- Email: [email protected]
MKNK2 is a valid target of miR-125b in breast cancer
Gene Reports 5 (2016) 92–97
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
MKNK2 is a valid target of miR-125b in breast cancer Yilin Wang a,1, Xiaolong Zhang a,1, Jian Ding a, Zou Chao a, Andreas Dress a,b, Lihui Lai a,⁎ a b
Institute of Molecular and Chemical Biology, East China Normal University, Shanghai, China CAS-MPG Partner Institute and Key Lab for Computational Biology, SIBS, CAS, Shanghai, China
a r t i c l e
i n f o
Article history: Received 25 August 2016 Accepted 22 September 2016 Available online 26 September 2016 Keywords: MiR-125b MKNK2 Breast cancer
a b s t r a c t MAP kinase interacting serine/threonine kinase 2 (MKNK2) belongs to the protein kinase superfamily which phosphorylates and activates eukaryotic initiation factor 4E (elF4E), a rate limiting factor in protein synthesis that enhances the translation of some proteins involved in cell cycle, apoptosis and angiogenesis regulation. In this study, we found that the expression levels of MKNK2 are inversely correlated with the expression levels of miR-125b in human breast cancer development. We showed that miR-125b inhibited tumor cell growth and invasion by regulating MKNK2 at posttranscriptional level. Thus, both miR-125b and MKNK2 could be therapeutically targeted in breast cancer. © 2016 Published by Elsevier Inc.
1. Introduction MicroRNAs(miRNAs) are endogenous small RNA molecules (approximately 19–25 nt) which regulate gene expression at the posttranscriptional level (Li et al., 2010). Recent studies have revealed that miRNAs are dysregulated in various cancers. MiRNAs have been demonstrated to play important roles in different aspects of tumorigenesis, including cell proliferation, apoptosis, invasion, metastasis, and angiogenesis (Kent and Mendell, 2006; Porkka et al., 2007). Therefore, expression signatures of miRNA in recent studies have been suggested to be useful diagnostic, prognostic and therapeutic biomarkers for tumors. Breast cancer (BC) is the major cause of cancer death in women, which is characterized by changes in tissue structure and gene expression (Castaneda et al., 2011). Recent studies described that the expression of several miRNAs were associated with BC pathogenesis. In the expanding list of cancer related miRNAs, miR-125b has been shown widespread dysregulated in various types of tumors, including breast cancer (Bloomston et al., 2007; Bousquet et al., 2008; Calin and Croce, 2006; Iorio et al., 2005; Park et al., 2009; Selcuklu et al., 2009; Wong et al., 2008). The mature sequences of miR-125a/b differ by 3 nucleotides, but bear identical seed sequences and have shared targets (Griffiths-Jones et al., 2008). MiR-125b is one of the most consistently dysregulated miRNAs in breast cancer. An early study reported that decreased expression of
Abbreviations: miRNA, MicroRNA; BC, breast cancer; MKNK2, MAP kinase interacting serine/threonine kinase 2; UTR, untranslated region; miR-125b, miRNA-125b. ⁎ Corresponding author at: Institute of Molecular and Chemical Biology, East China Normal University, 3663 North Road, Shanghai 200062, China. E-mail address: [email protected] (L. Lai). 1 These two authors contribute equally.
http://dx.doi.org/10.1016/j.genrep.2016.09.008 2452-0144/© 2016 Published by Elsevier Inc.
miR-125b was further found in ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) positive breast tumors (Mattie et al., 2006). Recent studies demonstrated that miR-125b functioned as a tumor suppressor in regulating the cell cycle and proliferation (Iorio et al., 2005). As a potential tumor suppressor factor, miR-125b was found to target several proto-oncogenes in cancers, such as BCL3 (Bcell CLL/lymphoma 3) in ovarian cancer (Guan et al., 2011) and E2F3 (E2F transcription factor 3) in bladder cancer (Huang et al., 2011). In breast cancer, v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1, a member of ETS transcription factor family), Mucin I, v-raf-1 murine leukemia viral oncogene homolog 1 (Raf1) and ERBB2/3 have been identified as the direct targets of miR-125b (Zhang et al., 2011; Rajabi et al., 2010; Hofmann et al., 2009; Scott et al., 2007). The human MAP kinase-interacting kinases comprise a group of four proteins derived from two genes (MKNK1 and MKNK2) by alternative splicing (Buxade et al., 2008). The isoforms of MKNKs differ markedly in their activity and regulation, and in subcellular localization which are activated by ERK or p38 and phosphorylate eIF4E (Buxade et al., 2008). Recent studies demonstrated an oncogenic role for MKNK2 in HER2-overexpressing cancers (Chrestensen et al., 2007). Early reports showed that miR-125b had negative effects on cellular proliferation of BC cells in an ERBB2 dependent manner (Ueda et al., 2010). In this study, we examined the expression of miR-125b and MKNK2 in breast tumors and its effects on breast cancer cell functions. We found that miR-125b was significantly down-regulated in breast cancer tissues and cell lines, compared with the matched adjacent cancer tissue and the basal-like breast epithelial cell line. Increased exogenous and endogenous expression of miR-125b could repress the proliferation and invasion of breast cell lines by directly targeting 3′ UTR of oncogenic MKNK2 that promoted cell growth and inhibited apoptosis. Our data imply that both miR-125b and MNK1/2 would be the potential therapeutic targets for breast cancer.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
2. Materials and methods 2.1. Human tissue samples Human samples from both breast tumor and normal tissues were obtained from Peking Union Medical College Hospital and Shanghai Huashan hospital. All participants provided written informed consent approved by the institutional review board at the participating hospitals. Tissue samples were stored in frozen liquid nitrogen after collection. Detailed clinical pathology information for all samples was available. Cases were classified and selected based on diagnosis using the CoPath Anatomic Pathology system. 2.2. Cell culture and RNA preparation MDA-MB-231, MDA-MB-435, MDA-MB-453, ZR-75-30, SK-BR-3, T47D, MCF 7 human breast cancer cell lines and HEK-293T cell line were purchased from ATCC. And MCF10A, immortalized nontumorigenic mammary cell line, was obtained from the Nanjing Medical University. MDA-MB-231, MDA-MB-435, MDA-MB-453, T47D and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone) and ZR-75-30, SK-BR-3 were maintained in Roswell Park Memorial Institute 1640 medium (RPIM 1640; Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco). MCF7 cells were cultured in Minimum Essential Medium (MEM; Invitrogen) supplemented with 0.01 mg/mL bovine insulin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS; Gibco). MCF10A cells were cultured in DMEM/F12 (Invitrogen) supplemented with 0.5 μg/mL Hydrocortizone (Sigma-Aldrich), 0.1 μg/mL CholeraToxin (Sigma-Aldrich), 5% Horse Serum (Gibco), 0.2 μg/mL EGF (SigmaAldrich), 1 μg/mL Insulin (Sigma-Aldrich) and 1 mL Pen/Strep (Invitrogen). Total RNAs were extracted from tissues and cells in Trizol reagent (Invitrogen). 2.3. Lentivirus preparation, titration and infection HEK293T cells were seeded in 6 cm plate at 2.5 × 106 cells, and cotransfected with 5 μg of pLemiR-125b plasmid and 15 μL of the packaging mix stock using Arrest-In transfection reagent (Open Biosystems). The culture supernatants were collected 48 and 72 h after transfection, which were filtered through 0.45 μm filters and stored as virus stocks. For virus titration, TLA-HEK293T cells were seeded at the level of 5 × 104 cells per well with 24-well tissue culture plate in DMEM. The diluted virus were added to each well, and incubated at 37 °C for 4 h. Then, the transduction mix was removed from cultures, cells were cultured for 48 h for the virus titration. For virus infection, the virus were concentrated before infection using Amicon Centrifugal Filter Devices(Millipore), cells were incubated at 37 °C with both of virus and polybrene (8 mg/mL) for 6 h, then for 2 days to select puromycinresistant cells. 2.4. Oligonucleotide transfection Pre-miR-125b and pre-miR™ miRNA negative control precursors were purchased from Ambion (Austin). Cells were transfected at 24 h after plating using Lipofectamine 2000 reagent (Invitrogen). Transfection complexes were prepared according to the manufacturer's instructions, and added directly to the cells to a final oligonucleotide concentration of 100 nM. 2.5. Real-time polymerase chain reaction assays for mature miRNAs Total RNAs were extracted from tissue samples and cell lines for the analysis. The primer sequences were designed by Biomics Company for assaying the expression levels of miR-125b. Reverse transcription reaction was performed using 30 ng of total RNAs and 1 mM looped primers.
