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MiRNA-183-5p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting the PDCD4
Reproductive Biology 16 (2016) 225–233
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Original article
MiRNA-183-5p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting the PDCD4 Yan Chenga,* , Guixian Xianga , Yanbo Menga , Runzhi Dongb a b
Department of Pharmacology, Xingtai Medical College, Xingtai 054000, China Department of traditional Chinese medicine, Xingtai people’s hospital, Xingtai 054000, China
A R T I C L E I N F O
Article history: Received 25 February 2016 Received in revised form 23 June 2016 Accepted 11 July 2016 Available online 28 July 2016 Keywords: miR-183-5p PDCD4 Cell proliferation Cell cycle Apoptosis Breast cancer
A B S T R A C T
MicroRNAs are often aberrantly expressed in breast cancer and postulated to play a causal role in the onset and maintenance of breast cancer by binding to its target mRNA. Here, we evaluated the effects of miRNA-183-5p on cell proliferation and apoptosis which attempted to elucidate the potential role of miR-183-5p/PDCD4 axis in human breast cancer. We found that the miR-183-5p expression level was extremely promoted in breast cancer in comparison with the adjacent normal tissues. Overexpression of miR-183-5p significantly enhanced the cell proliferation and inhibited cell apoptosis in MCF-7 and MDAMB-231 cells. Moreover, PDCD4 was predicted as a putative target of miR-183-5p by bioinformatic approaches, and miR-183-5p negatively regulated the expression of PDCD4. Furthermore, knockdown of PDCD4 suppressed expression of p21 and p27, which was consistent with the result of the attachment of miR-183-5p. These data collectively demonstrate that miR-183-5p exerts oncomiRs effects in breast cancer, and may have broad impacts on the field of using antimiRs as anti-cancer drugs for breast cancer. ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
1. Introduction Breast cancer is the second leading cause of cancer-related deaths in women [1]. Approximately, there will be almost 1.2 million new cases of breast cancer each year [2]. Over the past decade, the utilization of mammography and new therapeutics development had significantly decreased the mortality rates, however the pathogenesis of breast cancer are still only partially understood [3]. MicroRNAs (miRNAs) are a class of endogenous short (about 19–25 nucleotides) noncoding RNAs. MiRNAs usually exerted its function via base pairing with the 30 -untranslated region (30 UTR) of target genes [4]. Accumulated evidence showed that the alteration or dysfunction of miRNAs might play vital roles in tumorigenesis through regulating the major regulators involved in cell cycle progression, apoptosis, autophagy, as well as migration & invasion [5–7]. Let-7 or miR-34 family members were indicated to exert pathogenic, diagnostic and prognostic roles in breast cancer [8–10].
* Corresponding author at: Department of Pharmacology, Xingtai Medical College, No. 618 Gangtie Road, Qiaoxi District, Xingtai 054000, Hebei Province, China. E-mail address: [email protected] (Y. Cheng).
MiR196a, acting as oncogenes, was reported to enhance cell proliferation in vitro by directly suppressing the expression of ANXA1 in breast cancer [11]. Recently, miRNA-mediated downregulation of programmed cell death 4 (PDCD4) was indicated to be involved in the posttranscriptional mechanisms of cancer cells [12]. The PDCD4 was identified as a tumor-related gene in humans, which is always repressed or lost in several tumor types [13–15]. To date, a set of miRNAs have been confirmed to target PDCD4, such as miR-96 in glioma cancer [16], miR-4262 in hepatocellular carcinoma [17], miR-499 in oropharyngeal cancer [18]. Although, accumulated evidence had demonstrated the correlation of miR-183 and PDCD4 in various tumors including papillary thyroid carcinoma [19], hepatocellular carcinoma [20], and gastric cancer [21], the exact mechanism of miR-183-5p/PDCD4 in cancer was still not clearly yet. The present study is devoted to investigate the role of miR-183-5p and PDCD4 in breast cancer and cells. Our findings suggested that the expression of miR-183-5p was significantly elevated and PDCD4 was inhibited simultaneously in breast cancer tissues. The introduction of miR-183-5p in vitro promoted breast cancer cell proliferation and inhibited cell apoptosis, which was consistent with the deficiency of PDCD4. Simultaneously, elevated expression of PDCD4 could abrogate the miR-183-5p promoted cell proliferation in breast
http://dx.doi.org/10.1016/j.repbio.2016.07.002 1642-431X/ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
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cancer cells. In summary, our evidence collectively suggested that miR-183-5p can exert oncogenic role by regulating the expression of PDCD4, which may provide a novel potential therapeutic target for breast cancer. 2. Materials and methods 2.1. Patient samples Written informed consents were obtained prior from all patients and the study was approved by the Institutional Review Board of the Xingtai Medical College, Xingtai, Hebei province, China. Eighteen pairs of breast cancer specimens and adjacent nontumor tissues were collected in the Xingtai people’s hospital (Xingtai, China) from December 2014 to December 2015. The age of patients was ranged from 35 to 58 (mean age 45). All cases had been confirmed by Hematoxylin-Eosin staining and immunohistochemical detection (ER, PR and HER2). Subsequently, the patients were divided into two groups (“well and moderate” vs “Poor or no”) according to the results of pathological differentiation (WHO classification). The clinical characteristics of patients are shown in Table 1. 2.2. Cell culture The breast cancer cell lines (MCF-7 and MDA-MB-231) and a non-malignant breast epithelial cell (MCF-10A) were obtained from ATCC and maintained in RPMI 1640 with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Grand Island, NY, USA) at a density of 5 104 cells per well in the 12-well plates.
