Monitoring microRNAs Using a Molecular Beacon in CD 133 + / CD 338 + Human Lung Adenocarcinoma-initiating A 549 Cells

Lung cancer is the leading cause of cancer-related mortality worldwide, and its total 5-year survival rate is approximately 15 % for non-small cell lung cancer (NSCLC) (Jemal et al., 2006). The current screening strategies based on chest radiography are inefficient for detecting lung cancer in its early stage (Henschke et al., 2006), and a lack of effective methods for early diagnosis has greatly impacted the prognosis and survival rate of this cancer. Thus, it is important to develop new diagnostic methods of early diagnosis. Tumor-initiating cells (TICs) possess a strong selfrenewal capacity and multiplex differentiation potential and are considered to be the main responsible for tumorigenesis, resistance to tumor therapy, metastasis, and recurrence (Guo et al., 2006; Adhikari et al., 2011). As TICs are thought to be the major basis for therapeutic intervention and a potential therapeutic target, TICs can provide new clues for the early diagnosis of cancer.


Introduction
Lung cancer is the leading cause of cancer-related mortality worldwide, and its total 5-year survival rate is approximately 15 % for non-small cell lung cancer (NSCLC) (Jemal et al., 2006).The current screening strategies based on chest radiography are inefficient for detecting lung cancer in its early stage (Henschke et al., 2006), and a lack of effective methods for early diagnosis has greatly impacted the prognosis and survival rate of this cancer.Thus, it is important to develop new diagnostic methods of early diagnosis.
Tumor-initiating cells (TICs) possess a strong selfrenewal capacity and multiplex differentiation potential and are considered to be the main responsible for tumorigenesis, resistance to tumor therapy, metastasis, and recurrence (Guo et al., 2006;Adhikari et al., 2011).As TICs are thought to be the major basis for therapeutic intervention and a potential therapeutic target, TICs can provide new clues for the early diagnosis of cancer.

RESEARCH ARTICLE
Monitoring microRNAs Using a Molecular Beacon in CD133 + / CD338 + Human Lung Adenocarcinoma-initiating A549 Cells Quan Yao 1 , Jian-Guo Sun 2 , Hu Ma 3 , An-Mei Zhang 2 , Sheng Lin 4 , Cong-Hui Zhu 2 , Tao Zhang 1 *, Zheng-Tang Chen 2 * Lung cancer TICs were first isolated by Eramo et al., who found CD133-positive tumorigenic cells in both SCLC and NSCLC tissues.CD133-positive lung CSCs (LCSCs) display a high expression level of ABCG2 (CD338), an ATP-binding cassette (ABC) transporter, which is thought to be responsible for resistance to such chemotherapeutic agents as cisplatin (Zhou et al., 2001;Bertolini et al., 2009).TICs are also present in established lung cancer cell lines, including H460 and A549, which show tumor-initiating capacity in mice and chemotherapeutic drug resistant due to the elevated expression of ABCG2 (Ho et al., 2007).
MicroRNAs (miRNAs) are small non-coding singlestranded RNA molecules (19 to 25 nt) that regulate gene expression at a post-transcriptional level (Bartel, 2004;Bushati and Cohen, 2007;Krol et al., 2010).miRNAs can regulate cancer cell differentiation, proliferation, survival, and metastasis, acting as oncogenes or tumor suppressors by regulating the expression of downstream genes through different cancer development and progression pathways (Chen, 2005;Garzon et al., 2009;Kwak et al., 2010;Zandberga et al., 2013).Accordingly, these molecules could constitute a powerful therapeutic strategy for interfering with cancer initiation and progression (Garofalo and Croce, 2011).Moreover, miRNAs have been implicated in the control of self-renewal in stem cells (Hatfield and Ruohola-Baker, 2008).Georgantas et al. found that miRNA-155 played a role in normal human myelopoiesis, lung differentiation, and erythropoieis, and a recent study has shown that a high level of hsa-miR-155 is associated with a poor outcome in lung cancer (Georgantas et al., 2007;Wang et al., 2009b).Thus, the detection of related miRNAs in TICs is an important strategy for the early diagnosis of lung cancer.A molecular beacon (MB) is a highly sensitive detection tool that can be used in the study of miRNA visualization in living cells and may provide technical support for tumor diagnosis.
In this study, TICs were isolated from A549 cells by applying an immune magnetic bead isolation system.miR-155 MBs were then used to detect miR-155 gene expression in TICs.As a non-invasive imaging method, MBs could be used to detect miRNAs in lung cancer.

