeting miR-93-5p-regulated Bcl-w may be a useful strategy for preventing premature senescence.

miR-181b-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization by directly targeting YWHAG.

Epithelial-mesenchymal transition (EMT) is essential for increased invasion and metastasis during cancer progression. Among the candidate EMT-regulating microRNAs that we previously identified, miR-181b-3p was found to induce EMT in MCF7 breast cancer cells, as indicated by an EMT-characteristic morphological change, increased invasiveness, and altered expression of an EMT marker. Transfection with a miR-181b-3p inhibitor reduced the expression of mesenchymal markers and the migration and invasion of highly invasive breast cancer cells. miR-181b-3p induced the upregulation of Snail, a master EMT inducer and transcriptional repressor of E-cadherin, through protein stabilization. YWHAG was identified as a direct target of miR-181b-3p, downregulation of which induced Snail stabilization and EMT phenotypes. Ectopic expression of YWHAG abrogated the effect of miR-181b-3p, including Snail stabilization and the promotion of invasion. In situ hybridization and immunohistochemical analyses indicated that YWHAG expression was inversely correlated with the expression of miR-181b-3p and Snail in human breast cancer tissues. Furthermore, transfection with miR-181b-3p increased the frequency of metastatic nodule formation in the lungs of mice in experimental metastasis assays using MDA-MB-231 cells. Taken together, our data suggest that miR-181b-3p functions as a metastasis activator by promoting Snail-induced EMT, and may therefore be a therapeutic target in metastatic cancers.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.

Ionizing radiation-inducible miR-494 promotes glioma cell invasion through EGFR stabilization by targeting p190B rhoGAP.

MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3'-untranslated region (3'UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy.

3-Hydroxy-3',4'-dimethoxyflavone suppresses Bcl-w-induced invasive potentials and stemness in glioblastoma multiforme.

3-Hydroxy-3',4'-dimethoxyflavone (HDMF) is a natural chemical product that is not currently regarded as a drug. In our study, we employed glioblastoma cells and cell biology and biochemistry approaches to investigate the potential of HDMF as a natural anticancer therapy option. FACS analysis showed that treatment concentration of HDMF does not exert cytotoxicity on U251 cells. Wound-healing and invasion assays showed that HDMF dose-dependently decreased the migratory and invasive potentials of these cells, likely by indirectly inhibiting MMP-3 activity as a result of the inhibition of p38 and ERK signaling proteins - an effect of HDMF also shown by Western blotting. HDMF inhibits Bcl-w-induced neurosphere formation and the expression of glioma stem cell markers, such as Musashi, Sox-2 and c-myc. These results indicate that HDMF suppresses migratory or invasive potentials and stemness and functions as a negative agent against the aggressiveness of glioblastoma cells. We propose that HDMF has potential as anticancer drug for inhibiting the aggressiveness of glioblastoma multiforme (GBM).

miR-765 as a promising biomarker for low-dose radiation-induced pulmonary fibrosis.

High-dose radiation (HDR) is widely used for cancer treatment, but the effectiveness of low-dose radiation (LDR) in the treatment of various diseases is controversial. Therefore, to safely utilize LDR for therapeutic purposes, further research on its numerous biological effects of LDR is required. Interest in the increased use of medical imaging devices or the effects of surrounding living environmental radiation on the human body, particularly on fibrosis, is rapidly increasing. Therefore, this study aimed to verify the relationship between LDR and pulmonary fibrosis by evaluating the changes in fibroblasts after LDR treatment and their associated signaling mechanisms. LDR increased the expression of fibrosis markers COL1A1 and α-SMA, cell proliferation, and migration by activating YAP1 and Twist in fibroblasts. Meanwhile, miRNA was employed as a tool to inhibit LDR-induced fibrosis and it was found that miR-765 simultaneously targeted COL1A1, α-SMA, and YAP1. At the cellular level, miR-765 reduced the proliferation and migration of fibroblasts by suppressing the expression of LDR-induced fibrosis factors COL1A1, α-SMA, and YAP1. The efficacy of miR-765 in vivo was confirmed using bleomycin (BLM)-induced fibrotic mouse model. The characteristics of pulmonary fibrosis were reduced after injection of miR-765-overexpressing cells into BLM-induced fibrotic mice. In addition, the suppression of miR-765 expression in the plasma of patients with pulmonary fibrosis confirmed the negative relationship between pulmonary fibrosis and miR-765 expression. Therefore, this study demonstrates that miR-765 is a potential novel diagnostic biomarker and major target for the development of therapeutic agents to inhibit pulmonary fibrosis.