5p and corresponding control (miR-NC/Anti-NC) were synthesized by GenePharma (Shanghai, China) and siRNA-PDCD and its corresponding control (siRNA-NC) were purchased from Ambion (Austin, Texas, USA). 1.0 106 cells were seeded overnight and transfected with 183-5p mimics(50 nM)/Anti-miR-183-5p (100 nM)/siRNA-PDCD (100 nM) or corresponding control the next day using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). 48 h later, the mRNA/protein was collected appropriately, which will subject to qRT-PCR/Western blot analysis subsequently. 2.4. Cell growth assay MTT assay and colony formation assay were applied to explore the effects of miR-183-5p and PDCD4 on the cell growth. For MTT assay, 3 103 cells per well were plated in 96-well plates and treated with miR-183-5p mimics or siRNA for 12, 24 and 48 h, respectively. The MTT (0.5 mg/ml; Sigma, St. Louis, MO, USA) was then added into each well (20 ml/well) and further incubated for 4 h, followed by resolving with 200 ml DMSO (Sigma, St. Louis, MO, USA). The absorbance was measured on a Microplate Reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA) at 570 nm. For colony formation assay, cells were seeded in 12-well plates at 100 cells per well and replaced with fresh medium every three days. After 14 days, the colonies were fixed with methanol, stained with crystal violet, photographed and counted under the microscope (IX71, Olympus, Tokyo, Japan). Each experiment was performed in triplicate. 2.5. Cell cycle and apoptosis assays
Breast Cancer Clinicopathological Characteristics
Number of Patients n = 18
MCF-7 and MDA-MB-231 cells were seeded in triplicate into six-well plates and treated with miR-183-5p mimics or control for 3 days. Subsequently, the cells were collected, fixed in 95% ethanol, incubated at 20 C overnight and washed with phosphate buffered saline (PBS). Finally, the cells were analyzed using a FACS Calibur flow cytometer (Beckman Coulter, Fullerton, CA, USA) after resuspending in 1 ml of FACS solution (PBS, 0.1% TritonX-100, 60 mg/ml propidium iodide (PI), 0.1 mg/ ml DNase free RNase, and 0.1% trisodium citrate). A total of 10,000 events were counted for each sample. Apoptosis rates were evaluated by flow cytometry (FACS Calibur, USA) with Annexin V-FITC assay kit (Invitrogen, Carlsbad, CA, USA). After transfection for 48 h, serum-containing medium was replaced by serum-free medium for additional 24 h to induce apoptosis. Cells were then harvested and washed twice with cold PBS. Subsequently, 500 ml PI buffer, 5 ml FITC annexin V, and 5 ml propidium iodide (PI) were added according to the established protocols [22].
Median Patient Age Median Tumor Size mm
45 [35–58] 42 [32–74]
2.6. Western blotting
Intrinsic Subtype Luminal A Luminal B HER2 Overexpressing Triple-Negative
10 2 3 3
ER Status ER Positive ER Negative
11 7
PR Status PR Positive PR Negative
10 8
HER2/neu Status HER2 Positive HER2 Negative
6 12
2.3. Plasmids, siRNA, and transfection The wildtype 30 UTR of PDCD4 was cloned into the pMIR-Report vector (Ambion, Austin, TX, USA) at downstream of the stop codon of the luciferase gene to generate the pMIR-PDCD4-30 UTR luciferase reporter plasmid. Mutagenesis of the pMIR-PDCD430 UTR was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The full-length human PDCD4 cDNA was PCR-amplified and cloned into EcoRl and BamHI sites of the pcDNA3 (Clontech, Mountain View, CA, USA) to generate pcDNA3-PDCD4. The miR-183-5p mimics/Anti-miR-183-
Table 1 The clinical characteristics of patients (n = 18).
The cell lysates were prepared with RIPA buffer (1xPBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% Sodium Deoxycholate, 1 mM Sodium Orthovanadate, 1% PMSF). And the proteins were separated electrophoretically by SDS-PAGE with 10% separation gel and 5% spacer gel. Proteins were transferred to polyvinylidene fluoride membrane at 250 mA for 1.5 h, and then the membrane was blocked with 5% non-fat milk overnight at 4 C. The membrane was incubated with antibody (PDCD4 or GAPDH, dilution 1:500; p21 and p27, dilution 1:1000; Santa Cruz, CA, USA,) at 4 C overnight. After washing, HRP-conjugated rabbit secondary monoclonal antibody (dilution 1:1000, Cell Signaling, Beverly, MA, USA) was added and further incubated at room temperature for 1 h. Densitometry analysis of the bands was performed using the software NIH imageJ (version 1.32j).
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2.7. RNA extraction and quantitative RT-PCR Total RNA was extracted from the cells using Trizol reagent (Life Technologies, USA) according to instruction. For the detection of miR-183-5p, total RNA was reverse-transcribed with a miR-183specific RT primer (RiboBio, China) followed by amplification with primers (RiboBio, China) on Roche Lightcycler (Roche, Basel, Switzerland). The U6 snRNA was used as internal reference. Realtime RT-PCR for PDCD4 was performed with specific primers (forward, 50 -TATGATGTGGAGGAGGTGGATGTGA-30 ; reverse, 50 CCTTTCATCCAAAGGCAAAACTACAC-30 ), and the relative expression level was normalized against GAPDH (forward, 50 -GGGAGCCAAAAGGGTCATCATCTC-30 ; and reverse, 50 - CCATGCCAGTGAGCTTCCCGTTC-3; Invitrogen, Life Technologies, USA). The fold changes were calculated using the comparative threshold cycle value (2DDCT) method.
2.8. Luciferase assay 5 104 cells were seeded in triplicate into six-well plates for 24 h and then treated with pMIR-PDCD4-30 UTR (wt/mut) and 183-5p mimics/Anti-miR-183-5p or corresponding control for 48 h incubation. Luciferase activity was measured using a dualluciferase reporter assay system (Promega, USA) and was measured with luminometer (SIRIUS, Pforzheim, Germany)
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described as previously [23]. Luciferase signal ratio (Rluc/Luc) was calculated for each construct. 2.9. Statistical analysis A Student0 s test was performed to analyze the significance of differences between the samples means obtained from three independent experiments. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. The expression profile of miR-183-5p in breast cancer tissues and cell lines To investigate the expression profile in breast cancer tissues and adjacent normal tissues, mRNA of the 18 pairs of tissue samples were extracted and measured with qRT-PCR. The results showed that miR-183-5p was generally upregulated in the breast cancer tissues compared with adjacent normal tissues (Fig. 1A). Interestingly, we observed that the expression of miR-183-5p was highly associated with tumor size and pathological differentiation. The expression of miR-183-5p was both substantially promoted in Poor or no differentiation tissues and tumor size greater than 5 cm compared to their counterpart (Fig. 1B and C, *p < 0.05). Furthermore, we employed MDA-MB-231, MCF-7 (two breast cancer cell
Fig. 1. Relative expression of miR-183-5p in breast cancer tissues and cell lines. (A) miR-183-5p was detected with qRT-PCR in breast cancer tissues and cell lines; (B) Tissues were separated with tumor size greater than 5 cm or less than 5 cm, and the expression of miR-183-5p was measured; (C) Tissues were separated as well/moderately differentiation tissues and poor/no differentiation tissues, and the expression of miR-183-5p was measured; (D) Relative expression of miR-183-5p in MCF-7, MDA-MB-231 and MCF-10A cells were measured with qRT-PCR. *p < 0.05. NC: Negative control; BC: Breast cancer.
