Oncogenic miR-20b-5p contributes malignant behavior via bidirectionally regulating CCND1 and E2F1 in breast cancer stem cells

Background Breast cancer is the leading cause of cancer mortality in women worldwide. Therefore, it is of great significance to identify the biological mechanism of tumorigenesis and explore the development of breast cancer to achieve a better prognosis for individuals suffering from breast cancer. MicroRNAs (miRNAs) have become a hot topic in cancer research, but the underlying mechanism remains unclear. Methods The miRNA profile between breast cancer stem cells (BCSCs, CD44 + CD24 −/low ) and control MCF-7 breast cancer cells was obtained in a previous study. Based on biological analysis, miR-20b-5p was hypothesized to be a key factor due to the malignant behavior of BCSCs. Then, agomir-20b-5p and antagomir-20b-5p were transfected into MCF-7 and T47D breast cancer cells to detect cell migration, wound healing and proliferation, and lentivirus vectors silencing or overexpressing miR-20b-5p were transfected into T47D-CSCs to detect proliferation and apoptosis. The effect of miR-20b-5p on xenograft growth was investigated in vivo by transfection of a lentivirus overexpression vector in T47D cells. The target genes were predicted by the online programs picTar, miRanda and TargetScan and verified by dual luciferase assay, and changes in protein expression were detected by western blot. Results miR-20b-5p had the highest degree in both the miRNA-gene network and miRNA-GO network to regulate BCSCs. Overexpression of miR-20b-5p significantly promoted the migration and wound healing ability of MCF-7 cells and T47D cells compared with the control ( P < 0.05). In addition, miR-20b-5p facilitated the proliferation of MCF-7 cells and T47D-CSCs ( P < 0.05) and inhibited the apoptosis of T47D-CSCs ( P < 0.05). Moreover, miR-20b-5p promoted xenograft growth compared with the control group ( P < 0.05). Accordingly, potential targets of both CCND1 and E2F1 were predicted by bioinformatics MCF-7 incubated µM EdU, flow cytometry EdU assay USA) manufacturer’s instructions. The five times. Stable transfection of a lentivirus overexpression system was used in the T47D-CSC proliferation assays. The lentiviral vector (GV369) overexpressing miR-20b-5p was purchased from GeneChem (China). The miR-20b-5p overexpression vector was labeled T47D-CSCs/LV-miR-20b-5p, and the control vector was labeled T47D-CSCs/LV-NC. The induced T47D-CSCs were suspended in EpiCult-B serum-free medium, and Lipofectamine 2000 (Invitrogen, USA) was added together with T47D-CSCs/LV-miR-20b-5p or T47D-CSCs/LV-NC. Then, the viruses were harvested. Cultured cells were infected with 5 µg/mL polybrene and lentivirus (MOI = 50) for 24 h, and then the cells were incubated with fresh medium for another 48 h to establish stable cell lines. The proliferation assay was conducted as described above. The test was repeated five times. lentivirus-miR-sponge T47D-CSC proliferation assays.


Background
Breast cancer is one of the most common cancers in women worldwide [1]. In recent years, although significant progress has been made in the diagnosis and treatment of malignant tumors, breast cancer remains the leading cause of cancer-related deaths in women, largely due to its high rate of relapse, metastasis, and drug resistance [2][3][4][5]. Therefore, it is of great significance to explore the mechanism of tumorigenesis in breast cancer and further identify key regulatory factors. Recent studies, including our previous findings, have demonstrated that miRNAs play an important role in regulating biological functions [6][7][8]. Because of their unique expression profile and special functions, miRNAs have been used as molecular markers to define the direction and processes of cell differentiation by regulating one or several target genes. miRNAs can also control the self-renewal or proliferation of stem cells [9]. Abnormal changes in miRNAs have been discovered in breast cancer, and miRNAs have been demonstrated to be associated with the tumorigenesis and progression of breast cancer [8,10]. Previous studies have confirmed that miRNAs play an important role in gene regulation in CD44 + CD24 −/low breast cancer stem cells (BCSCs). For example, we found that miR-200C was significantly downregulated in BCSCs and played a critical role in their biological features [8]. In addition, let-7a plays an important role in BCSCs' self-renewal by inhibiting the expression of H-Ras [11,12].
In our previous research, we performed miRNA profiling between sorted CD44 + CD24 −/low BCSCs and the control MCF-7 breast cancer cells [8]. Finally, miR-20b-5p was chosen for further study due to having the highest degree of regulation. We found that miR-20b-5p belongs to the miR-106a-363 cluster, which together with the miR-17-92 cluster and miR-106b-25 cluster forms a large family of highly similar miRNAs called the miR-17 family [13]. In many human malignancies, members of the miR-17 family have been reported to accumulate in tumor cells and are speculated to exert oncogenic effects [14,15], and miR-20b serves as a potential oncogene in gastric cancer [16], breast cancer [17][18] and hepatocellular carcinoma [19]. However, there has been no exploration of the regulatory role of miR-20b in BCSCs, and the underlying mechanism of miR-20b-5p remains unclear. This study aimed to investigate the role of the miR-20b-5p, a subtype of miR-20b family, in the regulation of the malignant behavior of BCSCs and to identify its target genes.

