MiRNA-142-3p increases radiosensitivity in human umbilical cord blood mononuclear cells by inhibiting the expression of CD133

This study is to explore the molecular regulation mechanism of CD133 which is associated with malignancy and poor prognosis of blood system diseases. CD133+HUCB-MNC (human umbilical cord blood mononuclear cells) and CD133−HUCB-MNC were isolated and amplificated from umbilical cord blood, and then were exposed to different doses of radiation and subjected to a clonogenic assay. CCK-8 kit was used to detect cell viability, Annexin V-FITC/PI cell apoptosis detection kit was used for the detection of apoptotic cells and the BrdU assay was performed by flow cytometry. The expression of protein was analyzed by western blots. The profile of miRNA expression in response to radiation was examined and validated by RT-PCR. miR-142-3p inhibited the expression of CD133 in umbilical cord blood mononuclear cells to increase radiosensitivity. CD133+HUCB-MNC cells were more radioresistant compared with CD133−HUCB-MNC cells. CD133+HUCB-MNC cells showed higher p-AKT and p-ERK levels after radiation. And miR-142-3p acted on 3′UTR of CD133 mRNA to inhibit CD133 expression. Moreover, miRNA-142-3p mimic increased radiosensitivity in CD133+HUCB-MNC cells. Our results elucidated a novel regulation pathway in hematopoietic stem cells and suggested a potential therapeutic approach for blood system diseases therapy.

Radiotherapy is widely used for cancer treatment and the most common side effect is the bone marrow suppression 1 . Human umbilical cord blood mononuclear cells (hUCB-MNCs) is a suitable source of progenitor and stem cells, including subcomponents such as hematopoietic stem cells (HSCs), mesenchymal stem cell (MSCs), and endothelial progenitor cells (EPCs). Umbilical cord blood stem cells possess multi-differentiation potentials as mesoblast precursor 2 which can differentiate into leukocytes, adipocytes, osteoblasts, muscle tendons and cardiocytes under the proper induction conditions 3 . Umbilical cord blood stem cells can differentiate into endothelial cells or MSCs both in vitro and in vivo and improve the poorly functioning organs 4 . Intracranial injection of hUCB-MNC during the hyperacute phase of ischemic stroke could improve cerebrovascular function and reduce infarct volume and behavioral deficits 5 .
The CD133 is a transmembrane glycoprotein which is considered as a significant cancer-associated cell surface marker. The expression of CD133 has been elevated in plenty of cancer cell types. CD133 + colon cancer cells showed chemoresistance to 5-fluorouracil by increasing the survivin expression 6 . CD133 facilitates the CSC-like properties by stabilizing EGFR-AKT signaling in Hepatocellular carcinoma cells (HCC) 7 . CD133 is a positive marker for a specific class of human cord blood-derived CD34-negative HSCs 8 . Radiotherapy leads to myelosuppression, while CD133 could resist radiotherapy-induced bone marrow suppression 9 . CD133+ cells were the source of most of the stem cells present in the HUCB-MNC, and CD133 was critical for the radiosensitivity of HUCB-MNCs 10 .
MicroRNAs (miRNAs) are key regulators for some cellular processes. Specific expression signatures have been found in different blood cell lineages and stages of HSC differentiation during hematopoiesis 11 . MiRNAs are small, non-coding RNAs found in the eukaryotes that control the expression of a large number of genes 12 involved in commitment and differentiation of hematopoietic stem cells and tumorigenesis 13  has been a growing body of evidence supporting the role of miRNA in the regulation of CSCs recently 14 . For example, microRNA-139-5p regulates the proliferation of hematopoietic progenitors and is repressed during BCR-ABL-mediated leukemogenesis 15 .
Therefore, alterations in miRNAs can contribute to the inhibition of HSCs differentiation. MicroRNA-134-3p is a new potential inhibitor of human ovarian CSCs by targeting the RAB27A 16 . Wei-Wei Shen et al. reported that miR-142-3p functioned as a tumor suppressor by targeting CD133, ABCG2, and Lgr5 in colon cancer cells 17 . In this study, we identified miR-142-3p as a key modulator of CD133+ HUCB-MNC cells through pathways involving CD133, AKT and ERK pathways.