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lines) and MCF-10A (non-malignant breast epithelial cell) to investigate the functional role of miR-183-5p in breast cancer cells. The evidence validated that the level of miR-183-5p was enhanced in MDA-MB-231 and MCF-7 cells compared to MCF-10A cells (Fig. 1D, *p < 0.05). 3.2. miR-183-5p promotes the proliferation of breast cancer cells To elucidate the role of miR-183-5p in breast cancer cell lines, MCF-7 and MDA-MB-231 cells were transfected with miR-183-5p mimics, which showed that the levels of miR-183-5p were obviously enhanced (Fig. 2A, *p < 0.05). MTT and colony formation assays demonstrated that cell proliferation of miR-183-5p mimics group was promoted in both MCF-7 and MDA-MB-231cells in comparison with control (Fig. 2B–D, *p < 0.05). Furthermore, FACS
assay and Annexin V-FITC/PI were also used to detect the effect of miR-183-5p on cell cycle and apoptosis. Our findings showed that the proportion of cells in G0/G1 phase was decreased while the proportion of cells in S phase was increased after transfection with miR-183-5p mimics (Fig. 2E, *p < 0.05). Moreover, the cell apoptosis of miR-183-5p mimics group was extremely suppressed compared with control (Fig. 2F, *p < 0.05). 3.3. miR-183-5p targets and inhibits PDCD4 expression in breast cancer cells To explore whether miR-183-5p can target PDCD4 in breast cancer, we performed bioinformatic approaches (miRanda, miRbase and TargetScan) and 30 UTR luciferase reporter assay. The result of bioinformatics analysis revealed that the binding sites of
Fig. 2. MiR-183-5p can promote the proliferation of breast cancer cells. (A) Transfection efficiency of miR-183-5p mimics was measured by qRT-PCR 48 h after transfection; (B) Cell growth was measured by colony formation assay14 days later; (C and D) Cell growth viability was measured by MTT assay 12, 24 and 48 h after transfection; (E) Cell cycle was determined by FACS analysis 48 h after transfection; (F) Cell apoptosis was measured by Annexin V-FITC/PI apoptosis assay 48 h after transfection. *p < 0.05.
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Fig. 3. miR-183-5p targets and suppresses PDCD4 expression. (A) Schematic binding sites of miR-183-5p in the 30 UTR region of PDCD4, the mutated binding sites was also showed; (B) PDCD4 is a direct target of miR-183-5p demonstrated by 30 UTR luciferase assay; (C) The mRNA of PDCD4 was measured by Real-time PCR in cells (MCF-7 and MDA-MB-231) transfected with miR-183-5p mimics or anti-miR-183-5p; (D and E) The protein expression of PDCD4 was measured by Western blot assay in cells (MCF-7 and MDA-MB-231) transfected with miR-183-5p mimics or anti-miR-183-5p. *p < 0.05.
30 UTR of PDCD4 was located at the miR-183-5p seed region (Fig. 3A). Simultaneously, the results of 30 UTR luciferase reporter assay sufficiently demonstrated that miR-183-5p mimics promisingly repressed the luciferase intensity of wild-type PDCD4 30 UTR whereas anti-miR-183-5p increased the luciferase intensity of wild-type PDCD4 30 UTR. However, miR-183-5p mimics or antimiR-183-5p had no statistical effect on the mutant PDCD4 30 UTR (Fig. 3B, *p < 0.05). In addition, we performed Real-time PCR and western blot to explore the relationship between miR-183-5p and PDCD4, the data strongly indicated that the mRNA and protein level of endogenous PDCD4 were mainly suppressed in cells transfected with miR-183-5p mimics. On the contrary, the mRNA and protein level of endogenous PDCD4 were essentially increased in cells transfected with Anti-miR-183-5p (Fig. 3C–E, *p < 0.05).
3.4. miR-183-5p promoted cell proliferation is mediated by PDCD4 The MTT and colony formation assays were utilized to detect the role of PDCD4 in breast cancer cells lines. The result revealed that the cell proliferation was promoted in MCF-7 and MDA-MB231cells transfected with PDCD4-siRNA (Fig. 4A and B). Furthermore, we applied western blot to explore whether miR-183-5p induced cell growth was mediated by PDCD4. The results showed that the expression of PDCD4 was suppressed in miR-183-5p mimics group and miR-183-5p repressed PDCD4 expression can be rescued by PDCD4 overexpression (Fig. 4C, *p < 0.05). Simultaneously, colony formation assay further identified that miR-183-5p induced cell proliferation can be abrogated by the PDCD4 overexpression (Fig. 4D). Previous evidence provided by Qiu
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Fig. 4. miR-183-5p promoted cell proliferation can be rescued by the expression of PDCD4. (A) Cell viability of different time point (24 h, 48 h, 72 h) was measured with MTT assay in cells transfected with PDCD4-siRNA; (B) Cell growth was measured by colony formation assay in the cells transfected with PDCD4-siRNA; (C) The expression of PDCD4 was measured by Western blot in the cells co-transfected with pcDNA3-PDCD4 (or pcDNA3) as well as miR-183-5p mimics (or miR-NC); (D) Cell growth was measured by Cell colony formation assay in the cells co-transfected with pcDNA3-PDCD4 (or pcDNA3) as well as miR-183-5p mimics (or miR-NC); (E) The expression of P21 and P27 were measured by Western blot in the cells co-transfected with PDCD4-siRNA (or siRNA-NC) and miR-183-5p mimics (or miR-NC). *p < 0.05.
suggested that miR-183 could directly target protein phosphatase2A (PP2A) mRNA 30 UTRs, resulting in reduced expression of p21 and p27 [24]. Therefore, we performed function-lost assay with PDCD4-siRNA to investigate whether p21 and p27 involved in the miR-183-5p/PDCD4 axis in human breast cancer. From Fig. 4E we can see, miR-183-5p can suppress the expression of p21 and p27, which was consistent with the results of PDCD4 knockdown (Fig. 4E). All these findings collectively confirmed that p21 and p27 were involved in the miR-183-5p/PDCD4 axis in breast cancer.
3.5. PDCD4 expression was downregulated in breast cancer and inversely correlated with miR-183-5p As the above results suggested PDCD4 is a direct target of miR183-5p and miR-183-5p could promote cell proliferation. Therefore, we performed Real-time PCR and western blot to explore the expression pattern of PDCD4 in breast tumor tissues and cells. The data validated that PDCD4 mRNA/protein level was downregulated in breast cancer tissues and cell lines compared with
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Fig. 5. The expression levels of PDCD4 in breast cancer tissues. (A) Relative mRNA expression of PDCD4 was measured by qRT-PCR in breast cancer tissues and adjacent normal tissues; (B) Relative mRNA expression of PDCD4 was measured by qRT-PCR in MCF-7 and MDA-MB-231 cells compared with MCF-10A cells; (C) Relative expression of PDCD4 protein was measured by western blot in MCF-7 and MDA-MB-231 cells compared with MCF-10A cells; (D) The expression of PDCD4 was inversely correlated with the expression of miR-183-5p. *p < 0.05.