93
Real-time PCR was performed using the MX3000p Real-Time PCR Detection System (Stratagene) according to the standard SYBR Green Assay protocol. The 25 μL PCR reaction included 2 μL reverse transcription product, 1xPCR Master Mix (Takara), 1.5 mmol/L forward primer, and 0.7 mmol/L reverse primer. Thermal-cycling conditions of the reactions in a 96-well plate were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Data was examined by comparing relative miRNA expression to U6 RNA. Relative miRNA expression was determined using the 2-Ct method. All samples were analyzed in triplicate. 2.6. Western blot analysis For each sample, 25 μg of total protein extracts were separated on SDS-PAGE gels, and transferred to PVDF membrane. Antibody against MKNK2 (Cell Signaling Technology) was diluted at 1:200. PARP (Cell Signaling Technology) was diluted at 1:300 and EIF4E which was phosphorylated at S209 with a dilution of 1:500 were incubated at room temperature for 2 h with an AP-conjugated goat anti-rabbit secondary antibody (WesternDot™ 625 Goat Anti-Rabbit Western Blot Kit; Invitrogen). The results were visualized using BCIP/NBT (Amresco). As a loading control, beta-actin expression levels were measured by rabbit poyclonal anti-actin antibody (Santa Cruz Biotechnology). Bands were quantified with Labworks Instrument software (UVP LLC). 2.7. MiRNA target prediction and luciferase activity assay MiRNA target prediction was performed by in-lab developed algorithm KeyTar. KeyTar miRNA target prediction is based on miRNA: target sequence, structure, and function (unpublished). The primer sequences for amplifying the human 3′UTR region of MKNK2 gene as follows: MKNK2-3′UTR-F: 5′-GAATTCTCCGACCTTGACCTTAAAC-3′, and MKNK2-3′UTR-R: 5′-CTCGAGTCAGTTCACCTGGTACATT-3′, and cloned into the EcoRI and XhoI sites of the pGL3-control vector (Promega). Nucleotide-substitution mutations were carried out using PCR-based methods at the 3′UTR regions of MKNK2. Primers were as below: Mut 3′UTR of MKNK2: 5′-GAGTCCCCTCAGTCTGACCGTAGCCACG-3′ and 5′AAGGGGCACTCGGGTGCCCCAAGGG-3′. All constructs were verified by sequencing. For luciferase assay, the HEK293T and ZR-75-30 cells were seeded in 96-well plates, and transfected with 20 ng luciferase reporter plasmid, 1 ng pRL-TK vector expressing the Renilla luciferase (Promega), and 5 pmoles of miR125b or miRNA negative control precursor. Transfection was performed using Lipofectamine 2000 (Invitrogen). After transfection for 24–36 h, firefly and renilla luciferase activities were measured using the DualLuciferase Reporter Assay (Promega). All transfections were repeated twice in triplicate. 2.8. In vitro invasion assay ZR-75-30 and MDA-MB-453 cells were transfected with miR-125b or a scrambled control precursors. Forty-eight hours after transfection, cells (5 × 104) were plated in the top chamber with polymerized collagen-coated membrane (24-well insert; pore size, 8 μm; Chemicon ECM551) in medium with 0.1% FBS. Complete medium was used as a chemo attractant in the lower chamber. The left cells were incubated for 24 h, and unattached cells were removed cautiously by cotton swab. Cells that had invaded the lower surface of the membrane were stained with the Cell Stain Solution and counted accordingly. They were finally extracted and detected quantitatively by a standard microplate reader (at 560 nm). 2.9. Cell viability and cell proliferation analysis Cell growth assay with ZR-75-30 and MDA-MB-453 cells which were transfected with siMKNK2 and negative control (siNC) or pre-
94
Y. Wang et al. / Gene Reports 5 (2016) 92–97
miR-125b and pre-miR™ miRNA negative control precursors. In vitro proliferative kinetics were assayed by seeding 4 × 104 cells per well in 48-well plates and allowed to grow for different times. Total cell numbers in triplicate were measured every day, as indicated, by trypsinization and manual counting with a hemocytometer up to the 2th day.
2.10. Northern blot analysis Total RNAs were extracted using Trizol (Invitrogen) according to the manufacturer's instructions. The collected RNA samples (25 μg each) were electrophorized on 15% acrylamide and 8 M urea denature gels, then transferred onto Hybond N+ membrane (Amersham Biosciences). The membranes were agitated at 80 °C for 2 h before hybridization and were then hybridized with oligonucleotide probes corresponding to the complementary sequences of the following mature miRNAs: miR-21, 5′-TCAACATCAGTCTGATAAGCTA-3′; miR-143, 5′-GAGCTACAGTGCTT CATCTCA-3′ and miR-125b, 5′-TCACAAGTTAGGGTCTCAGGGA-3′. Probes were 5-end labeled using the polynucleotide kinase in the presence of [γ-32P] ATP (GE Healthcare). Hybridization was carried out at 39 °C in ULTRA hyb™-Oligo hybridization buffer (Ambion) for 16 h. Membranes were washed at 42 °C thrice with SSC (2×) plus 0.1% SDS. Northern blots were re-hybridized after stripping in 1% SDS for 30 min at 65 °C. In this part of the experiment, the U6 RNA probe (5′-GCTAAT CTTCTCTGTATCGTTCCAATTTT-3′) was used as a control for normalizing the RNA expression levels. Images were photo-documented using the scanner Storm 860 (Molecular Dynamics). Oligonucleotide probes were synthesized by Biosune (Shanghai, China).
2.11. Statistical analysis
Fig. 1. MiR-125b is underexpressed in breast cancer. (A). The expression levels of miR125b were analyzed in breast cancer cells by Northern blotting. The samples were assayed sequentially with radio-labeled probes specific to miR-125b, miR-143, miR-21 and small nuclear RNA U6. The U6 was used as the loading control. (B). The relative expression of miR-125b in a nonneoplastic control versus tumor cell lines has been normalized to the internal control U6 signal.
3.3. MiR-125b directly targets Mknk2 To understand molecular mechanism of miR-125b further in breast cancer cells, we found that 3′UTR of MKNK2 contained the highly conserved putative binding sites of miR-125b (Fig. 3a) using KeyTar miRNA target prediction algorithm. We constructed luciferase reporters carrying the 3′UTR of pGL3-MKNK2 with the putative and mutated miR125b binding sites (MKNK2-wt and MKNK2-mut). Luciferase assays revealed that increased expression of miR-125b caused a remarkable reduction of the luciferase activity in the MKNK2 gene reporter in miR125b oligo transfected cells, compared with mutated gene reporter (Fig. 3b). Moreover, Western blot analysis of MKNK2 in ZR-75-30 and MDA-MB-453 cell lines showed that transfection of miR-125b oligo decreased MKNK2 levels (Fig. 3c). These findings indicated that miR-125b could modulate MKNK2 at the translational level in breast cancer.
Data comparisons were performed using analysis of variance following Dunnett's method. The two-tailed Student's t-test was used to evaluate significance of the differences between cancer tissues or cell lines and normal controls. Statistical significance was determined as P b 0.05.
3. Results 3.1. MiR-125b is downregulated in human breast cancer To investigate whether miR-125b is involved in the development of breast cancer, we first examined the expression levels of miR-125b in human breast cancer cell lines including MCF7, T47D, ZR-75-30, MDAMB-453, SKBR3, MDA-MB-231, MDA-MB-435 by Northern Blot. The expression levels of miR-125b were greatly decreased in breast cancer cell lines, as compared with the basal-like breast epithelial cell line, MCF10A (Fig. 1a). The relative expression of miR-125b in a nonneoplastic control versus tumor cell lines has been normalized to the internal control U6 signal (Fig. 1b). Collectively, our results indicate that miR-125b is downregulated in breast cancer.
3.2. MiR-125b inhibits breast cancer cell growth and invasion in vitro To further investigate the role of miR-125b in breast cancer, we transfected the ZR-75-30 and MDA-MB-453 cells with precursor miR125b or miRNA scramble control (miR-scr) and examined the effect of miR-125b on cell growth and invasion. We found that over-expression of miR-125b decreased breast cancer cell invasion as indicated by invasion assay when compared with a miR-scr (Fig. 2a). In addition, cell growth assay results showed that over-expression of miR-125b exhibited a significant reduction of cell numbers when compared with the cells transfected with the miR-scr (Fig. 2b).
Fig. 2. MiR-125b inhibits breast cancer cell invasion and growth in vitro. (A). Matrigel invasion assay with MDA-MB-453 and ZR-75-30 cells transfected with miRNA scramble control or miR-125b oligo (left panel, 200×). Colorimetric measurement (relative level of optical density measured at 560 nm) of the stained cells for the miR-scr and miR125b transfected MDA-MB-453 and ZR7530 cells (right panel). miR-125b showed significant repression of tumor cell invasion comparing with the control. The data are expressed in mean ± S.E. (n = 3). (B). Cell growth assay with MDA-MB-453 and ZR7530 cells transfected with miRNA scramble control or miR-125b oligo. Total cell numbers in triplicate were assessed up to the 4th day. Cells transfected with miR-125b oligo exhibited a significant reduction in viable cells comparing with the negative control. a cells transfected with miRNA scramble control; b cells transfected with miR125b oligo. n = 3, ** p b 0.01,* p b 0.05.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
Fig. 3. MiR-125b directly targets MKNK2. (A). Schematic diagram of putative miR-125b banding sites in the 3′UTR region of MKNK2 gene. The possible binding sites of the target genes were searched using an in-house algorithm KeyTar (Liu et al., 2011). The conservation of the corresponding sites in the genomes of 28 species was analyzed using UCSC Genome Browser (NCBI36/hg18, http://genome.ucsc.edu/) and indicated in red box. (B). Luciferase assay. HEK 293t cells were transfected with miR-125b oligo or miRNA scramble control. Overexpression of miR-125b inhibited luciferase expression significantly when compared with the negative control. n = 3; ** p b 0.01. MKNK2-wt: MKNK2 wild type, MKNK2-Mut: MKNK2 mutation in miR-125b binding site indicated by underlined sequence as shown in panel A (upper panel). (C). Western blot analysis for MKNK2 levels in cells which were transfected with miR-125b oligo or miRNA scramble control. Significantly decreased levels of MKNK2 were shown after transfection. Actin was served as the internal control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Endogenous expression of miR-125b further confirms that miR-125b directly targets MKNK2 To confirm whether or not MKNK2 is a direct target of miR-125b further, we subsequently transfected ZR-75-30 and MDA-MB-453 cells with lentiviral vehicles which ubiquitously expressed miR-125b or a scrambled control precursors (Fig. 4a, upper panel). Semi-RT-qPCR analysis showed that stable-expression of miR-125b in both ZR-75-30 and MDA-MB-453 cells (Fig. 4a, lower panel). Consistent with results of luciferase assays observed in transient transfected cells, endogenous over-expression of miR-125b significantly reduced luciferase activities of MKNK2 gene reporter, but did not change the activity of mutated gene reporter (Fig. 4b). Furthermore, inhibition of endogenous expression of miR-125b restored the luciferase activity of MKNK2 gene reporter compared to mutated gene reporter in ZR-75-30 cells transfected with miR-125b antisense (Fig. 4b). Similarly, Western blot analysis revealed that the expression of MKNK2 was decreased by endogenous over-expression of miR-125b. In addition, we also found that up-regulation of miR-125b significantly reduced phosphorylation of EIF4 by MKNK2 and increased the cleavage of PARP, which resulted in cell apoptosis (Fig. 4c). In a word, our results further confirmed that miR-125b could target MKNK2 by directly binding to its 3’UTR in breast cancer in both sides.