Discussion
Umbilical cord blood is considered as an alternative source for stem cell transplantation and therapy due to its hematopoietic and mesenchymal properties 20 . For both HSCs and leukemia stem cells (LSCs), specific gene expression programs distinguish them from their differentiated progenies 21 . Human HSCs are a primary target of radiation-induced leukemogenesis and provide a correlation cellular model for evaluating the risk of cancer.
The CD133 is considered as a tumor marker in many cancer types. The therapeutic capacity of HUCB-MNC and stem cells is documented in animal models of focal cerebral ischemia, CD133+HUCB-MNC reduced neuronal apoptosis in the range of normoxic controls 18 . Radiotherapy leads to myelosuppression, but CD133 could resist radiotherapy-induced bone marrow suppression. CD133 can be phosphorylated on cytoplasmic tyrosine-828 by the Src family kinases 22 . And the Src family kinases can be activated rapidly by multiple cell surface receptors combination and regulate many cellular processes that include proliferation, differentiation and adhesion 23 . In our study, the results indicated that CD133+HUCB-MNC cells were more radioresistant compared with CD133−HUCB-MNC cells due to an increase in the proliferation rate, a decrease in the radiation induced apoptosis and prolonged S phase after radiation exposure, and CD133+HUCB-MNC cells showed higher p-AKT and p-ERK levels after radiation. CD133+ is a marker of human progenitor and hematopoietic stem cells; repair signaling in CD133+UCBC was different from CD133− UCBC and PBL. These differences included higher 53BP1 recruitment and lower endogenous DSB levels 24 . CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells by activating of PI3K/Akt pathway 25 . CD133+cells are used for treatment of leukemia, liver regeneration, neurodegenerative diseases and myocardial infarction 26 .
MicroRNAs are post-transcriptional regulators which bind to complementary sequences on target messenger RNA, usually leading to translational repression or target degradation and gene silencing 27 . MiRNAs were proved to play important roles in control of multifaceted of HSC and LSC biology 28 . Recent studies have showed that roles of miRNAs in reaction to physiologic and pathologic stress such as DNA damage in fully developed tissues. For example, micro-RNA30c negatively regulates REDD1 expression in human hematopoietic and osteoblast cells after irradiation 1 . Our results of a luciferase assay showed that miR-142-3p silenced CD133 by binding to the 3′-UTR of CD133 mRNA, and miRNA-142-3p mimic increased radiosensitivity in CD133+ HUCB-MNC cells. Therapeutic delivery of miR142-3p in ATRT cells suggested effective reduction in its lethality by repressing tumor growth, inhibiting invasion, enhancing radiosensitivity, and prolonging survival time in orthotropic-transplanted immunocompromised mice 29 . Moreover miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC 21 .

Conclusions
In conclusion, CD133+HUCB-MNC cells were more radioresistant compared with CD133−HUCB-MNC cells. CD133+HUCB-MNC cells showed higher p-AKT and p-ERK levels after radiation. The results of a luciferase assay showed that miR-142-3p silenced CD133 by binding to the 3′-UTR of CD133 mRNA. Overexpression of miR-142-3p in human CD133+HUCB-MNC cells can not only inhibit the expression of CD133 but also can inhibit the proliferation in vitro, arrest cell cycle progression, and promote apoptosis. This study gains an insight into the mechanism of radiotherapy resistance and gives a new strategy for blood system disease therapies in the future. Identification of the markers under the control of miRNA-142-3p and their regulation in CD133+HUCB-MNC cells may help develop efficient targeted treatments, improved diagnostic methods, and better prognostic evaluation.

