miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP, CK2-, CCNJ, and MEGF9

abstract: ...MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. To explore the dysregulation of miRNAs in breast cancer, a genome-wide expression profiling of 939 miRNAs was performed in 50 breast cancer patients. A total of 35 miRNAs were aberrantly expressed between breast cancer tissue and adjacent normal breast tissue and several novel miRNAs were identified as potential oncogenes or tumor suppressor miRNAs in breast tumorigenesis. miR-125b exhibited the largest decrease in expression. Enforced miR-125b expression in mammary cells decreased cell proliferation by inducing G2/M cell cycle arrest and reduced anchorage-independent cell growth of cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3-UTRs of ENPEP, CK2-, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 4056%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2- was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer.

...PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, USA 24098452 3789742 PONE-D-13-28054 10.1371/journal.pone.0076247 Research Article miR-125b Acts as a Tumor Suppressor in Breast Tumorigenesis via Its Novel Direct Targets ENPEP, CK2-, CCNJ, and MEGF9 Novel Targets of miR-125b in Breast Tumorigenesis Feliciano Andrea 1 Castellvi Josep 1 Artero-Castro Ana 1 Leal Jose A. 1 Romagosa Cleof 1 Hernndez-Losa Javier 1 Peg Vicente 1 Fabra Angels 2 Vidal Francisco

... cells of mammary origin. miR-125b was found to perform its tumor suppressor function via the direct targeting of the 3-UTRs of ENPEP, CK2-, CCNJ, and MEGF9 mRNAs. Silencing these miR-125b targets mimicked the biological effects of miR-125b overexpression, confirming that they are modulated by miR-125b. Analysis of ENPEP, CK2-, CCNJ, and MEGF9 protein expression in breast cancer patients revealed that they were overexpressed in 56%, 4056%, 20%, and 32% of the tumors, respectively. The expression of ENPEP and CK2- was inversely correlated with miR-125b expression in breast tumors, indicating the relevance of these potential oncogenic proteins in breast cancer patients. Our results support a prognostic role for CK2-, whose expression may help clinicians predict breast tumor aggressiveness. In particular, our results show that restoration of miR-125b expression or knockdown of ENPEP, CK2-, CCNJ, or MEGF9 may provide novel approaches for the treatment of breast cancer. This work was supported by

... as cyclin J (CCNJ) and multiple EGF-like-domains 9 (MEGF9). Moreover, miR-125b also downregulates other proteins whose roles in tumorigenesis are not well defined, such as glutamyl aminopeptidase or aminopeptidase A (ENPEP), and casein kinase 2-alpha (CK2-). Finally, we analyzed the protein expression of ENPEP, CK2-, CCNJ, and MEGF9 in breast cancer patients. The prognostic role of ENPEP, CK2-, CCNJ, and MEGF9 proteins and their importance in breast tumorigenesis are examined. Materials and

... described procedure [ 24 ]. Primers are detailed in Table S1 . For the luciferase assay, a reporter vector consisting of the luciferase coding sequence followed by the wild-type or mutant 3-UTR of putative targets of miR-125b was constructed. The 3-UTRs of ENPEP (945 nt), CK2- (1245 nt), CCNJ (916 nt), and MEGF9 (2061 nt), which contain the predicted miR-125b binding sites, were amplified by PCR from the normal tissues and cloned into the previously described pCI vector [ 25 ]. Primers are described in Table S1 . In all cases, the cloned PCR products were validated by sequencing (data not shown). Site-directed mutagenesis The QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to introduce point mutations into the 3-UTRs of human ENPEP, CK2-, CCNJ, and MEGF9. The following point mutations were introduced: at position 202 of the 3-UTR-ENPEP, at positions 210 and 450 of the 3-UTR-CK2-, at position 714 of the 3-UTR-CCNJ, and at three different positions416, 654, and 1721of the 3-UTR-MEGF9. A different mutant plasmid was constructed for each binding site located at the 3-UTR mRNA predicted for miR-125b. Primers are described in Table S1 . The PCR reaction was prepared by adding 5 l of 10X reaction buffer, 20 ng of dsDNA template, 125 ng each of the sense and antisense primers, 1 l of deoxyribonucleotide triphosphate mix, 3 l of QuickSolution, 1 l of Pfu Ultra HF DNA polymerase (2.5 U/l), and double-distilled water to a final volume of 50 l. The PCR was

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Gene: MEGF9 - multiple EGF-like-domains 9 - (NCBI)

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