Screening for MiRNAs Related to Laryngeal Squamous Carcinoma Stem Cell Radiation

Cancer stem cells are considered to be the source of tumor development and radiation resistance (Hambardzumyan et al., 2006; Blazek et al., 2007; Kang et al., 2008; Ghotra et al., 2009). In recent years, people have successfully separated and identified dozens of cancer stem cells; on the basis of isolated and identified tumor stem cells, some research has gone deep into the level of gene regulation in cancer stem cells. MicroRNA (miRNA) is a newly discovered short non-coding sequencing singlestranded RNA molecule in recent years. It is currently the most important class of gene regulatory molecules in known tumor cells. Specific miRNAs play a unique and important regulation role in characteristics of cancer stem cells (Schetter et al., 2008; Avissar et al., 2009; Hime and Somers, 2009; Mueller et al., 2009; Valeri et al., 2009). including the regulation of genes with biological characteristics such as radiation tolerance gene. It can be inferred that radiation-specific miRNAs play an important role in the regulation of radiation resistance related genes of cancer stem cells. Radiotherapy is one of clinically important treatments of laryngeal cancer. However, the radiation resistance of


Introduction
Cancer stem cells are considered to be the source of tumor development and radiation resistance (Hambardzumyan et al., 2006;Blazek et al., 2007;Kang et al., 2008;Ghotra et al., 2009).In recent years, people have successfully separated and identified dozens of cancer stem cells; on the basis of isolated and identified tumor stem cells, some research has gone deep into the level of gene regulation in cancer stem cells.MicroRNA (miRNA) is a newly discovered short non-coding sequencing singlestranded RNA molecule in recent years.It is currently the most important class of gene regulatory molecules in known tumor cells.Specific miRNAs play a unique and important regulation role in characteristics of cancer stem cells (Schetter et al., 2008;Avissar et al., 2009;Hime and Somers, 2009;Mueller et al., 2009;Valeri et al., 2009).including the regulation of genes with biological characteristics such as radiation tolerance gene.It can be inferred that radiation-specific miRNAs play an important role in the regulation of radiation resistance related genes of cancer stem cells.
Radiotherapy is one of clinically important treatments of laryngeal cancer.However, the radiation resistance of

Screening for MiRNAs Related to Laryngeal Squamous Carcinoma Stem Cell Radiation
Chang-Xin Huang 1 *, Ying Zhu 2 , Guang-Liang Duan 1 , Ji-Fen Yao 1 , Zhao-Yang Li 1 , Da Li 3 , Qing-Qing Wang 3 laryngeal cancer cells is still a difficult problem which seriously affects radiotherapy.Currently, studies of miRNA regulation on the radiotherapy resistance level of laryngeal cancer stem cells have not been reported both home and abroad.Because of the fast, parallel, high-throughput detection characteristics, we selected oligonucleotide microarrays of miRNA expression profiles for comparative analysis of miRNA expression level in laryngeal cancer stem cells before and after radiation, differential miRNA screening and clustering analysis; part of the differentially expressed miRNAs was verified with quantitative real-time RT-PCR method.This laid the foundation for further study of the relationship of miRNA with laryngeal cancer radiation related biological characteristics and provided basic information on the development of laryngeal cancer stem cell radiosensitizer based on miRNA regulation and targeting.

Materials
Hep-2 laryngeal squamous carcinoma cell line was purchased from Shanghai Institute of Cell Institute.CDl33 + monoclonal antibody was purchased from Chemicon company; goat anti-rabbit IgG/FITC was purchased from Toxin Tech.