3.5. Re-expression of MKNK2 reversed miR-125b dependent suppression in miR-125b expression cells To examine the effect of MKNK2 in breast cancer cells, we first performed Western blot analysis for MKNK2 expression levels in breast cancer cells and found that MKNK2 was up-regulated obviously (Fig. 5a). Then we conducted MKNK2 siRNA assay and found that cell viability was significantly decreased in MKNK2 siRNA-transfected cells, and not in siRNA scramble control transfected cells (Fig. 5b). Western
95
Fig. 4. Endogenous expression of miR-125b further confirms that miR-125b directly targets MKNK2. (A). Expression of miR-125b in ZR7530 or MDA-MB-453 cells which were transfected with lentivirus encoding miR-125b or miRNA scramble. Successful lentivirus transfection was visualized by red fluorescence in the upper panel. Marked miR-125b expression in miR-125b transfected cells was detected by semi-quantitative RT-PCR(lower panel). (B). Luciferase assay with miR-125b-overexpressed breast cancer cells. Compared with negative control ZR7530-mir-scr, over-expression of miR-125b inhibited luciferase expression (upper panel), while knockdown of miR-125b using miR125b antisense (miR-125bAS) increased luciferase expression significantly (lower panel). n = 3; * p b 0.05, ** p b 0.01. MKNK2-wt, MKNK2 coding sequence wild type; MKNK2-mt, MKNK2 coding sequence with mutation as indicated in Fig. 3a. (C). Western blot analysis of MKNK2 and its downstream genes. Significant decreased levels of MKNK2 was observed after 48 h infection. Cleaved PARP was also observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
blot analysis further confirmed that MKNK2 was down-regulated using the siRNA (Fig. 5c). Importantly, we found that re-expression of MKNK2 reversed miR-125b dependent suppression in miR-125b expression cells (Fig. 5d).
3.6. MiR-125b levels were correlated inversely with breast cancer malignancy and expression levels of target genes Finally, we tested a set of patient samples to verify whether MKNK2 protein expression correlates with miR-125b expression. As expected, in breast cancer samples, especially in HER2 positive ones, MKNK2 was over-expressed compared with two non-tumor breast tissues. What's more, the phosphorylation level of eIF4E was obviously increased (Fig. 6a). Then we performed qRT-PCR and found that miR125b was down-regulated dramatically in 28 invasive ductal carcinoma tissues compared with 12 normal tissues samples (a mixture containing 10 non-tumor breast samples and two non-tumor breast individuals) (Fig. 6b). But no significant difference of miR-125b expression was found between estrogen receptor (ER) positive (n = 8) versus ER negative (n = 7) breast cancers.
96
Y. Wang et al. / Gene Reports 5 (2016) 92–97
Fig. 5. Knockdown of MKNK2 inhibits breast cancer cell growth. (A). Western blotting analysis for MKNK2 levels in breast cancer cells. MCF10A was used as the normal control. Actin was used as the internal control. (B). Cell growth assay. In vitro proliferative kinetics were assayed by seeding 4 × 104 cells per well in 48-well plates and allowed to grow for different times. Total cell numbers in triplicate were assessed every day, as indicated. Cells transfected with siMKNK2 exhibit a significant reduction in viable cells comparing with cells transfected with the negative control (siNC). a cells transfected with a siRNA scramble control (siNC); b cells transfected with siMKNK2 oligo. n = 3, * p b 0.05. (C). Western blotting analysis for MKNK2 expression in cells which were transfected with siMKNK2 oligo or siNC. (D). Re-expression of MKNK2 reversed miR-125b mediated repression of breast cancer cell growth significantly. a miR-scr-453 cells transfected with a control plasmid; b miR-125b-453 cells transfected with a control plasmid and c miR-125b-453 cells transfected with a plasmid that express MKNK2. n = 3; * p b 0.05, ** p b 0.01.
4. Discussion Breast cancer is the most frequently diagnosed life-threatening cancer in women. Recent studies are focused on identifying novel therapeutic targets by better understanding the molecular mechanism of breast cancer. In this study, we demonstrated that miR-125b was down-
regulated in breast cancer and inhibited cell growth and invasion by directly targeting MKNK2. The decreased or increased expression of genes usually implied their functions as either tumor suppressors or oncogenes. Hence, we examined the expression of miR-125b in breast tumor cell lines and tumor tissue samples using two classical methods with Northern blot and qRT-
Fig. 6. MKNK2 expression is correlated with miR-125b status in Her2 positive breast cancer. (A). Western blotting analysis for MKNK2 levels in breast cancer tissues. Two human nontumor breast tissues were used as the normal control. Actin was used as the internal control. (B). qRT-PCR analysis of miR-125b levels in normal tissues from human non-tumor breast tissues (n = 12, a mixture containing 10 non-tumor breast samples and two non-tumor breast individuals) and invasive ductal carcinoma tissues (n = 28). U6 rRNA was used as a loading control. miR-125b levels inversely were correlated with malignancy in human breast tumors. p b 0.01. There were no significant differences between each stage of breast cancer (P N 0.05). No significant difference of miR-125b expression was found between estrogen receptor (ER) positive (n = 8) versus ER negative (n = 7) breast cancers.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
PCR methods. We found that miR-125b is dysregulated in both tumor cell lines and tumor tissues, which was in accordance with recent studies which indicating that miR-125b functioned as a tumor suppressor (Zhang et al., 2011; Rajabi et al., 2010; Rajabi et al., 2010; Hofmann et al., 2009; Scott et al., 2007). In ovarian cancer, ectopic expression of miR-125b was found to induce tumor cell cycle arrest, reduction in proliferation and clone formation (Guan et al., 2011). In this study, we demonstrated that over-expression of miR-125b oligo in ZR-75-30 and MDA-MB-453 cell lines decreased breast cancer cell growth in vitro significantly. Since miR-125b directly targets Bcl2 family members or positive and negative regulators in the p53 pathway (Jia et al., 2012; Shi et al., 2011; Xia et al., 2009; Gong et al., 2012; Jiang et al., 2011; Le et al., 2011; Boulares et al., 1999), over-expression of miR125b might disrupts the pathway and then activates caspases cascade. Caspase-3 is an integrator of apoptosis signals both in vitro and in vivo, which was activated by initiator caspases and then in turn cleaves PARP, subsequently leading to downstream of death signal(Boulares et al., 1999; Shi et al., 2012). In this study, the significant cleavage increasement of PARP was detected in cells with endogenous expression of miR-125b. Consistent with other studies, our data further confirmed that miR-125b played an important role in breast cancer cell apoptosis. Several miR-125b targets have been recently identified in a variety of cancers including STAT3(Liu et al., 2011), E2F2(Wu et al., 2012), MAZ(Smits et al., 2012), BCL3(Guan et al., 2011), c-Raf(Hofmann et al., 2009), Bcl-w, Mcl-1(Jia et al., 2012; Gong et al., 2012), Bmf (Xia et al., 2009), FGFR2 (Xu et al., 2011) and MUC1 (Rajabi et al., 2010; Schroeder et al., 2001). In addition, decreased expression of miR-125b was further detected in ERBB2 positive breast tumors and overexpression of miR-125b could impair their anchorage-dependent growth and reduce migration and invasion capacities by suppressing ERBB2 and ERBB3 (Mattie et al., 2006; Scott et al., 2007). In a recent study, MKNK2 was reported as a possible target of miR-125b in neuroblastoma cells (Le et al., 2009). In this work, we identified MKNK2 as a direct target of miR-125b in breast cancer by systemic studies and demonstrated that MKNK2 is a key mediator of tumor cell proliferation. It has been found that MKNK2 is up-regulated and sustained activation in HER2-overexpressing breast cancer cells (Chrestensen et al., 2007). In this study, we found that miR-125b modulated MKNK2 expression by directly targeting the binding site within its 3′UTR. Knockdown of MKNK2 by siRNA inhibited breast cancer cell growth and invasion. Moreover, Re-expression of MKNK2 reversed miR-125b mediated repression of breast cancer cell growth significantly. Together, miR-125b inhibits breast cancer cell growth and invasion by directly targeting MKNK2. In summary, our data revealed an important role of miR-125b in breast cancer and suggested that both miR-125b and MKNK2 are important therapeutic targets in breast cancer.