the corresponding controls (Fig. 5A–C). Furthermore, we also investigated the relationship between PDCD4 and miR-183-5p, as we can see form Fig. 5D the expression level of PDCD4 was inversely correlated with the level of miR-138-5p. 4. Discussion Breast cancer, accounting for approximately 25% of tumors in females, can be dived into three major subtypes: ER+ tumors (including luminal A and luminal B subtypes), HER2 subtype, and triple negative/basal-like breast cancers [25]. The evidence obtained from comprehensive portrait of the genomic makeup of breast cancer also favors the current concept of traditionally classification of breast cancer [26]. Compared with Luminal A, Luminal B (ER+ and/or PR+, HER2-/HER2+ and Ki67+) is still difficult to select the appropriate therapy due to higher proliferative index, grade and worse prognosis [27]. HER2 subtype breast tumors are typically characterized with promoted ERBB2, which mainly received anti-HER2 therapy. Triple negative/basal-like breast cancers are the most malignant tumors among the three. Patients with triple negative/basal-like breast cancers are probably managed with cytotoxic chemotherapy, because there was no other effective therapy until now. Numerous studies have suggested the potential role of microRNAs as oncogenic or tumor suppressive factors in regulating cell cycle or apoptosis via controlling different target genes in cancers [28]. MicroRNAs are endogenous RNAs typically exert its role by complementarily to 30 untranslated region (30 UTR) of their target gene. A set of miRNAs such as miR-147 [29], miR-146a [30], miR138 [31], miR-340 [32], miR-221 [33] and miR-96 [34], have been found to play critical roles in tumors. Otherwise, previous
evidences indicated that abnormal expression miRNAs in triple negative breast cancer might affect the outcome of chemotherapy via regulating the translation of their target mRNA [35,36]. Here, we performed Real-time PCR analysis and found a remarkable up-regulation of miR-183-5p in breast cancer tissues and cells. MiR-183-5p, which is aberrantly expressed in different cancers, is closely associated with the aggressiveness of many tumors including breast cancer [36–39]. Especially, the expression level of the miR-183/182/96 cluster could devote to the prognostic of breast cancer [39]. In the current study, our findings suggested that miR-183-5p was upregulated in poor/no-differentiation tissues or tumor size greater than 5 cm. And overexpression of miR-183-5p significantly increased cell proliferation and suppressed the apoptosis of cells (MCF-7 and MDA-MB-231). MCF-7 and MDAMB-231cell lines had been reported to represent low and high invasive breast carcinoma with different proliferative potentials. But in our study there were no significant differences in the amplitude of miR-183-5p effects on cell proliferation, colony formation or cell cycle which needed to investigate in the future. It has been described that miRNAs exert their function via binding to the 30 untranslated regions (30 UTR) of their target mRNAs [40]. In the current study, the result of bioinformatics analysis revealed that the binding site of PDCD4 30 UTR was obviously located at miR-183-5p seed region. We also identified that miR-183-5p can suppress the mRNA and protein expression of PDCD4 via directly target its 30 UTR in breast cancer, which was consistent with previous studies [19–21], and the levels of PDCD4 was inversely correlated with miR-183-5p. To further confirm that miR-183-5p can directly target PDCD4, a knockdown plasmid of PDCD4 (PDCD4-siRNA) was used, which showed that PDCD4siRNA can promote the cell viability and growth (MCF-7 and MDA-
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MB-231). Moreover, overexpression of PDCD4 rescued miR-183-5p induced PDCD4 downregulation and abrogated miR-183-5p induced cell proliferation. PDCD4 was frequently under expressed in a variety of tumors and function as a tumor suppressor via inhibiting cell cycle progression or promoting cell apoptosis [13,41–43]. Previous data reported that PDCD4 basically exerts its function through repressing cell cycle progression at G1 stage and PDCD4 loss results in cell cycle acceleration in ovarian cancer [44]. It also has been demonstrated that PDCD4 can induce the expression of p21 and p27 [45]. p21 and p27 are members of cyclin-dependent kinase inhibitors, which can inhibit cell cycle progression by inducing G1, G2/M phase arrest [46,47]. Simultaneously, Qiu et al. validated that miR-183 could directly target protein phosphatase2A (PP2A) mRNA 30 UTRs, and result in reduced expression levels of p21 and p27 [24]. Therefore, we determined to investigate the association of miR-183-5p, p21, and p27. Our data demonstrated miR-183-5p can suppress the expression of p21 and p27, which was consistent with the results of PDCD4 knockdown, further suggesting that p21 and p27 were involved in the miR-183-5p/ PDCD4 axis in breast cancer. In conclusion, the data here suggested that inhibition of miR183-5p might be a potential approach to repress the progression of breast cancer through restoring PDCD4 levels, which might provide us new insights into a potential therapeutic strategy for blocking proliferation in breast cancer. Conflict of interests The authors declare no conflict of interests. Acknowledgement No funding. References [1] Vergne Y, Matta J, Morales L, Vargas W, Alvarez-Garriga C, Bayona M. Breast cancer and DNA repair capacity: association with use of multivitamin and calcium supplements. Integr Med 2013;12:38–46. [2] Polyak K. Heterogeneity in breast cancer. J Clin Invest 2011;121:3786–8. [3] Youlden DR, Cramb SM, Dunn NA, Muller JM, Pyke CM, Baade PD. The descriptive epidemiology of female breast cancer: an international comparison of screening, incidence, survival and mortality. Cancer Epidemiol 2012;36(June (3)):237–48. [4] Iorio MV, Casalini P, Piovan C, Braccioli L, Tagliabue E. Breast cancer and microRNAs: therapeutic impact. Breast 2011;20(Suppl. (3)):S63–70. [5] Piovan C, Palmieri D, Di Leva G, et al. Oncosuppressive role of p53-induced miR-205 in triple negative breast cancer. Mol Oncol 2012;6(August (4)):458– 72. [6] Wang D, Liu D, Gao J, et al. TRAIL-induced miR-146a expression suppresses CXCR4-mediated human breast cancer migration. FEBS J 2013;280(July (14)):3340–53. [7] Yang S, Li Y, Gao J, et al. MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1. Oncogene 2013;32(September (36)):4294–303. [8] Tong J, Fu Y, Xu X, et al. TGF-beta1 stimulates human Tenon's capsule fibroblast proliferation by miR-200b and its targeting of p27/kip1 and RND3. Invest Ophthalmol Vis Sci 2014;55(April (4)):2747–56. [9] Barh D, Malhotra R, Ravi B, Sindhurani P. MicroRNA let-7: an emerging nextgeneration cancer therapeutic. Curr Oncol 2010;17(February (1)):70–80. [10] Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302(February (1)):1–12. [11] Yuan Y, Anbalagan D, Lee LH, et al. ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and C-Myc to reduce breast cancerproliferation. Oncotarget 2016(April). [12] Liwak-Muir U, Dobson CC, Naing T, et al. ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism. Oncotarget 2016;7(January (2)):1439–50. [13] Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma. Oncogene 2006;25(October (45)):6101–12.