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1.

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3'-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.

Targeting miR-5088-5p attenuates radioresistance by suppressing Slug.

Radiotherapy is widely used for cancer treatment, but paradoxically, it has been reported that surviving cancer cells can acquire resistance, leading to recurrence or metastasis. Efforts to reduce radioresistance are required to increase the effectiveness of radiotherapy. miRNAs are advantageous as therapeutic agents because it can simultaneously inhibit the expression of several target mRNAs. Therefore, this study discovered miRNA that regulated radioresistance and elucidated its signaling mechanism. Our previous study confirmed that miR-5088-5p was associated with malignancy and metastasis in breast cancer. As a study to clarify the relationship between radiation and miR-5088-5p identified as onco-miRNA, it was confirmed that radiation induced hypomethylation of the promoter of miR-5088-5p and its expression increased. On the other hand, miR-5088-5p inhibitors were confirmed to reduce radiation-induced epithelial-mesenchymal transition, stemness, and metastasis by reducing Slug. Therefore, this study showed the potential of miR-5088-5p inhibitors as therapeutic agents to suppress radioresistance.

miR-519d-3p suppresses tumorigenicity and metastasis by inhibiting Bcl-w and HIF-1α in NSCLC.

Bcl-w, a member of the Bcl-2 family, is highly expressed in various solid tumor, including lung cancer, suggesting that it is involved in cancer cell survival and carcinogenesis. Solid cancer-induced hypoxia has been reported to increase angiogenesis, growth factor, gene instability, invasion, and metastasis. Despite many studies on the treatment of non-small cell lung cancer (NSCLC) with a high incidence rate, the survival rate of patients has not improved because the cancer cells acquired resistance to treatment. This study investigated the correlation between Bcl-w expression and hypoxia in tumor malignancy of NSCLC. Meanwhile, microRNAs (miRNAs) are involved in a variety of key signaling mechanisms associated with hypoxia. Therefore, we discovered miR-519d-3p, which inhibits the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, and found that it reduces hypoxia-induced tumorigenesis. Spearman's correlation analysis showed that the expression levels of miR-519d-3p and Bcl-w/HIF-1α were negatively correlated, respectively. This showed that miR-519d-3p can be used as a diagnostic biomarker and target therapy for NSCLC.

Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2.

Breast cancer is the most common female cancer in the world. Despite the active research on metastatic breast cancer, the treatment of breast cancer patients is still difficult because the mechanism is not well known. Therefore, research on new targets and mechanisms for diagnosis and treatment of breast cancer patients is required. On the other hand, microRNA (miRNA) has the advantage of simultaneously regulating the expression of many target genes, so it has been proposed as an effective biomarker for the treatment of various diseases including cancer. This study analyzed the role and mechanism of DBC2 (deleted in breast cancer 2), which is known to inhibit its expression in breast cancer, and proposed microRNA (miR)-5088-5p, which regulates its expression. It was revealed that the biogenesis of miR-5088-5p was upregulated by hypomethylation of its promoter, promoted by Fyn, and was involved in malignancy in breast cancer. With the use of the cellular level, clinical samples, and published data, we verified that the expression patterns of DBC2 and miR-5088-5p were negatively related, suggesting the potential as novel biomarkers for the diagnosis of breast cancer patients.

Bcl-XL and STAT3 mediate malignant actions of gamma-irradiation in lung cancer cells.