References Bloomston, M., Frankel, W.L., Petrocca, F., Volinia, S., Alder, H., Hagan, J.P., Liu, C.G., Bhatt, D., Taccioli, C., Croce, C.M., 2007. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297 (17), 1901–1908. Boulares, A.H., Yakovlev, A.G., Ivanova, V., Stoica, B.A., Wang, G., Iyer, S., Smulson, M., 1999. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J. Biol. Chem. 274 (33), 22932–22940. Bousquet, M., Quelen, C., Rosati, R., Mansat-De Mas, V., La Starza, R., Bastard, C., Lippert, E., Talmant, P., Lafage-Pochitaloff, M., Leroux, D., et al., 2008. Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation. J. Exp. Med. 205 (11), 2499–2506. Buxade, M., Parra-Palau, J.L., Proud, C.G., 2008. The Mnks: MAP kinase-interacting kinases (MAP kinase signal-integrating kinases). Front. Biosci. 13, 5359–5373. Calin, G.A., Croce, C.M., 2006. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6 (11), 857–866. Castaneda, C.A., Agullo-Ortuno, M.T., Fresno Vara, J.A., Cortes-Funes, H., Gomez, H.L., Ciruelos, E., 2011. Implication of miRNA in the diagnosis and treatment of breast cancer. Expert. Rev. Anticancer. Ther. 11 (8), 1265–1275.
97
Chrestensen, C.A., Shuman, J.K., Eschenroeder, A., Worthington, M., Gram, H., Sturgill, T.W., 2007. MNK1 and MNK2 regulation in HER2-overexpressing breast cancer lines. J. Biol. Chem. 282 (7), 4243–4252. Gong, J., Zhang, J.P., Li, B., Zeng, C., You, K., Chen, M.X., Yuan, Y., Zhuang, S.M., 2012. MicroRNA-125b promotes apoptosis by regulating the expression of Mcl-1, Bcl-w and IL-6R. Ontogeny. Griffiths-Jones, S., Saini, H.K., van Dongen, S., Enright, A.J., 2008. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36 (Database issue), D154–D158. Guan, Y., Yao, H., Zheng, Z., Qiu, G., Sun, K., 2011. MiR-125b targets BCL3 and suppresses ovarian cancer proliferation. Int. J. Cancer 128 (10), 2274–2283. Hofmann, M.H., Heinrich, J., Radziwill, G., Moelling, K., 2009. A short hairpin DNA analogous to miR-125b inhibits C-Raf expression, proliferation, and survival of breast cancer cells. Mol. Cancer Res. 7 (10), 1635–1644. Huang, L., Luo, J., Cai, Q., Pan, Q., Zeng, H., Guo, Z., Dong, W., Huang, J., Lin, T., 2011. MicroRNA-125b suppresses the development of bladder cancer by targeting E2F3. Int. J. Cancer 128 (8), 1758–1769. Iorio, M.V., Ferracin, M., Liu, C.G., Veronese, A., Spizzo, R., Sabbioni, S., Magri, E., Pedriali, M., Fabbri, M., Campiglio, M., et al., 2005. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65 (16), 7065–7070. Jia, H.Y., Wang, Y.X., Yan, W.T., Li, H.Y., Tian, Y.Z., Wang, S.M., Zhao, H.L., 2012. MicroRNA125b functions as a tumor suppressor in hepatocellular carcinoma cells. Int. J. Mol. Sci. 13 (7), 8762–8774. Jiang, F., Liu, T., He, Y., Yan, Q., Chen, X., Wang, H., Wan, X., 2011. MiR-125b promotes proliferation and migration of type II endometrial carcinoma cells through targeting TP53INP1 tumor suppressor in vitro and in vivo. BMC Cancer 11, 425. Kent, O.A., Mendell, J.T., 2006. A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Ontogeny 25 (46), 6188–6196. Le, M.T., Xie, H., Zhou, B., Chia, P.H., Rizk, P., Um, M., Udolph, G., Yang, H., Lim, B., Lodish, H.F., 2009. MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets. Mol. Cell. Biol. 29 (19), 5290–5305. Le, M.T., Shyh-Chang, N., Khaw, S.L., Chin, L., Teh, C., Tay, J., O'Day, E., Korzh, V., Yang, H., Lal, A., et al., 2011. Conserved regulation of p53 network dosage by microRNA-125b occurs through evolving miRNA-target gene pairs. PLoS Genet. 7 (9), e1002242. Li, M., Li, J., Ding, X., He, M., Cheng, S.Y., 2010. microRNA and cancer. AAPS J. 12 (3), 309–317. Liu, L.H., Li, H., Li, J.P., Zhong, H., Zhang, H.C., Chen, J., Xiao, T., 2011. MiR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3. Biochem. Biophys. Res. Commun. 416 (1–2), 31–38. Mattie, M.D., Benz, C.C., Bowers, J., Sensinger, K., Wong, L., Scott, G.K., Fedele, V., Ginzinger, D., Getts, R., Haqq, C., 2006. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol. Cancer 5, 24. Park, N.J., Zhou, H., Elashoff, D., Henson, B.S., Kastratovic, D.A., Abemayor, E., Wong, D.T., 2009. Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin. Cancer Res. 15 (17), 5473–5477. Porkka, K.P., Pfeiffer, M.J., Waltering, K.K., Vessella, R.L., Tammela, T.L., Visakorpi, T., 2007. MicroRNA expression profiling in prostate cancer. Cancer Res. 67 (13), 6130–6135. Rajabi, H., Jin, C., Ahmad, R., McClary, C., Joshi, M.D., Kufe, D., 2010. Mucin 1 oncoprotein expression is suppressed by the miR-125b oncomir. Genes Cancer 1 (1), 62–68. Schroeder, J.A., Thompson, M.C., Gardner, M.M., Gendler, S.J., 2001. Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogenactivated protein kinase activation in the mouse mammary gland. J. Biol. Chem. 276 (16), 13057–13064. Scott, G.K., Goga, A., Bhaumik, D., Berger, C.E., Sullivan, C.S., Benz, C.C., 2007. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J. Biol. Chem. 282 (2), 1479–1486. Selcuklu, S.D., Yakicier, M.C., Erson, A.E., 2009. An Investigation of microRNAs Mapping to Breast Cancer. Shi, X.B., Xue, L., Ma, A.H., Tepper, C.G., Kung, H.J., White, R.W., 2011. miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes. Prostate 71 (5), 538–549. Shi, L., Zhang, S., Feng, K., Wu, F., Wan, Y., Wang, Z., Zhang, J., Wang, Y., Yan, W., Fu, Z., et al., 2012. MicroRNA-125b-2 confers human glioblastoma stem cells resistance to temozolomide through the mitochondrial pathway of apoptosis. Int. J. Oncol. 40 (1), 119–129. Smits, M., Wurdinger, T., van het Hof, B., Drexhage, J.A., Geerts, D., Wesseling, P., Noske, D.P., Vandertop, W.P., de Vries, H.E., Reijerkerk, A., 2012. Myc-associated zinc finger protein (MAZ) is regulated by miR-125b and mediates VEGF-induced angiogenesis in glioblastoma. FASEB J. 26 (6), 2639–2647. Ueda, T., Sasaki, M., Elia, A.J., Chio, I.I., Hamada, K., Fukunaga, R., Mak, T.W., 2010. Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development. Proc. Natl. Acad. Sci. U. S. A. 107 (32), 13984–13990. Wong, T.S., Liu, X.B., Wong, B.Y., Ng, R.W., Yuen, A.P., Wei, W.I., 2008. Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue. Clin. Cancer Res. 14 (9), 2588–2592. Wu, N., Xiao, L., Zhao, X., Zhao, J., Wang, J., Wang, F., Cao, S., Lin, X., 2012. MiR-125b regulates the proliferation of glioblastoma stem cells by targeting E2F2. FEBS Lett. 586 (21), 3831–3839. Xia, H.F., He, T.Z., Liu, C.M., Cui, Y., Song, P.P., Jin, X.H., Ma, X., 2009. MiR-125b expression affects the proliferation and apoptosis of human glioma cells by targeting Bmf. Cell. Physiol. Biochem. 23 (4–6), 347–358. Xu, N., Brodin, P., Wei, T., Meisgen, F., Eidsmo, L., Nagy, N., Kemeny, L., Stahle, M., Sonkoly, E., Pivarcsi, A., 2011. MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2. J. Invest. Dermatol. 131 (7), 1521–1529. Zhang, Y., Yan, L.X., Wu, Q.N., Du, Z.M., Chen, J., Liao, D.Z., Huang, M.Y., Hou, J.H., Wu, Q.L., Zeng, M.S., et al., 2011. miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer. Cancer Res. 71 (10), 3552–3562.