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Contents lists available at ScienceDirect
Reproductive Biology journal homepage: www.elsevier.com/locate/repbio
Original article
MiRNA-183-5p promotes cell proliferation and inhibits apoptosis in human breast cancer by targeting the PDCD4 Yan Chenga,* , Guixian Xianga , Yanbo Menga , Runzhi Dongb a b
Department of Pharmacology, Xingtai Medical College, Xingtai 054000, China Department of traditional Chinese medicine, Xingtai people’s hospital, Xingtai 054000, China
A R T I C L E I N F O
Article history: Received 25 February 2016 Received in revised form 23 June 2016 Accepted 11 July 2016 Available online 28 July 2016 Keywords: miR-183-5p PDCD4 Cell proliferation Cell cycle Apoptosis Breast cancer
A B S T R A C T
MicroRNAs are often aberrantly expressed in breast cancer and postulated to play a causal role in the onset and maintenance of breast cancer by binding to its target mRNA. Here, we evaluated the effects of miRNA-183-5p on cell proliferation and apoptosis which attempted to elucidate the potential role of miR-183-5p/PDCD4 axis in human breast cancer. We found that the miR-183-5p expression level was extremely promoted in breast cancer in comparison with the adjacent normal tissues. Overexpression of miR-183-5p significantly enhanced the cell proliferation and inhibited cell apoptosis in MCF-7 and MDAMB-231 cells. Moreover, PDCD4 was predicted as a putative target of miR-183-5p by bioinformatic approaches, and miR-183-5p negatively regulated the expression of PDCD4. Furthermore, knockdown of PDCD4 suppressed expression of p21 and p27, which was consistent with the result of the attachment of miR-183-5p. These data collectively demonstrate that miR-183-5p exerts oncomiRs effects in breast cancer, and may have broad impacts on the field of using antimiRs as anti-cancer drugs for breast cancer. ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
1. Introduction Breast cancer is the second leading cause of cancer-related deaths in women [1]. Approximately, there will be almost 1.2 million new cases of breast cancer each year [2]. Over the past decade, the utilization of mammography and new therapeutics development had significantly decreased the mortality rates, however the pathogenesis of breast cancer are still only partially understood [3]. MicroRNAs (miRNAs) are a class of endogenous short (about 19–25 nucleotides) noncoding RNAs. MiRNAs usually exerted its function via base pairing with the 30 -untranslated region (30 UTR) of target genes [4]. Accumulated evidence showed that the alteration or dysfunction of miRNAs might play vital roles in tumorigenesis through regulating the major regulators involved in cell cycle progression, apoptosis, autophagy, as well as migration & invasion [5–7]. Let-7 or miR-34 family members were indicated to exert pathogenic, diagnostic and prognostic roles in breast cancer [8–10].
* Corresponding author at: Department of Pharmacology, Xingtai Medical College, No. 618 Gangtie Road, Qiaoxi District, Xingtai 054000, Hebei Province, China. E-mail address: [email protected] (Y. Cheng).
MiR196a, acting as oncogenes, was reported to enhance cell proliferation in vitro by directly suppressing the expression of ANXA1 in breast cancer [11]. Recently, miRNA-mediated downregulation of programmed cell death 4 (PDCD4) was indicated to be involved in the posttranscriptional mechanisms of cancer cells [12]. The PDCD4 was identified as a tumor-related gene in humans, which is always repressed or lost in several tumor types [13–15]. To date, a set of miRNAs have been confirmed to target PDCD4, such as miR-96 in glioma cancer [16], miR-4262 in hepatocellular carcinoma [17], miR-499 in oropharyngeal cancer [18]. Although, accumulated evidence had demonstrated the correlation of miR-183 and PDCD4 in various tumors including papillary thyroid carcinoma [19], hepatocellular carcinoma [20], and gastric cancer [21], the exact mechanism of miR-183-5p/PDCD4 in cancer was still not clearly yet. The present study is devoted to investigate the role of miR-183-5p and PDCD4 in breast cancer and cells. Our findings suggested that the expression of miR-183-5p was significantly elevated and PDCD4 was inhibited simultaneously in breast cancer tissues. The introduction of miR-183-5p in vitro promoted breast cancer cell proliferation and inhibited cell apoptosis, which was consistent with the deficiency of PDCD4. Simultaneously, elevated expression of PDCD4 could abrogate the miR-183-5p promoted cell proliferation in breast
http://dx.doi.org/10.1016/j.repbio.2016.07.002 1642-431X/ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
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cancer cells. In summary, our evidence collectively suggested that miR-183-5p can exert oncogenic role by regulating the expression of PDCD4, which may provide a novel potential therapeutic target for breast cancer. 2. Materials and methods 2.1. Patient samples Written informed consents were obtained prior from all patients and the study was approved by the Institutional Review Board of the Xingtai Medical College, Xingtai, Hebei province, China. Eighteen pairs of breast cancer specimens and adjacent nontumor tissues were collected in the Xingtai people’s hospital (Xingtai, China) from December 2014 to December 2015. The age of patients was ranged from 35 to 58 (mean age 45). All cases had been confirmed by Hematoxylin-Eosin staining and immunohistochemical detection (ER, PR and HER2). Subsequently, the patients were divided into two groups (“well and moderate” vs “Poor or no”) according to the results of pathological differentiation (WHO classification). The clinical characteristics of patients are shown in Table 1. 2.2. Cell culture The breast cancer cell lines (MCF-7 and MDA-MB-231) and a non-malignant breast epithelial cell (MCF-10A) were obtained from ATCC and maintained in RPMI 1640 with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Grand Island, NY, USA) at a density of 5 104 cells per well in the 12-well plates.