Construction of laryngeal stem cells
Conventional isolation culture of stem cell microspheres: Hep-2 laryngeal squamous carcinoma cell line was cultured in medium containing serum l640; serum-free medium (SFM) was prepared freshly, that is EGF, bFGF, and glucose were added in serumfree DMEM/F12 medium.Laryngeal cancer cells in growth stage were subject to trypsin digestion and SFM resuspention into 10 3 /ml single cell suspension, followed by routine culture in glass culture flasks.Culture bottles were placed upright in an incubator and shaken a few times a day.The microsphere formation was observed.The medium was changed every 2 days at half the amount and passaged every 6 days.Microspheres were collected during passage, and were subject to accutase digestion and pipetting into single cells.After washing in SFM they were subcultured for amplification and enrichment of laryngeal stem cells.All growing cells ball were collected and made into single cell PBS suspension.Fluorescentlabeled monoclonal antibody was added and incubated.CDl33 + cells were screened out as laryngeal cancer stem cells using FACS.

Radiation treatment of laryngeal cancer stem cells
Radiation processing method for tumor cells commonly used in similar studies was applied: Laryngeal cancer stem cells SFM suspension with cells adjusted to 1 × 10 6 /ml, was inoculated in 6-well culture plate.After short-term culture, the cultured cells were irradiated with linear accelerator.The condition was vertical 6MV-X-ray irradiation and 2 Gy/min dose rate.During irradiation, the bottom of the culture plate was added 1.5 cm tissue equivalent filler and the source skin distance (SSDs) was 100cm.The irradiation was carried out twice with 5Gy/ times and the irradiation interval was 72 h.

Screening of different miRNA
miRNA The Ambion miRNA isolation kit was used for enrichment and purification of miRNA: Conventionally extracted total RNA and 5 times volume of lysis/binding buffer solution was mixed well and was added 1/10 volume of miRNA homogenate additives.The mixture was incubated on ice and was added 1/3 volume of anhydrous ethanol.It was passed through filter tube after mixing.The filtrate was added 2/3 volume of anhydrous ethanol and preheated to 95 °C followed by second filtration.Labeled reactant was prepared with bovine intestinal alkaline phosphatase, and miRNA was fluorescently labeled with T4 ligase and purified with column.Fluorescent miRNA sample was added 10 × GE blockers and 2 × HiRPM hybridization buffer, and the miRNA chip (Denmark Exiqon Company) hybridization was carried out with hybrid instrument and its rotary oven.Upon finish each chip was washed in GE buffer and scanned with Agilent microarray scanner to obtain the hybridization signal.

Chip signal reading and data processing
LuxScan3.0 image analysis software (CapitalBio, China) was used for mircoarray image analysis.
After overall normalization of data and logarithmic transformation, Significance Analysis of Microarrays (SAM, version 2.1) was used to select differentially expressed genes.The screening requirement was FDR was controlled in less than 5%, and Fold change was not less than 2.0.Finally, Cluster3.0(Stanford University) was adopted for cluster analysis.
Fluorescence quantitative PCR RNA-tailing and primer extension RT-PCR was applied for quantitative real-time detection of miRNAs in stem cells: First establishing 20 μl tailing reaction system: 1 μg total RNA, poly A polymerase (PAP) 40 U, 1 mmol/L ATP at 37 °C water bath for 1h.Then, an equal volume of phenol-chloroform (1:1) extraction followed by centrifugation at 12000 r/min for 10 min to afford the supernatant.1/l0 Volume of 3 mol/L NaAc and 2 volumes of anhydrous ethanol was added to precipitate RNA.RNA after tailing was dissolved in 10μl RNA-free enzyme water.Reverse transcription reaction was carried out with transcription kit, and the reverse transcription specific primer was the universal primer sequence binding to the polyA end in RACE kit.According miRNA sequences to be detected, PCR upstream primer was designed and synthesized.The common downstream primer: 5'-GCTGTCAACGATACGCTACGTAACG-3'; 5SRNA was selected as internal reference; upstream primer: 5'-CCATACCACCCTGAACGC 3'; downstream primer: 5'-AGCCTACAGCACCCGGTAT-3'. Real-time PCR amplification adopted crystalline core EvaGreen fluorescence quantitative PCR Universal kit of CapitalBio Company.In PCR amplification, primer concentration was 150 nmol/L.PCR cycling condition was: 94°C predenaturation for 5 min, 94 °C denaturation for 30 's, 58°C annealing for 30S, 72 °C extension for 45S, 74 °C plate reading, a total of 40 cycles, three duplicate for each miRNA.The specificity of the PCR reaction was confirmed with product melting curve, and the relative content of mRNA was calculated according to the formula: Relative fold chang = 2 -∆ (∆Ct) .where in ∆Ct = Ct(target)-Ct(5sRNA); ∆(∆Ct) = ∆Ct(test) -∆Ct(control).