Flow cytometry analysis
Spheres were dissociated into single cells, washed, and incubated with monoclonal antibodies specific for human APC-conjugated CD133/1and PE-conjugated ABCG2 (CD338, Miltenyi Biotec).The appropriate dilution and procedures were performed according to the manufacturer's instructions.After incubation for 30 min, the cells were washed again and analyzed using flow cytometry.

Immunofluorescence
Spheroids were centrifuged onto slides using a cytospin, fixed with 4% paraformaldehyde for 20 min, and blocked with normal serum for 30 min at room temperature.The slides were then incubated with rabbit monoclonal anti-CD133 (Abcam) and goat polyclonal anti-ABCG2 antibodies (Santa Cruz ) at dilution of 1:300 and stored overnight at 4°C protected from light.After washing, the slides were incubated with FITC-conjugated goat anti-rabbit IgG (Beyotime Biotec, China) and Cy3conjugated donkey anti-goat IgG (Biolegend) fluorescent antibodies at a dilution of 1:400 for 30 min.After DAPI staining for nuclei, the slides were examined by confocal microscopy.

Cell proliferation assays
The A549 cells and TICs were plated in a 96well plate (2000 cells/well) and supplemented with the corresponding growth medium.At the end of the treatments, 10µl of the WST-1 Cell proliferation Assay Kit (Beyotime Biotec, China) was added to each well, and the cells were incubated in a cell culture incubator for 2 hours.The plate was shaken for 1 minute on a shaker, and the absorbance was measured using a microplate reader at 450 nm; the reference wavelength was set to more than 600 nm.The absorbance of each well was measured for five days.

Generation of subcutaneous lung cancer xenografts
All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Xinqiao Hospital and Chengdu General Military General Hospital Institute for Biological Sciences.Four-week-old male nude mice were purchased from the Chinese Academy of Medical Sciences (Beijing, China).To generate xenografts, CD133 + /CD338 + cells and A549 cells were injected subcutaneously on the right side respectively.1×10 3 , 1×10 4 , and 1×10 5 CD133 + /CD338 + cells in 100 µl suspensions were injected; 1×10 4 , 1×10 5 , and 1×10 6 A549 cells were injected.The mice were observed twice a week and sacrificed at six weeks.

Statistical analysis
The data are presented as the mean ± SD.A statistical analysis was performed using an independent-samples t-test, and differences were considered statistically significant at p<0.05.

Isolation and identification of CD133 + /CD338 + cells from the A549 cell line
Normal A549 cells grew as monolayer squamous cells, attaching to the culture flask (Figure 1A).CD133 + / CD338 + cells were sorted grew into spheres in a serumfree medium (Figure 1B).The CD133 + /CD338 + cells were identified using flow cytometry, and we found that the expression of CD133 and CD338 was approximately 35.4% on the CD133 + /CD338 + cells (Figure 1C).

Immunofluorescence and cell proliferation assays
Immunofluorescence of the 5th sphere progeny showed that most of the spheroids were positive for CD133 and CD338 (Figure 2A).Cell proliferation assays demonstrated that a strong proliferation capacity of the CD133 + /CD338 + cells are stronger than A549 cells (Figure 2B).

miR-155 gene expression in CD133 + /CD338 + cells
The results of the MB specificity for the detection of miRNA-155 were the same as previously published research (Yao et al., 2012), indicating that the two MBs were suitable for our ensuring We found that miR-155 MBs could combine with the miR-155 gene and generate green fluorescent signals in CD133 + /CD338 + cells (Figure 4A), as detected in the cell membrane of living CD133 /CD338 + cells.Hochest 33342 staining was also detected in the cell membrane of living CD133 + /CD338 + cells for a dye efflux capacity.Moreover, the control MB did not generate a green fluorescent signal in the CD133 + / CD338 + cells (Figure 4A):the relative fluorescence intensity was markedly increased in the presence of miR-155 MB compared to in the presence of RS MB (Figure 4B).Overall, miR-155 gene expression in the CD133 + / CD338 + cells was higher than that in the A549 cells (Figure 4C).