5p and corresponding control (miR-NC/Anti-NC) were synthesized by GenePharma (Shanghai, China) and siRNA-PDCD and its corresponding control (siRNA-NC) were purchased from Ambion (Austin, Texas, USA). 1.0 106 cells were seeded overnight and transfected with 183-5p mimics(50 nM)/Anti-miR-183-5p (100 nM)/siRNA-PDCD (100 nM) or corresponding control the next day using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). 48 h later, the mRNA/protein was collected appropriately, which will subject to qRT-PCR/Western blot analysis subsequently. 2.4. Cell growth assay MTT assay and colony formation assay were applied to explore the effects of miR-183-5p and PDCD4 on the cell growth. For MTT assay, 3 103 cells per well were plated in 96-well plates and treated with miR-183-5p mimics or siRNA for 12, 24 and 48 h, respectively. The MTT (0.5 mg/ml; Sigma, St. Louis, MO, USA) was then added into each well (20 ml/well) and further incubated for 4 h, followed by resolving with 200 ml DMSO (Sigma, St. Louis, MO, USA). The absorbance was measured on a Microplate Reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA) at 570 nm. For colony formation assay, cells were seeded in 12-well plates at 100 cells per well and replaced with fresh medium every three days. After 14 days, the colonies were fixed with methanol, stained with crystal violet, photographed and counted under the microscope (IX71, Olympus, Tokyo, Japan). Each experiment was performed in triplicate. 2.5. Cell cycle and apoptosis assays
Breast Cancer Clinicopathological Characteristics
Number of Patients n = 18
MCF-7 and MDA-MB-231 cells were seeded in triplicate into six-well plates and treated with miR-183-5p mimics or control for 3 days. Subsequently, the cells were collected, fixed in 95% ethanol, incubated at 20 C overnight and washed with phosphate buffered saline (PBS). Finally, the cells were analyzed using a FACS Calibur flow cytometer (Beckman Coulter, Fullerton, CA, USA) after resuspending in 1 ml of FACS solution (PBS, 0.1% TritonX-100, 60 mg/ml propidium iodide (PI), 0.1 mg/ ml DNase free RNase, and 0.1% trisodium citrate). A total of 10,000 events were counted for each sample. Apoptosis rates were evaluated by flow cytometry (FACS Calibur, USA) with Annexin V-FITC assay kit (Invitrogen, Carlsbad, CA, USA). After transfection for 48 h, serum-containing medium was replaced by serum-free medium for additional 24 h to induce apoptosis. Cells were then harvested and washed twice with cold PBS. Subsequently, 500 ml PI buffer, 5 ml FITC annexin V, and 5 ml propidium iodide (PI) were added according to the established protocols [22].
Median Patient Age Median Tumor Size mm
45 [35–58] 42 [32–74]
2.6. Western blotting
Intrinsic Subtype Luminal A Luminal B HER2 Overexpressing Triple-Negative
10 2 3 3
ER Status ER Positive ER Negative
11 7
PR Status PR Positive PR Negative
10 8
HER2/neu Status HER2 Positive HER2 Negative
6 12
2.3. Plasmids, siRNA, and transfection The wildtype 30 UTR of PDCD4 was cloned into the pMIR-Report vector (Ambion, Austin, TX, USA) at downstream of the stop codon of the luciferase gene to generate the pMIR-PDCD4-30 UTR luciferase reporter plasmid. Mutagenesis of the pMIR-PDCD430 UTR was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The full-length human PDCD4 cDNA was PCR-amplified and cloned into EcoRl and BamHI sites of the pcDNA3 (Clontech, Mountain View, CA, USA) to generate pcDNA3-PDCD4. The miR-183-5p mimics/Anti-miR-183-
Table 1 The clinical characteristics of patients (n = 18).
The cell lysates were prepared with RIPA buffer (1xPBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% Sodium Deoxycholate, 1 mM Sodium Orthovanadate, 1% PMSF). And the proteins were separated electrophoretically by SDS-PAGE with 10% separation gel and 5% spacer gel. Proteins were transferred to polyvinylidene fluoride membrane at 250 mA for 1.5 h, and then the membrane was blocked with 5% non-fat milk overnight at 4 C. The membrane was incubated with antibody (PDCD4 or GAPDH, dilution 1:500; p21 and p27, dilution 1:1000; Santa Cruz, CA, USA,) at 4 C overnight. After washing, HRP-conjugated rabbit secondary monoclonal antibody (dilution 1:1000, Cell Signaling, Beverly, MA, USA) was added and further incubated at room temperature for 1 h. Densitometry analysis of the bands was performed using the software NIH imageJ (version 1.32j).
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2.7. RNA extraction and quantitative RT-PCR Total RNA was extracted from the cells using Trizol reagent (Life Technologies, USA) according to instruction. For the detection of miR-183-5p, total RNA was reverse-transcribed with a miR-183specific RT primer (RiboBio, China) followed by amplification with primers (RiboBio, China) on Roche Lightcycler (Roche, Basel, Switzerland). The U6 snRNA was used as internal reference. Realtime RT-PCR for PDCD4 was performed with specific primers (forward, 50 -TATGATGTGGAGGAGGTGGATGTGA-30 ; reverse, 50 CCTTTCATCCAAAGGCAAAACTACAC-30 ), and the relative expression level was normalized against GAPDH (forward, 50 -GGGAGCCAAAAGGGTCATCATCTC-30 ; and reverse, 50 - CCATGCCAGTGAGCTTCCCGTTC-3; Invitrogen, Life Technologies, USA). The fold changes were calculated using the comparative threshold cycle value (2DDCT) method.
2.8. Luciferase assay 5 104 cells were seeded in triplicate into six-well plates for 24 h and then treated with pMIR-PDCD4-30 UTR (wt/mut) and 183-5p mimics/Anti-miR-183-5p or corresponding control for 48 h incubation. Luciferase activity was measured using a dualluciferase reporter assay system (Promega, USA) and was measured with luminometer (SIRIUS, Pforzheim, Germany)
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described as previously [23]. Luciferase signal ratio (Rluc/Luc) was calculated for each construct. 2.9. Statistical analysis A Student0 s test was performed to analyze the significance of differences between the samples means obtained from three independent experiments. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. The expression profile of miR-183-5p in breast cancer tissues and cell lines To investigate the expression profile in breast cancer tissues and adjacent normal tissues, mRNA of the 18 pairs of tissue samples were extracted and measured with qRT-PCR. The results showed that miR-183-5p was generally upregulated in the breast cancer tissues compared with adjacent normal tissues (Fig. 1A). Interestingly, we observed that the expression of miR-183-5p was highly associated with tumor size and pathological differentiation. The expression of miR-183-5p was both substantially promoted in Poor or no differentiation tissues and tumor size greater than 5 cm compared to their counterpart (Fig. 1B and C, *p < 0.05). Furthermore, we employed MDA-MB-231, MCF-7 (two breast cancer cell
Fig. 1. Relative expression of miR-183-5p in breast cancer tissues and cell lines. (A) miR-183-5p was detected with qRT-PCR in breast cancer tissues and cell lines; (B) Tissues were separated with tumor size greater than 5 cm or less than 5 cm, and the expression of miR-183-5p was measured; (C) Tissues were separated as well/moderately differentiation tissues and poor/no differentiation tissues, and the expression of miR-183-5p was measured; (D) Relative expression of miR-183-5p in MCF-7, MDA-MB-231 and MCF-10A cells were measured with qRT-PCR. *p < 0.05. NC: Negative control; BC: Breast cancer.
