PDF file, 6751K, Figure S2. (A) SK1 mRNA expression in MCF7 cells treated with 10nM of estradiol (E2). MCF7 were treated with 10nM E2 or ethanol (negative control). After 3, 6, 12, 24 and 48 hours of treatment, SK1 mRNA levels were measured by RT-qPCR. For each time point, SK1 mRNA levels represent the ratio between the values obtained for E2 and ethanol. (B) miR-515-5p expression in ZR75-1 treated with 10nM of estradiol (E2). MCF7 were treated with 10nM E2 or ethanol (negative control). After 3, 6, 12, 24 and 48 hours of treatment, miR 515-5p levels were measured by RT-qPCR. For each time point, miR 515-5p levels represent the ratio between the values obtained for E2 and ethanol. (C) SK1 mRNA levels and phospho-ERK 1/2 protein expression in MCF7 cells treated with TAM. MCF7 cells were treated with 100nM TAM and ethanol. After 48 hours, SK1 mRNA levels were measured by RT-qPCR and (D) phospho-ERK 1/2 protein levels were analyzed by immuno-blotting. Data presented are the mean plus-minus SEM (*P<0.05; **P<0.01).

PDF file, 2594K, Figure S5. miR-515-5p levels are confirmed to be up-regulated in ERalpha-negative breast tumors in an independent dataset. GEO2R analysis of miR-515-5p expression in ERalpha-negative, n=82 and ERalpha-positive, n=128 using the dataset GSE22220 (19). miR-515-3p expression was also analyzed as a control (ns, non-significant; ***P<0.001).

PDF file, 150K, Table S1. Patient clinicopathological information (BC, breast cancer; IDC, invasive ductal carcinoma; IMC, invasive mixed carcinoma; ILC, invasive lobular carcinoma, ER, estrogen receptor; PgR, progesterone receptor; Her2, human epidermal growth factor receptor 2 respectively); Table S2. Primers used for the SK1 3' UTR amplification. ; Table S3. Details of the miR-515-5p promoter region cloning into the pGl2- basic plasmid. ; Table S4. Primers and their respective sequence. ; Table S5. Antibodies and their respective preparation. ; Table S6. Bibliographic references of the microRNAs predicted to target the SK1 3'UTR. ;

PDF file, 12967K, RNA-seq of MCF7 overexpressed with miR-515-5p.

PDF file, 10214K, Figure S1. (A) miRNAs predicted to target SK1 3'UTR using TargetScan. miRNAs targeting SK1 3'UTR were predicted using the web-based software, TargetScan. The miRNAs highlighted were selected for further analysis and experiments. (B) The effect of miR-206 and miR-515-5p overexpression on SK1 mRNA levels and (C) phospho-ERK 1/2 expression. MCF7 cells were transfected with 50nM of precursor miRNAs. After 48 hours, SK1 mRNA levels were measured by RT-qPCR and phospho-ERK 1/2 protein levels were analyzed by Immuno-blotting. Data presented are the mean plus-minus SEM (***P<0.001). (D) miRNA-515-5p directly interact with SK1 3'UTR. Cells were co-transfected with 50nM of pre-miRs, 100ng of pMIR-SK1 3'UTR or pMIR-SK1 3'UTRMUT and 50ng of pRLTK (Renilla luciferase vector). After 24 hours, both Firefly and Renilla luciferase activities were measured. Data were normalized to the negative control result (miR NC) and presented as the mean of three independent experiments plus-minus SEM. P values were calculated by t-test comparing individual miR values with miR NC values. (*P<0.05; **P<0.01; ***P<0.001).

PDF file, 7560K, Figure S3. SylArray enrichment analysis showing that the down-regulated genes upon miR-515-5p expression are enriched with miR-515-5p 'seeds'.

PDF file, 11170K, Figure S4. AUBPs (HuR and AUF1) mRNA expression is not altered upon estrogen treatment. GEO2R analysis of HuR mRNA levels (ELAVL1 gene) AUF1 mRNA levels (HNRNPD gene) using 8 published expression profiling datasets.

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers. miR‐515‐5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR‐515‐5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis. miR‐515‐5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR‐515‐5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis.

BACKGROUND & AIMS:There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS:We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS:We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS:In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

Abstract Sphingosine kinase 1 (SK1) plays an important role in estrogen-dependent breast tumorigenesis, but its regulation is poorly understood. A subset of microRNAs (miRNA, miR) is regulated by estrogen and contributes to cellular proliferation and cancer progression. Here, we describe that miR-515-5p is transcriptionally repressed by estrogen receptor α (ERα) and functions as a tumor suppressor in breast cancer. Its downregulation enhances cell proliferation and estrogen-dependent SK1 activity, mediated by a reduction of miR-515-5p posttranscriptional repression. Enforced expression of miR-515-5p in breast cancer cells causes a reduction in SK1 activity, reduced cell proliferation, and the induction of caspase-dependent apoptosis. Conversely, opposing effects occur with miR-515-5p inhibition and by SK1 silencing. Notably, we show that estradiol (E2) treatment downregulates miR-515-5p levels, whereas the antiestrogen tamoxifen causes a decrease in SK1, which is rescued by silencing miR-515-5p. Analysis of chromatin immunoprecipitation sequencing (ChIP-Seq) data reveals that miR-515-5p suppression is mediated by a direct interaction of ERα within its promoter. RNA-sequencing (RNA-Seq) analysis of breast cancer cells after overexpressing miR-515-5p indicates that it partly modulates cell proliferation by regulating the Wnt pathway. The clinical implications of this novel regulatory system are shown as miR-515-5p is significantly downregulated in ER-positive (n = 146) compared with ER-negative (n = 98) breast cancers. Overall, we identify a new link between ERα, miR-515-5p, proliferation, and apoptosis in breast cancer tumorigenesis. Cancer Res; 73(19); 5936–48. ©2013 AACR.