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MicroRNA-421-targeted PDCD4 regulates breast cancer cell proliferation

MicroRNAs (miRNAs) are expressed aberrantly in various types of cancer, and negatively regulate the expression of target genes which may be useful in therapeutic strategies in several biological processes. In the present study, the expression levels and the effects of miRNA (miR)-421 in breast cancer tissues and MCF-7 and MDA-MB-231 cells were evaluated to elucidate therapeutic targets in breast cancer cells. The putative targets of miR-421 were predicted by bioinformatics approaches, and the expression levels of miR-421 were measured in MCF-7 and MDA-MB-231 cells by reverse transcription-quantitative polymerase chain reaction analysis following miR-421 knockdown. The rates of cell proliferation, migration capacity, invasiveness and apoptosis were determined in miR-421 inhibitor-transfected MCF-7 and MDA-MB-231 cells. The expression levels of target proteins regulated by miR-421 in MCF-7 and MDA-MB-231 cells were analyzed by western blot analysis. miR-421 was increased significantly in breast cancer tissues and cells, and was regulated by miR-421 antisense oligonucleotides. The knockdown of miR-421 in MCF-7 and MDA-MB-231 cells decreased cell proliferation, migration capacity and invasiveness, and promoted apoptosis compared with control groups. The expression of target protein programmed cell death 4 (PDCD4) were decreased in MCF-7 and MDA-MB-231 cells transfected with miR-421 inhibitors. These results suggested a correlation between miR-421 and PDCD4, and physiological functions of breast cancer cells, suggesting that miR-421 may be a potential strategy in the therapy of breast cancer.


Introduction
Breast cancer (Bc) is one of the most common types of cancer and main causes of cancer-associated mortality in women worldwide (1). The onset of Bc typically occurs between 20 and 60 years of age, and the incidence of this disease has markedly increased in china in recent years (2). due to improvements in diagnostic and therapeutic methods, including surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy, stem cell therapy and gene silencing, the mortality rate of Bc has markedly decreased (3)(4)(5). However, the molecular mechanisms of Bc remain to be fully elucidated. It is well known that an accumulation of hereditary and epigenetic changes, including oncogene activation, tumor suppressor gene inactivation (6), changes in intercellular complex signal networks, microenvironment (7,8) and epigenetic changes (9,10) contribute to the formation of malignant tumors. An increasing number of studies have indicated that epigenetic changes may represent important events in the pathogenesis of Bc (11)(12)(13), and that gene silencing at the post-transcriptional level may be an important epigenetic change (14, 15).
Although prognostic microRNAs (miRNAs), including miRNA (miR)-21, miR-489 and miR125b, have been identified in Bc (16), the underlying pathways that regulate the invasiveness of Bc remain to be elucidated. miRNAs are able to bind to their target mRNAs to induce degradation or suppress translation (17). miRNAs are involved in cellular processes, including cell differentiation, cell proliferation, apoptosis and tumor inhibition (18)(19)(20). Previous studies have shown that miRNAs are critical in the development and progression of Bc, for example, by functioning as tumor suppressors or oncogenes. miR-421 is a tumor suppressor that is aberrantly expressed in several types of cancer. For example, miR-421 inhibits the proliferation and metabolism of prostate cancer cells by targeting cullin 4B (21) and suppresses Bc metastasis by targeting metastasis associated 1 (22). Furthermore, the higher positive detection rate of miR-421 compared with serum carcino-embryonic antigen in gastric cancer indicates that miR-421 may serve as a superior diagnostic marker (23). A number of studies have reported that miR-421 acts as an oncogene, however, its underlying mechanisms in Bc remain to be fully elucidated.
The aim of the present study was to investigate the regulatory role of miR-421 in Bc and the underlying molecular mechanism responsible.      immunohistochemistry results are presented. Pdcd4 protein expression was detected in three sets of five normal and five BC tissues in each set, using immunohistochemistry (Fig. 1A-c), and was decreased substantially in Bc tissues compared with normal control tissues.