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lines) and MCF-10A (non-malignant breast epithelial cell) to investigate the functional role of miR-183-5p in breast cancer cells. The evidence validated that the level of miR-183-5p was enhanced in MDA-MB-231 and MCF-7 cells compared to MCF-10A cells (Fig. 1D, *p < 0.05). 3.2. miR-183-5p promotes the proliferation of breast cancer cells To elucidate the role of miR-183-5p in breast cancer cell lines, MCF-7 and MDA-MB-231 cells were transfected with miR-183-5p mimics, which showed that the levels of miR-183-5p were obviously enhanced (Fig. 2A, *p < 0.05). MTT and colony formation assays demonstrated that cell proliferation of miR-183-5p mimics group was promoted in both MCF-7 and MDA-MB-231cells in comparison with control (Fig. 2B–D, *p < 0.05). Furthermore, FACS
assay and Annexin V-FITC/PI were also used to detect the effect of miR-183-5p on cell cycle and apoptosis. Our findings showed that the proportion of cells in G0/G1 phase was decreased while the proportion of cells in S phase was increased after transfection with miR-183-5p mimics (Fig. 2E, *p < 0.05). Moreover, the cell apoptosis of miR-183-5p mimics group was extremely suppressed compared with control (Fig. 2F, *p < 0.05). 3.3. miR-183-5p targets and inhibits PDCD4 expression in breast cancer cells To explore whether miR-183-5p can target PDCD4 in breast cancer, we performed bioinformatic approaches (miRanda, miRbase and TargetScan) and 30 UTR luciferase reporter assay. The result of bioinformatics analysis revealed that the binding sites of
Fig. 2. MiR-183-5p can promote the proliferation of breast cancer cells. (A) Transfection efficiency of miR-183-5p mimics was measured by qRT-PCR 48 h after transfection; (B) Cell growth was measured by colony formation assay14 days later; (C and D) Cell growth viability was measured by MTT assay 12, 24 and 48 h after transfection; (E) Cell cycle was determined by FACS analysis 48 h after transfection; (F) Cell apoptosis was measured by Annexin V-FITC/PI apoptosis assay 48 h after transfection. *p < 0.05.
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Fig. 3. miR-183-5p targets and suppresses PDCD4 expression. (A) Schematic binding sites of miR-183-5p in the 30 UTR region of PDCD4, the mutated binding sites was also showed; (B) PDCD4 is a direct target of miR-183-5p demonstrated by 30 UTR luciferase assay; (C) The mRNA of PDCD4 was measured by Real-time PCR in cells (MCF-7 and MDA-MB-231) transfected with miR-183-5p mimics or anti-miR-183-5p; (D and E) The protein expression of PDCD4 was measured by Western blot assay in cells (MCF-7 and MDA-MB-231) transfected with miR-183-5p mimics or anti-miR-183-5p. *p < 0.05.
30 UTR of PDCD4 was located at the miR-183-5p seed region (Fig. 3A). Simultaneously, the results of 30 UTR luciferase reporter assay sufficiently demonstrated that miR-183-5p mimics promisingly repressed the luciferase intensity of wild-type PDCD4 30 UTR whereas anti-miR-183-5p increased the luciferase intensity of wild-type PDCD4 30 UTR. However, miR-183-5p mimics or antimiR-183-5p had no statistical effect on the mutant PDCD4 30 UTR (Fig. 3B, *p < 0.05). In addition, we performed Real-time PCR and western blot to explore the relationship between miR-183-5p and PDCD4, the data strongly indicated that the mRNA and protein level of endogenous PDCD4 were mainly suppressed in cells transfected with miR-183-5p mimics. On the contrary, the mRNA and protein level of endogenous PDCD4 were essentially increased in cells transfected with Anti-miR-183-5p (Fig. 3C–E, *p < 0.05).
3.4. miR-183-5p promoted cell proliferation is mediated by PDCD4 The MTT and colony formation assays were utilized to detect the role of PDCD4 in breast cancer cells lines. The result revealed that the cell proliferation was promoted in MCF-7 and MDA-MB231cells transfected with PDCD4-siRNA (Fig. 4A and B). Furthermore, we applied western blot to explore whether miR-183-5p induced cell growth was mediated by PDCD4. The results showed that the expression of PDCD4 was suppressed in miR-183-5p mimics group and miR-183-5p repressed PDCD4 expression can be rescued by PDCD4 overexpression (Fig. 4C, *p < 0.05). Simultaneously, colony formation assay further identified that miR-183-5p induced cell proliferation can be abrogated by the PDCD4 overexpression (Fig. 4D). Previous evidence provided by Qiu
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Fig. 4. miR-183-5p promoted cell proliferation can be rescued by the expression of PDCD4. (A) Cell viability of different time point (24 h, 48 h, 72 h) was measured with MTT assay in cells transfected with PDCD4-siRNA; (B) Cell growth was measured by colony formation assay in the cells transfected with PDCD4-siRNA; (C) The expression of PDCD4 was measured by Western blot in the cells co-transfected with pcDNA3-PDCD4 (or pcDNA3) as well as miR-183-5p mimics (or miR-NC); (D) Cell growth was measured by Cell colony formation assay in the cells co-transfected with pcDNA3-PDCD4 (or pcDNA3) as well as miR-183-5p mimics (or miR-NC); (E) The expression of P21 and P27 were measured by Western blot in the cells co-transfected with PDCD4-siRNA (or siRNA-NC) and miR-183-5p mimics (or miR-NC). *p < 0.05.
suggested that miR-183 could directly target protein phosphatase2A (PP2A) mRNA 30 UTRs, resulting in reduced expression of p21 and p27 [24]. Therefore, we performed function-lost assay with PDCD4-siRNA to investigate whether p21 and p27 involved in the miR-183-5p/PDCD4 axis in human breast cancer. From Fig. 4E we can see, miR-183-5p can suppress the expression of p21 and p27, which was consistent with the results of PDCD4 knockdown (Fig. 4E). All these findings collectively confirmed that p21 and p27 were involved in the miR-183-5p/PDCD4 axis in breast cancer.
3.5. PDCD4 expression was downregulated in breast cancer and inversely correlated with miR-183-5p As the above results suggested PDCD4 is a direct target of miR183-5p and miR-183-5p could promote cell proliferation. Therefore, we performed Real-time PCR and western blot to explore the expression pattern of PDCD4 in breast tumor tissues and cells. The data validated that PDCD4 mRNA/protein level was downregulated in breast cancer tissues and cell lines compared with
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Fig. 5. The expression levels of PDCD4 in breast cancer tissues. (A) Relative mRNA expression of PDCD4 was measured by qRT-PCR in breast cancer tissues and adjacent normal tissues; (B) Relative mRNA expression of PDCD4 was measured by qRT-PCR in MCF-7 and MDA-MB-231 cells compared with MCF-10A cells; (C) Relative expression of PDCD4 protein was measured by western blot in MCF-7 and MDA-MB-231 cells compared with MCF-10A cells; (D) The expression of PDCD4 was inversely correlated with the expression of miR-183-5p. *p < 0.05.