Methods
Induction culture of BCSCs MCF-7 and T47D cells in this experiment were cultured in DMEM-H (HyClone, USA) containing 10% fetal bovine serum (FBS) at 37 °C in a 5% CO 2 incubator. Cells in the logarithmic growth phase, approximately 70-80% confluent, were harvested and digested into a single cell suspension by trypsin digestion.
The complete MammoCult™ medium contained 5% MammoCult™ proliferation supplement, 4 µg/mL heparin, and 0.48 µg/mL hydrocortisone. MCF-7 cells were resuspended in complete MammoCult™ medium, and T47D cells were resuspended in complete DMEM/F-12 medium. The cell suspension (4 × 10 3 ) was seeded into a 6-well plate and cultured at 37 °C and 5% CO 2 . After 7 days of culture, the spheres were collected and centrifuged at 350 g for 5 min, and the supernatant was discarded. Then, 1 mL of Accutase cell dispersion solution was added to digest the spheres, followed by adding 9 mL of sterile PBS solution and centrifugation at 350 g for 5 minutes. Finally, the supernatant was discarded, and the cells were collected. CD44 + CD24 −/low BCSCs were isolated from MCF-7 and T47D cells by staining with CD44-APC, CD24-PE and ESA-FITC (BD Pharmingen, USA) antibodies via FACS as described in our previous research [8,20]. miRNA profile and miRNA network Both human miRNA microarray fabrication and hybridization were performed as described previously [8]. The miRNA profiles of both BCSCs and control MCF-7 breast cancer cells were obtained from CapitalBio Corporation (Beijing, China). All microarray data were uploaded and submitted to the public repository Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc = GSE68271). The differential miRNA profiles between BCSCs and MCF-7 cells were obtained.
To identify the miRNAs that regulate BCSCs, we established a miRNA-gene network and miRNA-GO network through analyses of the significant target genes and Gene Ontology (GO) terms performed by Shanghai Qiming Corporation (Shanghai, China).

Target prediction by bioinformatics
Chromosome localization, sequence analysis and target prediction of the miRNAs were carried out by the online programs picTar (http://pictar.mdc-berlin.de/), miRanda (http://microrna.sanger.ac.uk), and TargetScan (http://www.targetscan.org). The mRNAs associated with cell proliferation and the cell cycle predicted by at least three algorithms were selected as putative targets. Then, the binding free energy (△G) of the hybridization between miRNAs and their 3′ UTR complementary sites was analyzed with the aid of Mfold software. The mRNAs that could bind with lower free energy at both 5′-70 bp and 3′-70 bp than their average random free energy were deemed to be accessible to specific miRNAs [21].

RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. After the concentration and purity of total RNA were determined, reverse transcription was performed using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China). For qPCR analysis, cDNA was amplified with a SYBR Premix Ex Taq (TaKaRa) kit by using an AB 7500 Real-time PCR system. The relative gene expression was calculated by the 2 −ΔΔCt method. For miR-20b-5p qRT-PCR, the primers for miR-20b-5p (Forward: 5'-CAAAGTGCTCATAGTGCAG GTAG-3', Reverse 5'-GCAAAGTGCTCATAGTGCAGG-3') and U6 (Forward 5'-CTCGCTTCGGCAGCACA-3', Reverse 5'-AACGCTTCACGAATTTGCGT-3') were used. For qRT-PCR of potential target genes, primers are listed in Table 1. Table 1 Primers of potential target genes Gene Forward primer Reverse primer Transwell migration assay 6 Transient transfection of agomir-20b-5p and antagomir-20b-5p in MCF-7 and T47D breast cancer cells was performed as follows. After digestion, breast cancer cells were mixed with 1 mL DMEM-H containing 10% FBS and subjected to resuspension followed by cell counting. Then, the 24-well plates were inoculated at 4 × 10 4 cells per well, containing 500 µL of medium, and placed in a 37 °C, 5% CO 2 incubator overnight. The medium was removed the next day, the cells were washed with PBS three times, and then 500 µL of Opti-MEM medium was added to each well. with fresh medium for another 48 h to establish stable cell lines. The proliferation assay was conducted as described above. The test was repeated five times.
Stable transfection of a lentivirus-miR-sponge construct was used in T47D-CSC proliferation assays.
The miR-20b-5p sponge shRNA lentiviral vector (Hanbio Biotechnology) or control lentiviral vector was incubated at a final concentration of 30 nM for 24-48 h. The proliferation assay in induced T47D-CSCs was conducted as described above. The test was repeated five times.