Previous reports suggest that, in addition to its therapeutic effects, ionizing radiation (IR) increases the invasiveness of surviving cancer cells. Here, we demonstrate that this activity of IR in lung cancer cells is mediated by a signaling pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and matrix metalloproteinase (MMP-2). The invasion-promoting doses of IR also increased and reduced the levels of vimentin and E-cadherin, respectively, both of which are markers for the epithelial-mesenchymal transition (EMT). Interestingly, all of these malignant actions of IR were mimicked by the overexpression of Bcl-X(L), a pro-survival member of the Bcl-2 family, in lung cancer cells. Moreover, both RNA and protein levels of Bcl-X(L) were elevated upon irradiation of the cells, and the prevention of this event using small-interfering RNAs of Bcl-X(L) reduced the ability of IR to promote invasion signals and EMT-associated events. This suggests that Bcl-X(L) functions as a signaling mediator of the malignant effects of IR. It was also demonstrated that IR enhances signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the reduction of STAT3 levels via RNA interference prevented IR-induced Bcl-X(L) accumulation, and thus all the tested Bcl-X(L)-dependent events. Overall, the data suggest that IR induces Bcl-X(L) accumulation via STAT3, which then promotes cancer cell invasion and EMT-associated markers. Our findings demonstrate a novel function of Bcl-X(L) in cancer, and also advance our understanding of the malignant actions of IR significantly.

Hypermethylation of miR-205-5p by IR Governs Aggressiveness and Metastasis via Regulating Bcl-w and Src.

Although radiotherapy has been successfully applied to treat many cancer types, surviving cancer cells often acquire therapeutic resistance, leading to increased risk of local recurrence and distant metastases via modification of the tumor microenvironment. Previously, we reported that high expression of Bcl-w in cancer patients is significantly correlated with poor survival as well as malignant activity. However, the relationship between ionizing radiation (IR)-induced resistance and Bcl-w expression in cancer cells is currently unclear. We showed that IR-induced Bcl-w contributes to EMT (epithelial-mesenchymal transition), migration, angiogenesis, stemness maintenance, and metastasis by promoting the expression of factors related to these phenotypes, both in vitro and in vivo. Meanwhile, IR enhanced hypermethylation of miR-205-5p CpG islands through Src activation, leading to decreased miR-205-5p expression and, in turn, potentially stimulating Bcl-w-mediated malignant activity and metastasis. The clinical applicability of Bcl-w and miR-205-5p from cells or animal models was confirmed using tissues and plasma of breast carcinoma patients. Based on the collective findings, we propose that miR-205-5ps as important negative mediators of resistance in radiotherapy could serve as useful potential targets of concurrently applied genetic therapy aimed to inhibit tumor aggressiveness and enhance the efficiency of radiotherapy in cancer patients.

miR-340-5p Suppresses Aggressiveness in Glioblastoma Multiforme by Targeting Bcl-w and Sox2.

Glioblastoma multiforme (GBM), a particularly aggressive type of malignant brain tumor, has a high mortality rate. Bcl-w, an oncogene, is reported to enhance cell survival, proliferation, epithelial-mesenchymal transition (EMT), migratory and invasive abilities, and stemness maintenance in a variety of cancer cell types, including GBM. In this study, we confirmed that Bcl-w-induced conditional medium (CM) enhances tumorigenic phenotypes of migration, invasiveness, and stemness maintenance. Notably, platelet-derived growth factor-A (PDGF-A) expression, among other factors of the tumor environment, was increased by CM of Bcl-w-overexpressing cells, prompting investigation of the potential correlation between Bcl-w and PDGF-A and their effects on GBM malignancy. Bcl-w and PDGF-A levels were positively regulated and increased tumorigenicity by Sox2 activation in GBM cells. miR-340-5p was further identified as a direct inhibitor of Bcl-w and Sox2. Overexpression of miR-340-5p reduced mesenchymal traits, cell migration, invasion, and stemness in GBM through attenuating Bcl-w and Sox2 expression. Our novel findings highlight the potential utility of miR-340-5p as a therapeutic agent for glioblastoma multiforme through inhibitory effects on Bcl-w-induced PDGF-A and Sox2 activation.