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
MKNK2 is a valid target of miR-125b in breast cancer Yilin Wang a,1, Xiaolong Zhang a,1, Jian Ding a, Zou Chao a, Andreas Dress a,b, Lihui Lai a,⁎ a b
Institute of Molecular and Chemical Biology, East China Normal University, Shanghai, China CAS-MPG Partner Institute and Key Lab for Computational Biology, SIBS, CAS, Shanghai, China
a r t i c l e
i n f o
Article history: Received 25 August 2016 Accepted 22 September 2016 Available online 26 September 2016 Keywords: MiR-125b MKNK2 Breast cancer
a b s t r a c t MAP kinase interacting serine/threonine kinase 2 (MKNK2) belongs to the protein kinase superfamily which phosphorylates and activates eukaryotic initiation factor 4E (elF4E), a rate limiting factor in protein synthesis that enhances the translation of some proteins involved in cell cycle, apoptosis and angiogenesis regulation. In this study, we found that the expression levels of MKNK2 are inversely correlated with the expression levels of miR-125b in human breast cancer development. We showed that miR-125b inhibited tumor cell growth and invasion by regulating MKNK2 at posttranscriptional level. Thus, both miR-125b and MKNK2 could be therapeutically targeted in breast cancer. © 2016 Published by Elsevier Inc.
1. Introduction MicroRNAs(miRNAs) are endogenous small RNA molecules (approximately 19–25 nt) which regulate gene expression at the posttranscriptional level (Li et al., 2010). Recent studies have revealed that miRNAs are dysregulated in various cancers. MiRNAs have been demonstrated to play important roles in different aspects of tumorigenesis, including cell proliferation, apoptosis, invasion, metastasis, and angiogenesis (Kent and Mendell, 2006; Porkka et al., 2007). Therefore, expression signatures of miRNA in recent studies have been suggested to be useful diagnostic, prognostic and therapeutic biomarkers for tumors. Breast cancer (BC) is the major cause of cancer death in women, which is characterized by changes in tissue structure and gene expression (Castaneda et al., 2011). Recent studies described that the expression of several miRNAs were associated with BC pathogenesis. In the expanding list of cancer related miRNAs, miR-125b has been shown widespread dysregulated in various types of tumors, including breast cancer (Bloomston et al., 2007; Bousquet et al., 2008; Calin and Croce, 2006; Iorio et al., 2005; Park et al., 2009; Selcuklu et al., 2009; Wong et al., 2008). The mature sequences of miR-125a/b differ by 3 nucleotides, but bear identical seed sequences and have shared targets (Griffiths-Jones et al., 2008). MiR-125b is one of the most consistently dysregulated miRNAs in breast cancer. An early study reported that decreased expression of
Abbreviations: miRNA, MicroRNA; BC, breast cancer; MKNK2, MAP kinase interacting serine/threonine kinase 2; UTR, untranslated region; miR-125b, miRNA-125b. ⁎ Corresponding author at: Institute of Molecular and Chemical Biology, East China Normal University, 3663 North Road, Shanghai 200062, China. E-mail address: [email protected] (L. Lai). 1 These two authors contribute equally.
http://dx.doi.org/10.1016/j.genrep.2016.09.008 2452-0144/© 2016 Published by Elsevier Inc.
miR-125b was further found in ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) positive breast tumors (Mattie et al., 2006). Recent studies demonstrated that miR-125b functioned as a tumor suppressor in regulating the cell cycle and proliferation (Iorio et al., 2005). As a potential tumor suppressor factor, miR-125b was found to target several proto-oncogenes in cancers, such as BCL3 (Bcell CLL/lymphoma 3) in ovarian cancer (Guan et al., 2011) and E2F3 (E2F transcription factor 3) in bladder cancer (Huang et al., 2011). In breast cancer, v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1, a member of ETS transcription factor family), Mucin I, v-raf-1 murine leukemia viral oncogene homolog 1 (Raf1) and ERBB2/3 have been identified as the direct targets of miR-125b (Zhang et al., 2011; Rajabi et al., 2010; Hofmann et al., 2009; Scott et al., 2007). The human MAP kinase-interacting kinases comprise a group of four proteins derived from two genes (MKNK1 and MKNK2) by alternative splicing (Buxade et al., 2008). The isoforms of MKNKs differ markedly in their activity and regulation, and in subcellular localization which are activated by ERK or p38 and phosphorylate eIF4E (Buxade et al., 2008). Recent studies demonstrated an oncogenic role for MKNK2 in HER2-overexpressing cancers (Chrestensen et al., 2007). Early reports showed that miR-125b had negative effects on cellular proliferation of BC cells in an ERBB2 dependent manner (Ueda et al., 2010). In this study, we examined the expression of miR-125b and MKNK2 in breast tumors and its effects on breast cancer cell functions. We found that miR-125b was significantly down-regulated in breast cancer tissues and cell lines, compared with the matched adjacent cancer tissue and the basal-like breast epithelial cell line. Increased exogenous and endogenous expression of miR-125b could repress the proliferation and invasion of breast cell lines by directly targeting 3′ UTR of oncogenic MKNK2 that promoted cell growth and inhibited apoptosis. Our data imply that both miR-125b and MNK1/2 would be the potential therapeutic targets for breast cancer.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
2. Materials and methods 2.1. Human tissue samples Human samples from both breast tumor and normal tissues were obtained from Peking Union Medical College Hospital and Shanghai Huashan hospital. All participants provided written informed consent approved by the institutional review board at the participating hospitals. Tissue samples were stored in frozen liquid nitrogen after collection. Detailed clinical pathology information for all samples was available. Cases were classified and selected based on diagnosis using the CoPath Anatomic Pathology system. 2.2. Cell culture and RNA preparation MDA-MB-231, MDA-MB-435, MDA-MB-453, ZR-75-30, SK-BR-3, T47D, MCF 7 human breast cancer cell lines and HEK-293T cell line were purchased from ATCC. And MCF10A, immortalized nontumorigenic mammary cell line, was obtained from the Nanjing Medical University. MDA-MB-231, MDA-MB-435, MDA-MB-453, T47D and HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone) and ZR-75-30, SK-BR-3 were maintained in Roswell Park Memorial Institute 1640 medium (RPIM 1640; Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco). MCF7 cells were cultured in Minimum Essential Medium (MEM; Invitrogen) supplemented with 0.01 mg/mL bovine insulin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS; Gibco). MCF10A cells were cultured in DMEM/F12 (Invitrogen) supplemented with 0.5 μg/mL Hydrocortizone (Sigma-Aldrich), 0.1 μg/mL CholeraToxin (Sigma-Aldrich), 5% Horse Serum (Gibco), 0.2 μg/mL EGF (SigmaAldrich), 1 μg/mL Insulin (Sigma-Aldrich) and 1 mL Pen/Strep (Invitrogen). Total RNAs were extracted from tissues and cells in Trizol reagent (Invitrogen). 2.3. Lentivirus preparation, titration and infection HEK293T cells were seeded in 6 cm plate at 2.5 × 106 cells, and cotransfected with 5 μg of pLemiR-125b plasmid and 15 μL of the packaging mix stock using Arrest-In transfection reagent (Open Biosystems). The culture supernatants were collected 48 and 72 h after transfection, which were filtered through 0.45 μm filters and stored as virus stocks. For virus titration, TLA-HEK293T cells were seeded at the level of 5 × 104 cells per well with 24-well tissue culture plate in DMEM. The diluted virus were added to each well, and incubated at 37 °C for 4 h. Then, the transduction mix was removed from cultures, cells were cultured for 48 h for the virus titration. For virus infection, the virus were concentrated before infection using Amicon Centrifugal Filter Devices(Millipore), cells were incubated at 37 °C with both of virus and polybrene (8 mg/mL) for 6 h, then for 2 days to select puromycinresistant cells. 2.4. Oligonucleotide transfection Pre-miR-125b and pre-miR™ miRNA negative control precursors were purchased from Ambion (Austin). Cells were transfected at 24 h after plating using Lipofectamine 2000 reagent (Invitrogen). Transfection complexes were prepared according to the manufacturer's instructions, and added directly to the cells to a final oligonucleotide concentration of 100 nM. 2.5. Real-time polymerase chain reaction assays for mature miRNAs Total RNAs were extracted from tissue samples and cell lines for the analysis. The primer sequences were designed by Biomics Company for assaying the expression levels of miR-125b. Reverse transcription reaction was performed using 30 ng of total RNAs and 1 mM looped primers.