Apoptosis assay in T47D-CSCs
The stable transfection of lentivirus overexpression and lentivirus-miR-sponge constructs was used to assay apoptosis in T47D-CSCs as described above. The induced T47D-CSCs were stained with antiannexin V/7-AAD antibodies in binding buffer for 15 min and then analyzed for apoptosis by flow cytometry. The test was repeated five times.

Animal experiments
The stable transfection of T47D cells overexpressing lentivirus was conducted as described above, Finally, immunoreactive proteins were visualized using a chemiluminescence detection system (FluorChem HD2, USA).

Bioinformatics analysis of target genes
We explored the target genes of miR-20b-5p related to cell proliferation and cell cycle and found 10 potential targets of miR-20b-5p, namely, CyclinD1 (CCND1), E2F1, MAPK1, STAT3, R2b23b, RAB5BR, RABEP1, TAOK3, PPARDR and XIAP. After transfection of agomir-20b-5p in MCF-7 cells, the qRT-PCR results showed that miR-20b-5p increased the expression of some of the potential target genes and inhibited the others (Fig. 2A). The dramatically increased expression of CCND1 and E2F1 was intriguing and unexpected, since miRNAs usually negatively regulate their target genes. Therefore, the contradictory preliminary results of qRT-PCR compelled us to choose CCND1 and E2F1 for subsequent validation and functional studies. The binding free energies between CCND1 and miR-20b-5p at both the 5′ UTR and 3′ UTR were − 14.50 kcal/mol and − 12.10 kcal/mol, respectively, lower than the average random free energy of CCND1 (0 kcal/mol). Likewise, the binding free energies between E2F1 and miR-20b-5p at both the 5′ UTR and 3′ UTR were − 18.10 kcal/mol and − 19.00 kcal/mol, respectively, lower than the average random free energy of E2F1 (− 3.60 kcal/mol) ( Fig. 2B) (Supplementary Fig. 2).

Verification of target genes
To verify the target genes of miR-20b-5p, we conducted three experiments.
First, we compared the correlation between miR-20b-5p expression and CCND1 and E2F1 mRNA levels in BCSCs and MCF-7 cells or T47D cells. The CD44 + CD24 −/low subpopulation was 2.58% and 64.3% of MCF-7 and induced MCF-7-CSCs, respectively (Fig. 4A). The expression of miR-20b-5p was 3.98 times and 2.21 times higher in BCSCs than in the control MCF-7 and T47D cells, respectively. Accordingly, the mRNA expression levels of E2F1 and CCND1 were higher in BCSCs than in MCF-7 and T47D cells, although the difference was not statistically significant (Fig. 6A).
Second, the potential targeting of CCND1 and E2F1-WT was further verified by dual luciferase assay.
Third, the protein expression levels of CCND1 and E2F1 were detected via western blot assay after agomir-20b-5p or antagomir-20b-5p transfection in MCF-7 cells. When the relative expression level of the control group was defined as 1, the relative expression levels of E2F1 and CCND1 were both 1.04 in the agomir-20b-5p group. The relative expression levels of E2F1 and CCND1 in the antagomir-20b-5p group were decreased to 0.46 and 0.45, respectively (Fig. 6C).