Bcl-w promotes gastric cancer cell invasion by inducing matrix metalloproteinase-2 expression via phosphoinositide 3-kinase, Akt, and Sp1.

Given a previous report that Bcl-w is expressed in gastric cancer cells, particularly in those of an infiltrative morphology, we investigated whether Bcl-w expression influences the invasiveness of gastric cancer cells. To accomplish this, Bcl-w was overexpressed in adherent types of gastric adenocarcinoma cell lines, and this was found to result in an increase in their migratory and invasive potentials. These effects were not induced when Bcl-2 was overexpressed in the same cell types. Consistently, Bcl-w, but not Bcl-2, overexpression increased matrix metalloproteinase-2 (MMP-2) expression, and synthetic or natural inhibitors of MMP-2 abolished Bcl-w-induced cell invasion. Bcl-w overexpression also activated phosphoinositide 3-kinase (PI3K), Akt, and Sp1, and the blocking effects of each of these components using pharmacologic inhibitors, dominant-negative mutants, or small interfering RNA abolished the ability of Bcl-w to induce MMP-2 and cell invasion. The inhibition of PI3K/Akt signaling also prevented Sp1 activation. Overall, our data suggest that Bcl-w, which was previously shown to enhance gastric cancer cell survivability, also promotes their invasiveness by inducing MMP-2 expression via the sequential actions of PI3K, Akt, and Sp1.

FBXL14 abolishes breast cancer progression by targeting CDCP1 for proteasomal degradation.

Understanding the molecular mechanisms that underlie the aggressive behavior and relapse of breast cancer may help in the development of novel therapeutic interventions. CUB-domain-containing protein 1 (CDCP1), a transmembrane adaptor protein, is highly maintained and required in the context of cellular metastatic potential in triple-negative breast cancer (TNBC). For this reason, gene expression levels of CDCP1 have been considered as a prognostic marker in TNBC. However, not rarely, transcript levels of genes do not reflect always the levels of proteins, due to the post-transcriptional regulation. Here we show that miR-17/20a control the FBXL14 E3 ligase, establishing FBXL14 as an upstream regulator of the CDCP1 pathway. FBXL14 acts as an novel interaction partner of CDCP1, and facilitates its ubiquitination and proteasomal degradation with an enhanced capacity to suppress CDCP1 protein stability that eventually prevents CDCP1 target genes involved in breast cancer metastasis. Our findings first time uncovers the regulatory mechanism of CDCP-1 protein stabilization, more predictable criteria than gene expression levels for prognosis of breast cancer patients.

Radiation-inducible miR-770-5p sensitizes tumors to radiation through direct targeting of PDZ-binding kinase.

Radiotherapy represents the most effective non-surgical modality in cancer treatment. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, and are involved in many biological processes and diseases. To identify miRNAs that influence the radiation response, we performed miRNA array analysis using MCF7 cells at 2, 8, and 24 h post irradiation. We demonstrated that miR-770-5p is a novel radiation-inducible miRNA. When miR-770-5p was overexpressed, relative cell number was reduced due to increased apoptosis in MCF7 and A549 cells. Transcriptomic and bioinformatic analyses revealed that PDZ-binding kinase (PBK) might be a possible target of miR-770-5p for regulation of radiosensitivity. PBK regulation mediated by direct targeting of miR-770-5p was demonstrated using luciferase reporter assays along with wild-type and mutant PBK-3'untranslated region constructs. Radiation sensitivity increased and decreased in miR-770-5p- and anti-miR-770-5p-transfected cells, respectively. Consistent with this result, transfection of short interfering RNA against PBK inhibited cell proliferation, while ectopic expression of PBK restored cell survival from miR-770-5p-induced cell death. In addition, miR-770-5p suppressed tumor growth, and miR-770-5p and PBK levels were inversely correlated in xenograft model mice. Altogether, these data demonstrated that miR-770-5p might be a useful therapeutic target miRNA that sensitizes tumors to radiation via negative regulation of PBK.