93
Real-time PCR was performed using the MX3000p Real-Time PCR Detection System (Stratagene) according to the standard SYBR Green Assay protocol. The 25 μL PCR reaction included 2 μL reverse transcription product, 1xPCR Master Mix (Takara), 1.5 mmol/L forward primer, and 0.7 mmol/L reverse primer. Thermal-cycling conditions of the reactions in a 96-well plate were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Data was examined by comparing relative miRNA expression to U6 RNA. Relative miRNA expression was determined using the 2-Ct method. All samples were analyzed in triplicate. 2.6. Western blot analysis For each sample, 25 μg of total protein extracts were separated on SDS-PAGE gels, and transferred to PVDF membrane. Antibody against MKNK2 (Cell Signaling Technology) was diluted at 1:200. PARP (Cell Signaling Technology) was diluted at 1:300 and EIF4E which was phosphorylated at S209 with a dilution of 1:500 were incubated at room temperature for 2 h with an AP-conjugated goat anti-rabbit secondary antibody (WesternDot™ 625 Goat Anti-Rabbit Western Blot Kit; Invitrogen). The results were visualized using BCIP/NBT (Amresco). As a loading control, beta-actin expression levels were measured by rabbit poyclonal anti-actin antibody (Santa Cruz Biotechnology). Bands were quantified with Labworks Instrument software (UVP LLC). 2.7. MiRNA target prediction and luciferase activity assay MiRNA target prediction was performed by in-lab developed algorithm KeyTar. KeyTar miRNA target prediction is based on miRNA: target sequence, structure, and function (unpublished). The primer sequences for amplifying the human 3′UTR region of MKNK2 gene as follows: MKNK2-3′UTR-F: 5′-GAATTCTCCGACCTTGACCTTAAAC-3′, and MKNK2-3′UTR-R: 5′-CTCGAGTCAGTTCACCTGGTACATT-3′, and cloned into the EcoRI and XhoI sites of the pGL3-control vector (Promega). Nucleotide-substitution mutations were carried out using PCR-based methods at the 3′UTR regions of MKNK2. Primers were as below: Mut 3′UTR of MKNK2: 5′-GAGTCCCCTCAGTCTGACCGTAGCCACG-3′ and 5′AAGGGGCACTCGGGTGCCCCAAGGG-3′. All constructs were verified by sequencing. For luciferase assay, the HEK293T and ZR-75-30 cells were seeded in 96-well plates, and transfected with 20 ng luciferase reporter plasmid, 1 ng pRL-TK vector expressing the Renilla luciferase (Promega), and 5 pmoles of miR125b or miRNA negative control precursor. Transfection was performed using Lipofectamine 2000 (Invitrogen). After transfection for 24–36 h, firefly and renilla luciferase activities were measured using the DualLuciferase Reporter Assay (Promega). All transfections were repeated twice in triplicate. 2.8. In vitro invasion assay ZR-75-30 and MDA-MB-453 cells were transfected with miR-125b or a scrambled control precursors. Forty-eight hours after transfection, cells (5 × 104) were plated in the top chamber with polymerized collagen-coated membrane (24-well insert; pore size, 8 μm; Chemicon ECM551) in medium with 0.1% FBS. Complete medium was used as a chemo attractant in the lower chamber. The left cells were incubated for 24 h, and unattached cells were removed cautiously by cotton swab. Cells that had invaded the lower surface of the membrane were stained with the Cell Stain Solution and counted accordingly. They were finally extracted and detected quantitatively by a standard microplate reader (at 560 nm). 2.9. Cell viability and cell proliferation analysis Cell growth assay with ZR-75-30 and MDA-MB-453 cells which were transfected with siMKNK2 and negative control (siNC) or pre-
94
Y. Wang et al. / Gene Reports 5 (2016) 92–97
miR-125b and pre-miR™ miRNA negative control precursors. In vitro proliferative kinetics were assayed by seeding 4 × 104 cells per well in 48-well plates and allowed to grow for different times. Total cell numbers in triplicate were measured every day, as indicated, by trypsinization and manual counting with a hemocytometer up to the 2th day.
2.10. Northern blot analysis Total RNAs were extracted using Trizol (Invitrogen) according to the manufacturer's instructions. The collected RNA samples (25 μg each) were electrophorized on 15% acrylamide and 8 M urea denature gels, then transferred onto Hybond N+ membrane (Amersham Biosciences). The membranes were agitated at 80 °C for 2 h before hybridization and were then hybridized with oligonucleotide probes corresponding to the complementary sequences of the following mature miRNAs: miR-21, 5′-TCAACATCAGTCTGATAAGCTA-3′; miR-143, 5′-GAGCTACAGTGCTT CATCTCA-3′ and miR-125b, 5′-TCACAAGTTAGGGTCTCAGGGA-3′. Probes were 5-end labeled using the polynucleotide kinase in the presence of [γ-32P] ATP (GE Healthcare). Hybridization was carried out at 39 °C in ULTRA hyb™-Oligo hybridization buffer (Ambion) for 16 h. Membranes were washed at 42 °C thrice with SSC (2×) plus 0.1% SDS. Northern blots were re-hybridized after stripping in 1% SDS for 30 min at 65 °C. In this part of the experiment, the U6 RNA probe (5′-GCTAAT CTTCTCTGTATCGTTCCAATTTT-3′) was used as a control for normalizing the RNA expression levels. Images were photo-documented using the scanner Storm 860 (Molecular Dynamics). Oligonucleotide probes were synthesized by Biosune (Shanghai, China).
2.11. Statistical analysis
Fig. 1. MiR-125b is underexpressed in breast cancer. (A). The expression levels of miR125b were analyzed in breast cancer cells by Northern blotting. The samples were assayed sequentially with radio-labeled probes specific to miR-125b, miR-143, miR-21 and small nuclear RNA U6. The U6 was used as the loading control. (B). The relative expression of miR-125b in a nonneoplastic control versus tumor cell lines has been normalized to the internal control U6 signal.
3.3. MiR-125b directly targets Mknk2 To understand molecular mechanism of miR-125b further in breast cancer cells, we found that 3′UTR of MKNK2 contained the highly conserved putative binding sites of miR-125b (Fig. 3a) using KeyTar miRNA target prediction algorithm. We constructed luciferase reporters carrying the 3′UTR of pGL3-MKNK2 with the putative and mutated miR125b binding sites (MKNK2-wt and MKNK2-mut). Luciferase assays revealed that increased expression of miR-125b caused a remarkable reduction of the luciferase activity in the MKNK2 gene reporter in miR125b oligo transfected cells, compared with mutated gene reporter (Fig. 3b). Moreover, Western blot analysis of MKNK2 in ZR-75-30 and MDA-MB-453 cell lines showed that transfection of miR-125b oligo decreased MKNK2 levels (Fig. 3c). These findings indicated that miR-125b could modulate MKNK2 at the translational level in breast cancer.
Data comparisons were performed using analysis of variance following Dunnett's method. The two-tailed Student's t-test was used to evaluate significance of the differences between cancer tissues or cell lines and normal controls. Statistical significance was determined as P b 0.05.
3. Results 3.1. MiR-125b is downregulated in human breast cancer To investigate whether miR-125b is involved in the development of breast cancer, we first examined the expression levels of miR-125b in human breast cancer cell lines including MCF7, T47D, ZR-75-30, MDAMB-453, SKBR3, MDA-MB-231, MDA-MB-435 by Northern Blot. The expression levels of miR-125b were greatly decreased in breast cancer cell lines, as compared with the basal-like breast epithelial cell line, MCF10A (Fig. 1a). The relative expression of miR-125b in a nonneoplastic control versus tumor cell lines has been normalized to the internal control U6 signal (Fig. 1b). Collectively, our results indicate that miR-125b is downregulated in breast cancer.
3.2. MiR-125b inhibits breast cancer cell growth and invasion in vitro To further investigate the role of miR-125b in breast cancer, we transfected the ZR-75-30 and MDA-MB-453 cells with precursor miR125b or miRNA scramble control (miR-scr) and examined the effect of miR-125b on cell growth and invasion. We found that over-expression of miR-125b decreased breast cancer cell invasion as indicated by invasion assay when compared with a miR-scr (Fig. 2a). In addition, cell growth assay results showed that over-expression of miR-125b exhibited a significant reduction of cell numbers when compared with the cells transfected with the miR-scr (Fig. 2b).
Fig. 2. MiR-125b inhibits breast cancer cell invasion and growth in vitro. (A). Matrigel invasion assay with MDA-MB-453 and ZR-75-30 cells transfected with miRNA scramble control or miR-125b oligo (left panel, 200×). Colorimetric measurement (relative level of optical density measured at 560 nm) of the stained cells for the miR-scr and miR125b transfected MDA-MB-453 and ZR7530 cells (right panel). miR-125b showed significant repression of tumor cell invasion comparing with the control. The data are expressed in mean ± S.E. (n = 3). (B). Cell growth assay with MDA-MB-453 and ZR7530 cells transfected with miRNA scramble control or miR-125b oligo. Total cell numbers in triplicate were assessed up to the 4th day. Cells transfected with miR-125b oligo exhibited a significant reduction in viable cells comparing with the negative control. a cells transfected with miRNA scramble control; b cells transfected with miR125b oligo. n = 3, ** p b 0.01,* p b 0.05.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
Fig. 3. MiR-125b directly targets MKNK2. (A). Schematic diagram of putative miR-125b banding sites in the 3′UTR region of MKNK2 gene. The possible binding sites of the target genes were searched using an in-house algorithm KeyTar (Liu et al., 2011). The conservation of the corresponding sites in the genomes of 28 species was analyzed using UCSC Genome Browser (NCBI36/hg18, http://genome.ucsc.edu/) and indicated in red box. (B). Luciferase assay. HEK 293t cells were transfected with miR-125b oligo or miRNA scramble control. Overexpression of miR-125b inhibited luciferase expression significantly when compared with the negative control. n = 3; ** p b 0.01. MKNK2-wt: MKNK2 wild type, MKNK2-Mut: MKNK2 mutation in miR-125b binding site indicated by underlined sequence as shown in panel A (upper panel). (C). Western blot analysis for MKNK2 levels in cells which were transfected with miR-125b oligo or miRNA scramble control. Significantly decreased levels of MKNK2 were shown after transfection. Actin was served as the internal control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Endogenous expression of miR-125b further confirms that miR-125b directly targets MKNK2 To confirm whether or not MKNK2 is a direct target of miR-125b further, we subsequently transfected ZR-75-30 and MDA-MB-453 cells with lentiviral vehicles which ubiquitously expressed miR-125b or a scrambled control precursors (Fig. 4a, upper panel). Semi-RT-qPCR analysis showed that stable-expression of miR-125b in both ZR-75-30 and MDA-MB-453 cells (Fig. 4a, lower panel). Consistent with results of luciferase assays observed in transient transfected cells, endogenous over-expression of miR-125b significantly reduced luciferase activities of MKNK2 gene reporter, but did not change the activity of mutated gene reporter (Fig. 4b). Furthermore, inhibition of endogenous expression of miR-125b restored the luciferase activity of MKNK2 gene reporter compared to mutated gene reporter in ZR-75-30 cells transfected with miR-125b antisense (Fig. 4b). Similarly, Western blot analysis revealed that the expression of MKNK2 was decreased by endogenous over-expression of miR-125b. In addition, we also found that up-regulation of miR-125b significantly reduced phosphorylation of EIF4 by MKNK2 and increased the cleavage of PARP, which resulted in cell apoptosis (Fig. 4c). In a word, our results further confirmed that miR-125b could target MKNK2 by directly binding to its 3’UTR in breast cancer in both sides.
