MicroRNA-499a decelerates glioma cell proliferation while accelerating apoptosis through the suppression of Notch1 and the MAPK signaling pathway

Accepted Manuscript Title: MicroRNA-499a decelerates glioma cell proliferation while accelerating apoptosis through the suppression of Notch1 and the MAPK signaling pathway Authors: Bang-Qing Wang, Bin Yang, Hua-Chao Yang, Jun-Yi Wang, Sen Hu, Yu-Shuai Gao, Xing-Yao Bu PII: DOI: Reference:

S0361-9230(17)30697-4 https://doi.org/10.1016/j.brainresbull.2018.06.005 BRB 9449

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

22-11-2017 12-5-2018 10-6-2018

Please cite this article as: Wang B-Qing, Yang B, Yang H-Chao, Wang J-Yi, Hu S, Gao Y-Shuai, Bu X-Yao, MicroRNA-499a decelerates glioma cell proliferation while accelerating apoptosis through the suppression of Notch1 and the MAPK signaling pathway, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MicroRNA-499a decelerates glioma cell proliferation while accelerating apoptosis through the suppression of Notch1 and the MAPK signaling pathway Running title: miR-499a in glioma cell proliferation & apoptosis

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Bang-Qing Wang 1, Bin Yang 1, Hua-Chao Yang 1, 2, Jun-Yi Wang 1, 2, Sen Hu 1, 2, Yu-Shuai Gao 1, Xing-Yao Bu 1, 2, *

Department of Neurosurgery, People's Hospital of Zhengzhou University (Henan Provincial

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People's Hospital), Zhengzhou 450003, P.R. China

Henan University of Chinese Medicine, Zhengzhou 450046, P.R. China

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* Correspondence to: Dr. Xing-Yao Bu, Department of Neurosurgery, People's Hospital of

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Zhengzhou University (Henan Provincial People's Hospital), No. 7, Weiwu Road, Zhengzhou

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450003, Henan Province, P.R. China; Henan University of Chinese Medicine, No. 156, Jinshui East

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Road, Zhengzhou 450046, Henan Province, P.R. China

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Highlights

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E-mail: [email protected]

(1) A study reported miR-499a targeting Notch1 in glioma cell proliferation & apoptosis.

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(2) miR-499a expresses highly and Notch1 expresses lowly in glioma cell.

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(3) miR-499a targets and inhibits Notch1.

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(4) miR-499a inhibits glioma cell proliferation by targeting Notch1via MAPK pathway.

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(5) Notch1 will be a new breakthrough in the diagnosis and treatment of glioma.

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Tel./Fax.: +86-18538297990

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ABSTRACT As the most common and lethal of intracranial tumors, glioma accounts for 81% of all malignant brain tumors. Research data has identified the role of microRNAs (miRs) as functional suppressers in the progression of Glioma. The present study aimed to, ascertain as to whether microRNA-499a (miR-499a) influences cell proliferation and apoptosis through the MAPK signaling pathway by

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targeting Notch1 in glioma. Both glioma and adjacent tissues between 2012~2016, were obtained from People's Hospital of Zhengzhou University (Henan Provincial People's Hospital). The

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collected glioma cells were treated with miR-449a mimic, miR-449a inhibitor, siRNA-Notch1, or SB230580 (an inhibitor of the MAPK signaling pathway). Verification of the targeting effect of

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miR-449a on Notch1 was provided by a dual-luciferase reporter gene assay. The expressions of

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miR-449a, Notch1, p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated

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protein kinases (ERK1/2), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (bax), CyclinD1,

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and phosphorylation of p38MAPK (p-p38MAPK) and ERK1/2 (p-ERK1/2) in tissues and cells were detected by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR)

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and western blot analysis methods. Cellular processes of proliferation, cell cycle and apoptosis were evaluated by MTT and BrdU assays as well as flow cytometry, respectively. Notch1 was

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subsequently identified to be a target gene of miR-499a. After the cells were treated with miR-449a

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mimic, siRNA-Notch1 or SB230580, decreased expressions of Notch1, Bcl-2, CyclinD1, ERK1/2 and p-ERK1/2, cell proliferation as well as cells arrested at the G0/G1 stage with elevated increased expressions levels of p38MAPK, p-p38MAPK, Bax, as well as increased cell apoptosis and number

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of cells arrested in S stage were assessed. Taken together, based on the evidence obtained from the present study, assertions were subsequently made suggesting that MiR-499a targeted-inhibition of Notch1 may be a promising future therapeutic strategy for glioma treatment, by means of overexpressing of miR-499a resulting in the inhibition of glioma cell proliferation and promotion of cell apoptosis through suppression of the MAPK signaling pathway by decreasing Notch1.

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Keywords: MicroRNA-449a; Glioma; Notch1; MAPK signaling pathway; Proliferation; Apoptosis

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1. Introduction Glioma represents the most commonly occurring malignant primary brain tumor, accompanied by devastating effects. Glioma is among the most lethal tumors of the central nervous system (CNS) (Russo et al. 2017). Statistics have indicated that glioma accounts for over 36% of all primary CNS cancers worldwide (Adamson et al. 2010). At present glioma is one of the most obstinate cancers,

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with its complexity making it a difficult condition to treat effectively, hence it is no surprise that it associated with high levels of mortality (Adamson et al. 2010). Studies have indicated the rick

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factors associated with the occurrence of the glioma include gender, age and race due to their significant influence on the morbidity of the condition (Ostrom et al. 2014). Despite the vast

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application of invasive surgery, medical radiotherapy and chemotherapy, glioma remains

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significantly associated with inevitable tumor recurrence and poor prognosis, with the prognoses

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among older individuals significantly poorer on account of their higher morbidity (Yin et al. 2013).