The successful separation construction of laryngeal squamous cancer stem cells
Conventional isolated culture of stem cell microspheres: Laryngeal cancer cells in growth stage were subject to trypsin digestion and SFM resuspention into 10 3 /ml single cell suspension followed by routine culture in glass culture flasks.Culture bottles were placed upright in an incubator, and were shaken a few times a day.The microsphere formation was observed.The medium was changed every 2 days at half the amount and passaged every 6 days.Microspheres were collected during passage (Figure 1).and were subject to accutase digestion and pipetting into single cells.After washing in the SFM they were subcultured for amplification and enrichment of laryngeal stem cells.All growing cells ball were collected and made into single cell PBS suspension.Fluorescent- CDl33 + cells were screened out with FACS as laryngeal cancer stem cells. 2 mL of stem cell suspension liquid was obtained with 1 × 10 7 /ml concentration of cells.
Figure 1 Spheroids of enriched laryngeal stem cells (× 200 times): Laryngeal cancer cells in growth stage were subject to trypsin digestion and SFM resuspention into 10 3 / ml single cell suspension followed by routine culture in glass culture flasks.Culture bottles were placed upright in an incubator, and were shaken a few times a day.The microsphere formation was observed.

Hybridization results
Trizol method was applied to isolate total RNA before and after laryngeal cancer stem cells irradiation.After enrichment miRNA was detected and showed A260/ A280 values were in the range of 1.8 to 2.1, in line with the requirements of the miRNA microarray.Such sample can be used for miRNA microarray experiments.After hybridization of labeled and purified miRNA with microarray chip, it was scanned to afford images at 543 nm channel.Positive control signals such as HEX, external standard, internal standard were normal and the negative control test was negative.The microarray chip had good repeatability of duplicate points and uniform signal without affecting the data pollution; leak rate of ≤ 0.3%, and the correction coefficient was stable.After overall normalization and log-transformation of data SAM software was used for selection of differentially expressed miRNA.The screening condition was: FDR less than 5% and Fold change ≥ 2.0.Fluorescently labeled miRNA microarray hybridization result is shown in Figure 2.
Figure 2 fluorescently labeled miRNA microarray hybridization: Trizol method was applied to isolate total RNA before and after laryngeal cancer stem cells irradiation.After enrichment miRNA was detected and showed A260/A280 values were in the range of 1.8 to 2.1, in line with the requirements of the miRNA microarray.Such sample can be used for miRNA microarray experiments.After hybridization of labeled and purified miRNA with microarray chip, it was scanned to afford images at 543 nm channel.

RT-PCR validation results of differentially expressed miRNA
Poly (A) polymerase was used at the 3 'end to add tail, and then single nucleotide anchor Olig-dT primer of 5' with a 40 nt extending sequence was used for reverse transcription.80 -83 bp cDNA was obtained followed by Eva Green quantitative real-time PCR amplification with specific primer.This method was applied to detect the expression of some miRNA that showed differential expression before and after irradiation in chips, and 5SRNA was used for normalization.The chip results and fluorescence quantitative RT-PCR results were compared and the results showed consistent expression of differential miRNA detected from chip and quantitative RT-PCR, suggesting test results of this experiment are true and reliable (Figure3A-B).
Figure 3 A-B Some miRNA expression detected by qRT-PCR and the chip: Eva Green quantitative real-time PCR amplification was applied to detect the expression of some miRNA that showed differential expression before and after irradiation in chips.The results and fluorescence quantitative RT-PCR results were compared and the results showed consistent expression of differential miRNA detected from chip and quantitative RT-PCR.