Expression of miR-421 in BC tissues and cell lines.
RT-qPcR analysis was performed to assess the expression of miR-421 in Bc tissues, Hs578bst cells, McF-7 cells and MdA-MB-231 cells ( Fig. 2A and B). The expression of miR-421 was significantly increased in the Bc tissues and cells compared with the normal controls. By contrast, the protein expression of Pdcd4 was markedly downregulated in Bc cells compared with the controls (Fig. 2c and d). These results suggested that the overexpression of miR-421 was associated with the downregulation of Pdcd4 in Bc.
PDCD4 is a direct target gene of miR-421. Pdcd4 was found as a potential target gene of miR-421. Luciferase reporter assays were performed to further confirm whether PDCD4 is a direct target of miR-421. The 3'-UTR of PDCD4 mRNAs, including putative binding sites of miR-421 together with their corresponding mutated sequences, were cloned into vectors and co-transfected into Bc cells with miR-421 mimics, inhibitors or mock miRNA (Fig. 3A). Transfection with miR-421 mimics suppressed the luciferase activity significantly in McF-7 cells (Fig. 3B) and MdA-MB-231 cells (Fig. 3c); by contrast, miR-421 inhibitors significantly enhanced the luciferase activity in McF-7 cells (Fig. 3B) and MdA-MB-231 cells (Fig. 3c). The functions of the miR-421 mimics and inhibitors were abrogated when mutated 3'-UTR PDCD4-vector constructs were used, suggesting that miR-421 directly targets the 3'-UTRs of PDCD4.

Discussion
miRNAs have previously been reported to be associated with key developmental pathways, and various miRNA disorders contribute to a number of human ailments, including inflammatory diseases, infection, developmental disabilities and cancer (25). Abnormal miRNA expression has been reported in a number of types of cancer, with miRNAs serving tumor-suppressive or oncogenic roles to modulate tumor cell growth, cell cycle progression, migration and metastasis (26). miR-421, which is located in Xq13.2, has been reported to be involved in the post-transcriptional regulation of gene expression by affecting the stability and translation of genes in multicellular organisms, while regulating the expression of multiple tumor-promoting genes (27). Previous studies have shown that miR-421 is significantly overexpressed in gastric cancer tissues and promotes gastric cancer cell proliferation by downregulating the expression of caspase-3 (28). Furthermore, miR-421 may act as a tumor promoter in pancreatic cancer by targeting dPc4/Smad4 (29).
In the present study, it was found that miR-421 was significantly upregulated in BC tissues and cells compared with normal controls. However, miR-421 knockdown inhibited the proliferation, migration and invasion abilities of Bc cells, and promoted apoptosis. Pdcd4 was predicted as a target gene of miR-421 using TargetScan software. Pdcd4 is a tumor suppressor protein, which is able to induce apoptosis in tumor cells (30,32) by binding to the translation initiation factor eukaryotic initiation factor-4A, inhibiting RNA-helicase activity and in turn suppressing protein translation (33). Furthermore, Pdcd4 is able to inhibit activator protein-1-mediated transactivation (34) and induce the expression of cyclin-dependent kinase inhibitor p21 (35). Pdcd4 is involved in tumorigenesis and tumor progression (36,37) and is downregulated in Bc (38). These results suggest that miRNA-421 may function as an onco-miRNA in Bc cells by regulating Pdcd4.
The results of the present study indicated that miR-421 regulates the proliferation, migration, invasion and apoptosis of Bc cells by targeting Pdcd4. Pdcd4 is important in a number of cellular physiological activities. In conclusion, Pdcd4 may represent a novel therapeutic target for Bc, and miR-421 may be used as a potential treatment for Bc.

Acknowledgements
Not applicable.

Funding
No funding was received.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Authors' contributions
YW and ZL completed all experiments and wrote the manuscript. JS designed and guided all experiments in the paper. In addition, all authors revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
All protocols were approved by the Ethics committee of the Second Affiliated Hospital of Xi'an Medical University (Xi'an, china) and written informed consent was provided by all participants.

Patient consent for publication
All patients involved in the present study consented to the publication of their data.