Discussion
Despite the variety of drastic strategies, such as chemotherapy and radiotherapy, for the treatment of lung cancer, the prognosis of these patients remains poor.The identification and characterization of the tumorigenic cell population responsible for lung cancer formation and metastasis may contribute to the development of more effective diagnoses and therapies aiming at improving the prognosis of lung cancer (Eramo et al., 2008).In this study, we isolated and identified the population of CD133 + /CD338 + cells as the lung cancer tumorigenic cell population.We found that CD133 + /CD338 + cells sorted by MACS showed a greater potential for proliferation and increased tumorigenicity in vivo compared to A549 cells.We were able to detect miR-155 using molecular beacon technique and found that the expression of miR-155 was higher in CD133 + /CD338 + cells than in A549 cells.Thus, miR-155 might serve as a biomarker in the diagnosis of lung cancer.
Recent studies have found that TICs exhibit some of the same specific surface molecular markers as some tumors.The cell surface marker CD133, a member of the cell membrane protein superfamily, was used to identify TICs as a specific marker in such solid tumors as a brain tumor (Singh et al., 2003;Singh et al., 2004), hepatocellular carcinoma (Suetsugu et al., 2006;Yin et al., 2007), pancreatic cancer (Hermann et al., 2007), prostate cancer (Collins et al., 2005), colon cancer (O'Brien et al., 2007;Ricci-Vitiani et al., 2007), and lung cancer (Eramo et al., 2008).Purified CD133 + TICs from these

Table 1. Subcutaneous Xenografts on Nude Mice (male, 4-week-old)
Cell number 1 × 10 3 1 × 10 4 1× 10 5 1 × 10 6 CD133+/CD338+ cells 0/3 3/3 3/3 A549 cells 0/3 0/3 3/3  doi.org/10.7314/APJCP.2014.15.1.161 Molecular Beacon Monitoring of microRNAs in Lung Adenocarcinoma-initiating A549 Cells cancers showed the potential of self-renewal capacity, differentiation, and proliferation and had the capability to form tumors in immunodeficient mice (Collins et al., 2005;Suetsugu et al., 2006;O'Brien et al., 2007).The CD133 + cells from fresh lung tumor samples were also found to have tumor-initiating properties in both small cell and non-small cell lung cancers (Eramo et al., 2008).However, some evidence has indicated that the ability of CD133 + expression to discriminate lung TICs may have been overestimated (Sullivan et al., 2010) and that CD133 + cells do not represent TICs (Meng et al., 2009).Thus, the CD133 marker should be combined with other cell surface markers for the isolation and identification of lung TICs.CD338 (ABCG2), a member of the ATP-binding cassette (ABC) transporter superfamily, is an important determinant of the side population (SP) phenotype (Zhou et al., 2001).SP cells expressing marked by CD338 have stem cell-like capabilities that have found in a variety of solid malignancies and NSCLC (Ho et al., 2007).Furthermore, CD338 can promote resistance to multiple drugs and has been observed in many types of TICs (Bertolini et al., 2009).Lung cancer stem cells, isolated with Hoechst 33342, were found to express significantly higher levels of CD338 (Seo et al., 2007).In our study, we found that the TICs we isolated strongly had highly expressed CD133 + /CD338 + , possessed proliferation and enhanced tumorigenesis capacities, and more highly expressed of stem cell-associated genes.These results demonstrate that CD133 + /CD338 + cells might be TICs with tumor-initiating capacity in NSCLC.
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure.In the absence of a complementary miRNA target, molecular beacons form a stem-loop structure and do not fluoresce.However when they hybridize to their complementary miRNA sequence, these beacons undergo a conformational change that enables them to fluoresce upon excitation (Santangelo et al., 2006).Due to their stem structure, the recognition of targets by molecular beacons is so specific that if the target differs even by a single nucleotide, the probe does not bind to it.Furthermore, molecular beacons can be modified with locked nucleic acids (LNAs) to ensure a greater stability of the probe and enable the simultaneous detection of multiple targets (Tyagi and Kramer, 1996;Tyagi et al., 1998) .
In summary, we successfully isolated and identified a cellular subpopulation expressing CD133 + /CD338 + that could represent TICs of the A549 cell line, and miR-155 was efficiently and specifically detected by a miR-155 MB in TICs.Molecular beacons have the potential to serve as powerful tools for quantifying miRNA expression in biological specimens.We believe that molecular beacons could be used to detect miRNAs in various tissues during carcinogenesis.