3.5. Re-expression of MKNK2 reversed miR-125b dependent suppression in miR-125b expression cells To examine the effect of MKNK2 in breast cancer cells, we first performed Western blot analysis for MKNK2 expression levels in breast cancer cells and found that MKNK2 was up-regulated obviously (Fig. 5a). Then we conducted MKNK2 siRNA assay and found that cell viability was significantly decreased in MKNK2 siRNA-transfected cells, and not in siRNA scramble control transfected cells (Fig. 5b). Western
95
Fig. 4. Endogenous expression of miR-125b further confirms that miR-125b directly targets MKNK2. (A). Expression of miR-125b in ZR7530 or MDA-MB-453 cells which were transfected with lentivirus encoding miR-125b or miRNA scramble. Successful lentivirus transfection was visualized by red fluorescence in the upper panel. Marked miR-125b expression in miR-125b transfected cells was detected by semi-quantitative RT-PCR(lower panel). (B). Luciferase assay with miR-125b-overexpressed breast cancer cells. Compared with negative control ZR7530-mir-scr, over-expression of miR-125b inhibited luciferase expression (upper panel), while knockdown of miR-125b using miR125b antisense (miR-125bAS) increased luciferase expression significantly (lower panel). n = 3; * p b 0.05, ** p b 0.01. MKNK2-wt, MKNK2 coding sequence wild type; MKNK2-mt, MKNK2 coding sequence with mutation as indicated in Fig. 3a. (C). Western blot analysis of MKNK2 and its downstream genes. Significant decreased levels of MKNK2 was observed after 48 h infection. Cleaved PARP was also observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
blot analysis further confirmed that MKNK2 was down-regulated using the siRNA (Fig. 5c). Importantly, we found that re-expression of MKNK2 reversed miR-125b dependent suppression in miR-125b expression cells (Fig. 5d).
3.6. MiR-125b levels were correlated inversely with breast cancer malignancy and expression levels of target genes Finally, we tested a set of patient samples to verify whether MKNK2 protein expression correlates with miR-125b expression. As expected, in breast cancer samples, especially in HER2 positive ones, MKNK2 was over-expressed compared with two non-tumor breast tissues. What's more, the phosphorylation level of eIF4E was obviously increased (Fig. 6a). Then we performed qRT-PCR and found that miR125b was down-regulated dramatically in 28 invasive ductal carcinoma tissues compared with 12 normal tissues samples (a mixture containing 10 non-tumor breast samples and two non-tumor breast individuals) (Fig. 6b). But no significant difference of miR-125b expression was found between estrogen receptor (ER) positive (n = 8) versus ER negative (n = 7) breast cancers.
96
Y. Wang et al. / Gene Reports 5 (2016) 92–97
Fig. 5. Knockdown of MKNK2 inhibits breast cancer cell growth. (A). Western blotting analysis for MKNK2 levels in breast cancer cells. MCF10A was used as the normal control. Actin was used as the internal control. (B). Cell growth assay. In vitro proliferative kinetics were assayed by seeding 4 × 104 cells per well in 48-well plates and allowed to grow for different times. Total cell numbers in triplicate were assessed every day, as indicated. Cells transfected with siMKNK2 exhibit a significant reduction in viable cells comparing with cells transfected with the negative control (siNC). a cells transfected with a siRNA scramble control (siNC); b cells transfected with siMKNK2 oligo. n = 3, * p b 0.05. (C). Western blotting analysis for MKNK2 expression in cells which were transfected with siMKNK2 oligo or siNC. (D). Re-expression of MKNK2 reversed miR-125b mediated repression of breast cancer cell growth significantly. a miR-scr-453 cells transfected with a control plasmid; b miR-125b-453 cells transfected with a control plasmid and c miR-125b-453 cells transfected with a plasmid that express MKNK2. n = 3; * p b 0.05, ** p b 0.01.
4. Discussion Breast cancer is the most frequently diagnosed life-threatening cancer in women. Recent studies are focused on identifying novel therapeutic targets by better understanding the molecular mechanism of breast cancer. In this study, we demonstrated that miR-125b was down-
regulated in breast cancer and inhibited cell growth and invasion by directly targeting MKNK2. The decreased or increased expression of genes usually implied their functions as either tumor suppressors or oncogenes. Hence, we examined the expression of miR-125b in breast tumor cell lines and tumor tissue samples using two classical methods with Northern blot and qRT-
Fig. 6. MKNK2 expression is correlated with miR-125b status in Her2 positive breast cancer. (A). Western blotting analysis for MKNK2 levels in breast cancer tissues. Two human nontumor breast tissues were used as the normal control. Actin was used as the internal control. (B). qRT-PCR analysis of miR-125b levels in normal tissues from human non-tumor breast tissues (n = 12, a mixture containing 10 non-tumor breast samples and two non-tumor breast individuals) and invasive ductal carcinoma tissues (n = 28). U6 rRNA was used as a loading control. miR-125b levels inversely were correlated with malignancy in human breast tumors. p b 0.01. There were no significant differences between each stage of breast cancer (P N 0.05). No significant difference of miR-125b expression was found between estrogen receptor (ER) positive (n = 8) versus ER negative (n = 7) breast cancers.
Y. Wang et al. / Gene Reports 5 (2016) 92–97
PCR methods. We found that miR-125b is dysregulated in both tumor cell lines and tumor tissues, which was in accordance with recent studies which indicating that miR-125b functioned as a tumor suppressor (Zhang et al., 2011; Rajabi et al., 2010; Rajabi et al., 2010; Hofmann et al., 2009; Scott et al., 2007). In ovarian cancer, ectopic expression of miR-125b was found to induce tumor cell cycle arrest, reduction in proliferation and clone formation (Guan et al., 2011). In this study, we demonstrated that over-expression of miR-125b oligo in ZR-75-30 and MDA-MB-453 cell lines decreased breast cancer cell growth in vitro significantly. Since miR-125b directly targets Bcl2 family members or positive and negative regulators in the p53 pathway (Jia et al., 2012; Shi et al., 2011; Xia et al., 2009; Gong et al., 2012; Jiang et al., 2011; Le et al., 2011; Boulares et al., 1999), over-expression of miR125b might disrupts the pathway and then activates caspases cascade. Caspase-3 is an integrator of apoptosis signals both in vitro and in vivo, which was activated by initiator caspases and then in turn cleaves PARP, subsequently leading to downstream of death signal(Boulares et al., 1999; Shi et al., 2012). In this study, the significant cleavage increasement of PARP was detected in cells with endogenous expression of miR-125b. Consistent with other studies, our data further confirmed that miR-125b played an important role in breast cancer cell apoptosis. Several miR-125b targets have been recently identified in a variety of cancers including STAT3(Liu et al., 2011), E2F2(Wu et al., 2012), MAZ(Smits et al., 2012), BCL3(Guan et al., 2011), c-Raf(Hofmann et al., 2009), Bcl-w, Mcl-1(Jia et al., 2012; Gong et al., 2012), Bmf (Xia et al., 2009), FGFR2 (Xu et al., 2011) and MUC1 (Rajabi et al., 2010; Schroeder et al., 2001). In addition, decreased expression of miR-125b was further detected in ERBB2 positive breast tumors and overexpression of miR-125b could impair their anchorage-dependent growth and reduce migration and invasion capacities by suppressing ERBB2 and ERBB3 (Mattie et al., 2006; Scott et al., 2007). In a recent study, MKNK2 was reported as a possible target of miR-125b in neuroblastoma cells (Le et al., 2009). In this work, we identified MKNK2 as a direct target of miR-125b in breast cancer by systemic studies and demonstrated that MKNK2 is a key mediator of tumor cell proliferation. It has been found that MKNK2 is up-regulated and sustained activation in HER2-overexpressing breast cancer cells (Chrestensen et al., 2007). In this study, we found that miR-125b modulated MKNK2 expression by directly targeting the binding site within its 3′UTR. Knockdown of MKNK2 by siRNA inhibited breast cancer cell growth and invasion. Moreover, Re-expression of MKNK2 reversed miR-125b mediated repression of breast cancer cell growth significantly. Together, miR-125b inhibits breast cancer cell growth and invasion by directly targeting MKNK2. In summary, our data revealed an important role of miR-125b in breast cancer and suggested that both miR-125b and MKNK2 are important therapeutic targets in breast cancer.