Discussion
To determine the normal function of miR-20b-5p, we searched for miR-20b expression in normal tissues using the public website miRNAmap (http://mirnamap.mbc.nctu.edu.tw/). The highest expression level was in the thymus, and the expression level was also high in the prostate, kidney, lung and other normal tissues (Supplementary Fig. 3).
Furthermore, we researched the reports on the miR-20b family in different tumors. The expression level of miR-20b was reported to be increased in gastric cancer [16], breast cancer [17][18] and hepatocellular carcinoma [19]. Wang B et al. [24] found that miR-20b promoted proliferation, migration, invasion and tumorigenicity in esophageal cancer cells. It was also reported that aberrant expression of miR-20b contributed to tumorigenesis and progression of breast cancer [25]. In the above literature, miR-20b is shown to play an oncogenic role. However, miR-20b includes both miR-20b-3p and miR-20b-5p, which might have different biological functions. Only one report showed that overexpressed miR-20b-5p in cancer tissue and patient serum had a regulatory effect on the proliferation and migration of breast cancer cells [26]. In addition, there has been no exploration of the regulatory role of miR-20b in BCSCs. In the current study, we focused on the function of miR-20b-5p and found that miR-20b-5p acted as an oncogene in the regulation of the malignant behavior of breast cancer and BCSCs.
Cancer stem cells (CSCs) have the ability of self-renewal and multidirectional differentiation [27].
Studies have shown that the molecular regulation of BCSCs is different from that of breast cancer cells [28]. BCSCs are associated with epithelial-mesenchymal transformation (EMT), miRNAs, the tumor microenvironment and other factors [29]. Our experimental results indicated that the upregulation of miR-20b-5p in MCF-7 and T47D cells significantly promoted the migration ability of breast cancer cells. Furthermore, overexpression and knockdown systems of miR-20b-5p in BCSCs were established. It was confirmed that miR-20b-5p promoted the proliferation of BCSCs and inhibited the apoptosis of BCSCs. As an integrated outcome of miR-20b-5p on breast cancer cells and stem cells, we observed that the xenografts of breast cancer cells were significantly enlarged by miR-20b-5p overexpression, which indicated that miR-20b-5p acted as an oncogene in vivo.
To further clarify the underlying mechanism of miR-20b-5p in the regulation of breast cancer cells and stem cells, we chose CCND1 and E2F1 as potential targets based on bioinformatics analysis and preliminary screening by qRT-PCR. CCND1, as an oncogene, is overexpressed in tumors and plays an important regulatory role in normal breast development, damage repair, maintenance of breast epithelial stem cell proliferation and self-renewal [30][31][32]. E2F1, a member of the E2F transcription factor family, regulates gene expression related to cell proliferation, differentiation and apoptosis and controls the cell cycle via a two-way regulatory mechanism. As a tumor suppressor gene or oncogene, E2F1 is closely related to tumor progression and drug resistance [33][34][35][36]. The literature shows that CCND1 and E2F1 are regulated by the miR-17-92 cluster, which is a miRNA cluster located on chromosome 13 that is composed of miR-17, miR-20a and miR-92a-1 [8,9]. This suggests that E2F1 is targeted by certain miRNAs and is involved in the regulation of tumor cells [37]. To confirm that miR-20b-5p regulated CCND1 and E2F1 expression, we performed three experiments, namely, a comparison between BCSCs and the control breast cancer cells, a dual luciferase assay and western blot analysis after overexpression or knockdown of miR-20b-5p. The dual-luciferase reporting system showed that CCND1 and E2F1 were real targets of miR-20b-5p. Moreover, our western blot analysis showed that antagomir-20b-5p significantly decreased the protein levels of both CCND1 and E2F1.
This seems a contradictory but interesting phenomenon. Our results revealed an indirect upregulation of CCND1 and E2F1 by miR-20b-5p. There likely are unknown targets (X and Y) negatively regulated by miR-20b-5p. The unknown targets (X and Y) could inhibit CCND1 and E2F1 protein expression.
Overall, miR-20b-5p regulated both CCND1 and E2F1 via bidirectional regulation, namely, direct downregulation and indirect upregulation. As a tug-of-war mechanism, the indirect promoting effect of miR-20b-5p on CCND1 and E2F1 may eclipse its direct downregulation of CCND1 and E2F1.
However, it is still necessary to reveal the underlying mechanism, which will be explored in further studies. The hypothesis involving the effect of miR-20b-5p on CCND1 and E2F1 is shown in a regulatory network (Supplementary Fig. 4).
As above, we conclude that miR-20b-5p has a cancer-promoting function in malignant behavior in both breast cancer cells and stem cells. We speculate that the underlying mechanism of miR-20b-5p contributing to the malignant behavior of breast cancer is that miR-20b-5p overall upregulates both CCND1 and E2F1 via bidirectional regulation.

Conclusion
As above, we conclude that miR-20b-5p has a cancer-promoting function in malignant behavior in both breast cancer cells and stem cells. We speculate that the underlying mechanism lies in miR-20b-5p overall enhanced both CCND1 and E2F1 targets via bidirectional regulation probably involving direct downregulation and indirect upregulation.

Ethic approval and consent to participate
The study was approved by the animal ethics committee of the Xinqiao Hospital of Army Medical University.

Consent for publication
Applicable.

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

Competing interests
The authors declare that they have no competing interests.

Funding
The work was supported by National Science Foundation of China (No: 30772108 and 81272910); and Chongqing Natural Science Foundation (No: cstc2012jjB10003). These are earmarked funds for cancer research. The funding bodies have no role in study design, data collection, data analysis, data interpretation, or manuscript preparation.

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