Isolation of primary umbilical cord blood mononuclear cells.
Blood samples (about 50 mL) were collected from three fresh placentas with attached umbilical cords by gravity flow. MNCs were isolated by density gradient centrifugation over Biocoll (Biochrom, Berlin, Germany) for 30 min at 400 × g and washed three times in phosphate buffered saline (PBS) (Biochrom) 2 . MNCs were counted and incubated for 30 min at 4 °C with anti-human CD133 (eBioscience, CA, USA). CD133+/− MNCs were purified by FACS (fluorescence-activated cell sorter) sorting using a FACSAria III flow cytometer (BD Biosciences, CA, USA). The FACS-sorting purity were more than 90%. The mononuclear cells were inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL-3, and 5 ng/mL Granulocyte-macrophage colony-stimulating factor (GM-CSF), and cultured in a humidified 5% CO 2 atmosphere at 37 °C. The medium was replaced by half at a 3-day interval until the attached cells grew to 80% confluence, and then used for the following experiments 30 . All experimental procedures were approved by Laboratory Animal Ethics Committee of Chinese PLA general hospital. The principles Hematopoietic colony formation assay. A volume of 1 × 10 5 /ml CD133+HUCB-MNC Cells or 1 × 10 5 /ml CD133−HUCB-MNC Cells were plated in 12-well plate culture flasks coated with methylcellulose and 10 ng/ml Granulocyte colony stimulating factor (G-CSF). After 24 h, the cells were irradiated with graded doses (0, 2, 4, 6, 8 Gy) using X-ray generator (Primus High-Energy Siemens) at a dose rate of 2 Gy/min. After 10 days of incubation in a humidified 5% CO 2 atmosphere at 37 °C, the colonies were fixed. Those colonies containing ≥50 cells were scored as viable colonies. The data were fit into the single-hit multi-target model, and the survival curve of each group was demonstrated by Graphpad prism 6.0 software (San Diego, CA, USA) 32 . The hematopoietic colony formation assays were performed in triplicate.
CCK8 Cell proliferation assay. CCK-8 kit (Beyotime, China) was used to detect cell viability 6 . Briefly, CD133+HUCB-MNC cells or CD133−HUCB-MNC cells were seeded into 96-well plate (Corning Costar, Cambridge, MA) at a density of 0.2 × 10 4 cells per well. After 24 h, the cells were irradiated with graded doses (0, 2, 4, 6, 8 Gy) using X-ray generator (Primus High-Energy Siemens) at a dose rate of 2 Gy/min. 14 h after radiation, 10 μL of CCK-8 solution was added, followed by incubation for 3 h at 37 °C. The optical density (OD) was measured at 450 nm to reflect the cell viability. Detection was done in 6 wells per group, and blank controls were also detected. The proliferation assays were performed in triplicate.

Detection of cell apoptosis by flow cytometry. Annexin V-FITC/PI cell apoptosis detection kit (BD
Biosciences, SanJose, CA) was used for the detection of apoptotic cells. Cells in logarithmic growth phase were used to prepare single cell suspension at 1 × 10 6 cells/ml. After addition of propidium iodide (PI) and FITC Annexin V, the apoptotic cells were detected by flow cytometry (BD Biosciences), and apoptosis rate was calculated. Experiment was done thrice, and data were analyzed with Flowjo (tree star) 33 .
BrdU labeled cell cycle analysis. Briefly, cells were incubated with 10 μM BrdU for 2 h and then were fixed with 4% paraformaldehyde. After rinsed with PBS, cells were treated with 2 N HCl for 30 min. Nonspecific binding was blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature. Cells were respectively incubated with mouse anti-BrdU (1:200, Millipore, MAB3222) overnight at 4 °C one after another. Then followed by three times washes in PBS, they were incubated with Goat Anti-Mouse IgG H&L (Cy5 ® ) preadsorbed (ab6563) for 1 h at room temperature. Immunofluorescence was observed using flow cytometry.
Statistical analysis. Statistical analysis was performed with SPSS17.0 statistical software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± SEM. One-way ANOVA in conjunction with least significant difference test was performed to determine statistical significance (P < 0.05). The difference was considered significantly if p < 0.05. *means p < 0.05, **means p < 0.01 and ***means p < 0.001. Data Availability. All data generated or analyzed during this study are included in this published article.