the corresponding controls (Fig. 5A–C). Furthermore, we also investigated the relationship between PDCD4 and miR-183-5p, as we can see form Fig. 5D the expression level of PDCD4 was inversely correlated with the level of miR-138-5p. 4. Discussion Breast cancer, accounting for approximately 25% of tumors in females, can be dived into three major subtypes: ER+ tumors (including luminal A and luminal B subtypes), HER2 subtype, and triple negative/basal-like breast cancers [25]. The evidence obtained from comprehensive portrait of the genomic makeup of breast cancer also favors the current concept of traditionally classification of breast cancer [26]. Compared with Luminal A, Luminal B (ER+ and/or PR+, HER2-/HER2+ and Ki67+) is still difficult to select the appropriate therapy due to higher proliferative index, grade and worse prognosis [27]. HER2 subtype breast tumors are typically characterized with promoted ERBB2, which mainly received anti-HER2 therapy. Triple negative/basal-like breast cancers are the most malignant tumors among the three. Patients with triple negative/basal-like breast cancers are probably managed with cytotoxic chemotherapy, because there was no other effective therapy until now. Numerous studies have suggested the potential role of microRNAs as oncogenic or tumor suppressive factors in regulating cell cycle or apoptosis via controlling different target genes in cancers [28]. MicroRNAs are endogenous RNAs typically exert its role by complementarily to 30 untranslated region (30 UTR) of their target gene. A set of miRNAs such as miR-147 [29], miR-146a [30], miR138 [31], miR-340 [32], miR-221 [33] and miR-96 [34], have been found to play critical roles in tumors. Otherwise, previous
evidences indicated that abnormal expression miRNAs in triple negative breast cancer might affect the outcome of chemotherapy via regulating the translation of their target mRNA [35,36]. Here, we performed Real-time PCR analysis and found a remarkable up-regulation of miR-183-5p in breast cancer tissues and cells. MiR-183-5p, which is aberrantly expressed in different cancers, is closely associated with the aggressiveness of many tumors including breast cancer [36–39]. Especially, the expression level of the miR-183/182/96 cluster could devote to the prognostic of breast cancer [39]. In the current study, our findings suggested that miR-183-5p was upregulated in poor/no-differentiation tissues or tumor size greater than 5 cm. And overexpression of miR-183-5p significantly increased cell proliferation and suppressed the apoptosis of cells (MCF-7 and MDA-MB-231). MCF-7 and MDAMB-231cell lines had been reported to represent low and high invasive breast carcinoma with different proliferative potentials. But in our study there were no significant differences in the amplitude of miR-183-5p effects on cell proliferation, colony formation or cell cycle which needed to investigate in the future. It has been described that miRNAs exert their function via binding to the 30 untranslated regions (30 UTR) of their target mRNAs [40]. In the current study, the result of bioinformatics analysis revealed that the binding site of PDCD4 30 UTR was obviously located at miR-183-5p seed region. We also identified that miR-183-5p can suppress the mRNA and protein expression of PDCD4 via directly target its 30 UTR in breast cancer, which was consistent with previous studies [19–21], and the levels of PDCD4 was inversely correlated with miR-183-5p. To further confirm that miR-183-5p can directly target PDCD4, a knockdown plasmid of PDCD4 (PDCD4-siRNA) was used, which showed that PDCD4siRNA can promote the cell viability and growth (MCF-7 and MDA-
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MB-231). Moreover, overexpression of PDCD4 rescued miR-183-5p induced PDCD4 downregulation and abrogated miR-183-5p induced cell proliferation. PDCD4 was frequently under expressed in a variety of tumors and function as a tumor suppressor via inhibiting cell cycle progression or promoting cell apoptosis [13,41–43]. Previous data reported that PDCD4 basically exerts its function through repressing cell cycle progression at G1 stage and PDCD4 loss results in cell cycle acceleration in ovarian cancer [44]. It also has been demonstrated that PDCD4 can induce the expression of p21 and p27 [45]. p21 and p27 are members of cyclin-dependent kinase inhibitors, which can inhibit cell cycle progression by inducing G1, G2/M phase arrest [46,47]. Simultaneously, Qiu et al. validated that miR-183 could directly target protein phosphatase2A (PP2A) mRNA 30 UTRs, and result in reduced expression levels of p21 and p27 [24]. Therefore, we determined to investigate the association of miR-183-5p, p21, and p27. Our data demonstrated miR-183-5p can suppress the expression of p21 and p27, which was consistent with the results of PDCD4 knockdown, further suggesting that p21 and p27 were involved in the miR-183-5p/ PDCD4 axis in breast cancer. In conclusion, the data here suggested that inhibition of miR183-5p might be a potential approach to repress the progression of breast cancer through restoring PDCD4 levels, which might provide us new insights into a potential therapeutic strategy for blocking proliferation in breast cancer. Conflict of interests The authors declare no conflict of interests. Acknowledgement No funding. References [1] Vergne Y, Matta J, Morales L, Vargas W, Alvarez-Garriga C, Bayona M. Breast cancer and DNA repair capacity: association with use of multivitamin and calcium supplements. Integr Med 2013;12:38–46. [2] Polyak K. Heterogeneity in breast cancer. J Clin Invest 2011;121:3786–8. [3] Youlden DR, Cramb SM, Dunn NA, Muller JM, Pyke CM, Baade PD. The descriptive epidemiology of female breast cancer: an international comparison of screening, incidence, survival and mortality. Cancer Epidemiol 2012;36(June (3)):237–48. [4] Iorio MV, Casalini P, Piovan C, Braccioli L, Tagliabue E. Breast cancer and microRNAs: therapeutic impact. Breast 2011;20(Suppl. (3)):S63–70. [5] Piovan C, Palmieri D, Di Leva G, et al. Oncosuppressive role of p53-induced miR-205 in triple negative breast cancer. Mol Oncol 2012;6(August (4)):458– 72. [6] Wang D, Liu D, Gao J, et al. TRAIL-induced miR-146a expression suppresses CXCR4-mediated human breast cancer migration. FEBS J 2013;280(July (14)):3340–53. [7] Yang S, Li Y, Gao J, et al. MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1. Oncogene 2013;32(September (36)):4294–303. [8] Tong J, Fu Y, Xu X, et al. TGF-beta1 stimulates human Tenon's capsule fibroblast proliferation by miR-200b and its targeting of p27/kip1 and RND3. Invest Ophthalmol Vis Sci 2014;55(April (4)):2747–56. [9] Barh D, Malhotra R, Ravi B, Sindhurani P. MicroRNA let-7: an emerging nextgeneration cancer therapeutic. Curr Oncol 2010;17(February (1)):70–80. [10] Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302(February (1)):1–12. [11] Yuan Y, Anbalagan D, Lee LH, et al. ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and C-Myc to reduce breast cancerproliferation. Oncotarget 2016(April). [12] Liwak-Muir U, Dobson CC, Naing T, et al. ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism. Oncotarget 2016;7(January (2)):1439–50. [13] Zhang H, Ozaki I, Mizuta T, et al. Involvement of programmed cell death 4 in transforming growth factor-beta1-induced apoptosis in human hepatocellular carcinoma. Oncogene 2006;25(October (45)):6101–12.
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