Discussion
There are a variety of separation and identification of tumor stem cells such as: microspheres isolation and culture method, side population cell sorting method.The most recognized cell molecular "marker" is CDl33+ (Mangoni et al., 2012).Our group has also carried out a series of preliminary studies of cancer stem cells: successful isolation, identification and amplification of laryngeal squamous carcinoma stem cells from Hep-2 laryngeal carcinoma cell line.This study intents to further arrive at the level of gene regulation of tumor stem cells and observe the changes in the most eyecatching miRNAs.Micro RNA (miRNA) is a newly discovered short non-coding sequencing single-stranded RNA molecule in recent years which is composed of 21 to 25 nucleotides.It is the most important class of gene regulatory molecules currently known in tumor cells.The discovery of miRNA has become another significant progress and research focus of oncology research in recent years (Yu et al., 2007;Silber et al., 2008;Friedman and Jones, 2009) which including studies on the relationship between miRNAs and radiosensitivity.Chaudhry et al. (2010) found there was differential expression of miRNA in TK6 and WTK1 cell lines (both human lymphoblastoma cells) that have different radiation resistance.The analysis showed that significantly differential expression of miRNA is closely related to different radiation resistance of TK6 and WTK1.Wang, et al. (2011) reported 12 differently expressed miRNAs in the radiotherapy sensitive and resistant non-small cell lung cancer samples.Comparing with radiotherapy resistant patients, five miRNAs (miRNA-126, miRNAlet-7a, miRNA-495, miRNA-451 and miRNA-128b) were significantly upregulated and seven miRNAs  were greatly downregulated in radiotherapy sensitive group.By further analysis, they believed that miRNA-126 promoted the cancer cells apoptosis induced by irradiation through the PI3K-Akt pathway.Furthermore, some recent studies focused on cancer radiosensitivity-modulated targets or miRNAs of cell signal transduction pathways.Shin et al. (2009) investigated the profile of miRNA expression following ionizing radiation in the human lung carcinoma cell line A549, and the expression profiles of radiationresponsive miRNAs were identified and confirmed by qRT-PCR and microarray analysis.Target prediction for radiation-responsive miRNAs suggested that their target genes could be related to apoptosis, regulation of cell cycle, and DNA damage and repair.Taken together, it is indicated that miRNA expression may be affected by radiation, and in turn involved in the regulation of radiation responses.In Josson's research (Josson et al., 2008).several miRNAs were significantly changed in prostate cancer cells in response to radiation treatment, with miR-521 downregulated and miR-34c upregulated.By altering the levels of DNA repair protein, CSA, miR-521 sensitized prostate cancer cells to radiation treatment.Lee et al. (2011) demonstrated that microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling, and Qu's experiment (Qu et al., 2012) testified that MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN.More studies (Lal et al., 2009;Hu et al., 2010;Ng et al., 2010;Moskwa et al., 2011) revealed that miRNA can impact the radiosensitivity by modulating the expression of key proteins in DNA damage repair pathways.
Currently there is no study on relationship of miRNAs with the radiosensitivity of laryngeal cancer stem cells, which are recognized to be the source of radiation resistance as described above.Specific miRNAs may play an important regulating role in radiation-related gene of cancer stem cells.
In this study, we successfully isolated, identified and amplified laryngeal squamous carcinoma stem cells, and further studied specific changes of miRNA before

Figure 1 .
Figure 1.Spheroids of Enriched Laryngeal Stem Cells (× 200 times).Laryngeal cancer cells in growth stage were subject to trypsin digestion and SFM resuspention into 103/ml single cell suspension followed by routine culture in glass culture flasks.Culture bottles were placed upright in an incubator, and were shaken a few times a day.The microsphere formation was observed