References Bloomston, M., Frankel, W.L., Petrocca, F., Volinia, S., Alder, H., Hagan, J.P., Liu, C.G., Bhatt, D., Taccioli, C., Croce, C.M., 2007. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297 (17), 1901–1908. Boulares, A.H., Yakovlev, A.G., Ivanova, V., Stoica, B.A., Wang, G., Iyer, S., Smulson, M., 1999. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J. Biol. Chem. 274 (33), 22932–22940. Bousquet, M., Quelen, C., Rosati, R., Mansat-De Mas, V., La Starza, R., Bastard, C., Lippert, E., Talmant, P., Lafage-Pochitaloff, M., Leroux, D., et al., 2008. Myeloid cell differentiation arrest by miR-125b-1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation. J. Exp. Med. 205 (11), 2499–2506. Buxade, M., Parra-Palau, J.L., Proud, C.G., 2008. The Mnks: MAP kinase-interacting kinases (MAP kinase signal-integrating kinases). Front. Biosci. 13, 5359–5373. Calin, G.A., Croce, C.M., 2006. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6 (11), 857–866. Castaneda, C.A., Agullo-Ortuno, M.T., Fresno Vara, J.A., Cortes-Funes, H., Gomez, H.L., Ciruelos, E., 2011. Implication of miRNA in the diagnosis and treatment of breast cancer. Expert. Rev. Anticancer. Ther. 11 (8), 1265–1275.
97
Chrestensen, C.A., Shuman, J.K., Eschenroeder, A., Worthington, M., Gram, H., Sturgill, T.W., 2007. MNK1 and MNK2 regulation in HER2-overexpressing breast cancer lines. J. Biol. Chem. 282 (7), 4243–4252. Gong, J., Zhang, J.P., Li, B., Zeng, C., You, K., Chen, M.X., Yuan, Y., Zhuang, S.M., 2012. MicroRNA-125b promotes apoptosis by regulating the expression of Mcl-1, Bcl-w and IL-6R. Ontogeny. Griffiths-Jones, S., Saini, H.K., van Dongen, S., Enright, A.J., 2008. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36 (Database issue), D154–D158. Guan, Y., Yao, H., Zheng, Z., Qiu, G., Sun, K., 2011. MiR-125b targets BCL3 and suppresses ovarian cancer proliferation. Int. J. Cancer 128 (10), 2274–2283. Hofmann, M.H., Heinrich, J., Radziwill, G., Moelling, K., 2009. A short hairpin DNA analogous to miR-125b inhibits C-Raf expression, proliferation, and survival of breast cancer cells. Mol. Cancer Res. 7 (10), 1635–1644. Huang, L., Luo, J., Cai, Q., Pan, Q., Zeng, H., Guo, Z., Dong, W., Huang, J., Lin, T., 2011. MicroRNA-125b suppresses the development of bladder cancer by targeting E2F3. Int. J. Cancer 128 (8), 1758–1769. Iorio, M.V., Ferracin, M., Liu, C.G., Veronese, A., Spizzo, R., Sabbioni, S., Magri, E., Pedriali, M., Fabbri, M., Campiglio, M., et al., 2005. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65 (16), 7065–7070. Jia, H.Y., Wang, Y.X., Yan, W.T., Li, H.Y., Tian, Y.Z., Wang, S.M., Zhao, H.L., 2012. MicroRNA125b functions as a tumor suppressor in hepatocellular carcinoma cells. Int. J. Mol. Sci. 13 (7), 8762–8774. Jiang, F., Liu, T., He, Y., Yan, Q., Chen, X., Wang, H., Wan, X., 2011. MiR-125b promotes proliferation and migration of type II endometrial carcinoma cells through targeting TP53INP1 tumor suppressor in vitro and in vivo. BMC Cancer 11, 425. Kent, O.A., Mendell, J.T., 2006. A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Ontogeny 25 (46), 6188–6196. Le, M.T., Xie, H., Zhou, B., Chia, P.H., Rizk, P., Um, M., Udolph, G., Yang, H., Lim, B., Lodish, H.F., 2009. MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets. Mol. Cell. Biol. 29 (19), 5290–5305. Le, M.T., Shyh-Chang, N., Khaw, S.L., Chin, L., Teh, C., Tay, J., O'Day, E., Korzh, V., Yang, H., Lal, A., et al., 2011. Conserved regulation of p53 network dosage by microRNA-125b occurs through evolving miRNA-target gene pairs. PLoS Genet. 7 (9), e1002242. Li, M., Li, J., Ding, X., He, M., Cheng, S.Y., 2010. microRNA and cancer. AAPS J. 12 (3), 309–317. Liu, L.H., Li, H., Li, J.P., Zhong, H., Zhang, H.C., Chen, J., Xiao, T., 2011. MiR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3. Biochem. Biophys. Res. Commun. 416 (1–2), 31–38. Mattie, M.D., Benz, C.C., Bowers, J., Sensinger, K., Wong, L., Scott, G.K., Fedele, V., Ginzinger, D., Getts, R., Haqq, C., 2006. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol. Cancer 5, 24. Park, N.J., Zhou, H., Elashoff, D., Henson, B.S., Kastratovic, D.A., Abemayor, E., Wong, D.T., 2009. Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clin. Cancer Res. 15 (17), 5473–5477. Porkka, K.P., Pfeiffer, M.J., Waltering, K.K., Vessella, R.L., Tammela, T.L., Visakorpi, T., 2007. MicroRNA expression profiling in prostate cancer. Cancer Res. 67 (13), 6130–6135. Rajabi, H., Jin, C., Ahmad, R., McClary, C., Joshi, M.D., Kufe, D., 2010. Mucin 1 oncoprotein expression is suppressed by the miR-125b oncomir. Genes Cancer 1 (1), 62–68. Schroeder, J.A., Thompson, M.C., Gardner, M.M., Gendler, S.J., 2001. Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogenactivated protein kinase activation in the mouse mammary gland. J. Biol. Chem. 276 (16), 13057–13064. Scott, G.K., Goga, A., Bhaumik, D., Berger, C.E., Sullivan, C.S., Benz, C.C., 2007. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J. Biol. Chem. 282 (2), 1479–1486. Selcuklu, S.D., Yakicier, M.C., Erson, A.E., 2009. An Investigation of microRNAs Mapping to Breast Cancer. Shi, X.B., Xue, L., Ma, A.H., Tepper, C.G., Kung, H.J., White, R.W., 2011. miR-125b promotes growth of prostate cancer xenograft tumor through targeting pro-apoptotic genes. Prostate 71 (5), 538–549. Shi, L., Zhang, S., Feng, K., Wu, F., Wan, Y., Wang, Z., Zhang, J., Wang, Y., Yan, W., Fu, Z., et al., 2012. MicroRNA-125b-2 confers human glioblastoma stem cells resistance to temozolomide through the mitochondrial pathway of apoptosis. Int. J. Oncol. 40 (1), 119–129. Smits, M., Wurdinger, T., van het Hof, B., Drexhage, J.A., Geerts, D., Wesseling, P., Noske, D.P., Vandertop, W.P., de Vries, H.E., Reijerkerk, A., 2012. Myc-associated zinc finger protein (MAZ) is regulated by miR-125b and mediates VEGF-induced angiogenesis in glioblastoma. FASEB J. 26 (6), 2639–2647. Ueda, T., Sasaki, M., Elia, A.J., Chio, I.I., Hamada, K., Fukunaga, R., Mak, T.W., 2010. Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) delays tumor development. Proc. Natl. Acad. Sci. U. S. A. 107 (32), 13984–13990. Wong, T.S., Liu, X.B., Wong, B.Y., Ng, R.W., Yuen, A.P., Wei, W.I., 2008. Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue. Clin. Cancer Res. 14 (9), 2588–2592. Wu, N., Xiao, L., Zhao, X., Zhao, J., Wang, J., Wang, F., Cao, S., Lin, X., 2012. MiR-125b regulates the proliferation of glioblastoma stem cells by targeting E2F2. FEBS Lett. 586 (21), 3831–3839. Xia, H.F., He, T.Z., Liu, C.M., Cui, Y., Song, P.P., Jin, X.H., Ma, X., 2009. MiR-125b expression affects the proliferation and apoptosis of human glioma cells by targeting Bmf. Cell. Physiol. Biochem. 23 (4–6), 347–358. Xu, N., Brodin, P., Wei, T., Meisgen, F., Eidsmo, L., Nagy, N., Kemeny, L., Stahle, M., Sonkoly, E., Pivarcsi, A., 2011. MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2. J. Invest. Dermatol. 131 (7), 1521–1529. Zhang, Y., Yan, L.X., Wu, Q.N., Du, Z.M., Chen, J., Liao, D.Z., Huang, M.Y., Hou, J.H., Wu, Q.L., Zeng, M.S., et al., 2011. miR-125b is methylated and functions as a tumor suppressor by regulating the ETS1 proto-oncogene in human invasive breast cancer. Cancer Res. 71 (10), 3552–3562.