Cellular components involved in the cell death induced by cisplatin in the absence of p53 activation.

Cisplatin (CDDP) is a DNA damaging agent and is widely used for treating cancer. While the role of p53 in CDDP-induced cell death has been stressed, evidence exists that CDDP can also kill p53-mutated cells. To investigate the latter mechanism, we performed a comparative study using three different human cell types, SNU-16 (a stomach cancer cell-line), U937 (a leukemic cell-line) and 293T (a kidney fibroblast cell-line), which are defective in terms of p53 activation. A focus was placed on Bcl-2 family proteins, reactive oxygen species (ROS), and mitogen-activated protein kinases. Our results suggest that the ability of CDDP to kill these cells can be mediated by JNK, p38 MAPK and ROS, but not by ERK. It was also found that CDDP can increase the ratio of pro-apoptotic/pro-survival Bcl-2 members. While the importance of these components was found to depend on cell type, JNK was commonly involved in the deaths of all cell types examined. Therefore, the JNK pathway appears to be an ideal target for the modulation of the lethal action of CDDP in multiple types of p53-mutated cells.

miR-5003-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization and direct targeting of E-cadherin.

One of the initial steps in metastatic dissemination is the epithelial-mesenchymal transition (EMT). Along this line, microRNAs (miRNAs) have been shown to function as important regulators of tumor progression at various stages. Therefore, we performed a functional screening for EMT-regulating miRNAs and identified several candidate miRNAs. Among these, we demonstrated that miR-5003-3p induces cellular features characteristic of EMT. miR-5003-3p induced upregulation of Snail, a key EMT-promoting transcription factor and transcriptional repressor of E-cadherin, through protein stabilization. MDM2 was identified as a direct target of miR-5003-3p, the downregulation of which induced Snail stabilization. E-cadherin was also demonstrated to be a direct target of miR-5003-3p, reinforcing the EMT-promoting function of miR-5003-3p. In situ hybridization and immunohistochemical analyses using tissue microarrays revealed that miR-5003-3p expression was higher in paired metastatic breast carcinoma tissues than in primary ductal carcinoma tissues, and was inversely correlated with the expression of MDM2 and E-cadherin. Furthermore, miR-5003-3p enhanced the formation of metastatic nodules in the lungs of mice in a tail vein injection experiment. Collectively, our results suggest that miR-5003-3p functions as a metastasis activator by promoting EMT through dual regulation of Snail stability and E-cadherin, and may therefore be a potential therapeutic target in metastatic cancers.

IL-4, a direct target of miR-340/429, is involved in radiation-induced aggressive tumor behavior in human carcinoma cells.

Radiotherapy induces the production of cytokines, thereby increasing aggressive tumor behavior. This radiation effect results in the failure of radiotherapy and increases the mortality rate in patients. We found that interleukin-4 (IL-4) and IL-4Rα (IL-4 receptor) are highly expressed in various human cancer cells subsequent to radiation treatment. In addition, IL-4 is highly overexpressed in metastatic carcinoma tissues compared with infiltrating carcinoma tissues. High expression of IL-4 in patients with cancer is strongly correlated with poor survival. The results of this study suggest that radiation-induced IL-4 contributes to tumor progression and metastasis. Radiation-induced IL-4 was associated with tumorigenicity and metastasis. IL-4 expression was downregulated by miR-340 and miR-429, which were decreased by ionizing radiation (IR). Radiation-regulated miR-340/429-IL4 signaling increased tumorigenesis and metastasis by inducing the production of Sox2, Vimentin, VEGF, Ang2, and MMP-2/9 via activating JAK, JNK, ß-catenin, and Stat6 in vitro and in vivo. Our study presents a conceptual advance in our understanding of the modification of tumor microenvironment by radiation and suggests that combining radiotherapy with genetic therapy to inhibit IL-4 may be a promising strategy for preventing post-radiation recurrence and metastasis in patients.