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The prognosis for glioma tumors is generally determined mainly based on the histologic grade, with a median survival of between 1 and 3 years (Hilario et al. 2014). Hence, biomarkers as tools that

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could improve the clinical outcomes of patients suffering from glioma, remains extremely important for the diagnosis of glioma. Various studies have highlighted the role of microRNAs (miRs) in

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relation to the incidence and progression of glioma have been paid increasing attention in recent

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times due to their relevance prediction value for cases of glioma (Bao et al. 2014). MiRs are short endogenous noncoding RNAs that are approximately 22 nucleotides in length,

capable of inhibiting the expressions of their cognate target genes by binding to 3' untranslated

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regions (3' UTRs), which result in translational repression or mRNA degradation (Du et al. 2014). MiR-499, is a miRs capable of regulating the expressions of certain genes, particularly in conditions of involving hypoxia or ischemia such as in cases of infarction, cancer or myocardial disorders (Okamoto et al. 2016). Evidence has been provided suggesting that cloned RNA is unique regarding its length, while miR-499a is correlated with the miR-499 family (Kochegarov et al. 2013). Studies

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have indicated that miR-499a could play an essential role by targeting mitogen-activated protein kinase (MAPK) 6 in the development and progression of Hepatitis B Virus related hepatocellular carcinogenesis (Xiang et al. 2014). Notch1 has been highlighted, due to its ability to enhance the differentiation process in certain tissues, while acting to maintain stem cell proliferation in other tissues. Notch1 could play a paradoxical role in various diseases, either as a tumor suppressor or an

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oncogene (Liu et al. 2011). Interestingly, a previous study investigating Notch 1 and glioma, subsequently revealed that Notch1 expression to be a critical role in glioma cell proliferation and

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survival (Li et al. 2011). A previous study demonstrated that the overexpression of miR-449a could influence Notch1 in the celiac small intestine (Capuano et al. 2011). Accumulating evidence continues to provide indications that the suppression of MAPK signaling activity could inhibit the

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ability of glioma cell proliferation and invasion, highlighting the potential of the MAPK signaling

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pathway as an effective therapeutic target for glioma treatment (Guo et al. 2013). Based on the

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aforementioned literature, a lack of elucidation regarding the role by which miR-499a interacts with

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Notch 1 in glioma exists; thus, the central objective of the present study was to explore the influence of miR-499a on the processes of cell proliferation and apoptosis by targeting Notch1

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through the MAPK signaling pathway in glioma.

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2. Materials and methods

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2.1 Ethical statements

The study was conducted with the approval of the Clinical Research Ethics Committee

(CREC) of People's Hospital of Zhengzhou University (Henan Provincial People's Hospital).

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Written informed consent documentation was obtained from each participant. 2.2 Study subjects A total of 76 glioma tissue specimens (case group) were extracted between 2012 and 2016 from patients at the People's Hospital of Zhengzhou University (Henan Provincial People's Hospital), among which there were 44 males and 32 females aged between 11 - 66 years. The 5

adjacent tissues (confirmed with no tumor under a microscope) more than 10 mm away from the carcinomas of 69 patients were collected to serve as the control group. The inclusion criteria were as follows: (1) patients were yet to receive any chemoradiotherapy prior to surgery; (2) patients were classified into I-IV grade according to the World Health Organization (WHO) classification; (3) patients received surgery due to the existence of a malignant tumor rather than other causes,

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such as brain trauma. There were 36 cases diagnosed with glioma at stage I-II and 40 cases between stage III-IV in the case group. The control group was comprised of 32 cases diagnosed with glioma

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between stage I-II and 37 cases between stage III-IV. 2.3 Hematoxylin-eosin (HE) staining

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All formaldehyde-fixed and paraffin-embedded specimens were cut into 4 μm serial sections

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followed by the performance of HE staining. The specimens were de-waxed twice with (15 min

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each), dehydrated with absolute ethyl alcohol twice, once with 90% ethanol, and another time in

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80% ethanol, followed by washing with running water for 5 min. Next, the specimens were stained with hematoxylin for 5 min allowing them to turn back to blue after washing, differentiated with

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1% hydrochloric acid-ethanol (1 s~3 s) and turned back to blue after washing (1 s~30 s). Eosin staining was then performed for 2 min, after that, the specimens were flushed with water,

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dehydrated once with 80% ethanol (5 min each), another time with 90% ethanol (5 min each), and

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de-waxed with xylene twice (15 min each). The specimens were then sealed with neutral balsam. An optical microscope (DMM-300D, Shanghai Caikon Optical Instrument Co., Ltd., Shanghai, China) was employed to observe both the glioma tissues and adjacent tissues, with their respective

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histopathological changes compared. 2.4 Immunohistochemistry (IHC) Ten percent formalin-fixed and paraffin-embedded specimens were cut into 4 μm serial sections and then placed in an incubator at 60°C for 1 h. The specimens were conventionally dewaxed with xylene, dehydrated with gradient ethanol, and then incubated in 3% H2O2 (BD5024, 6

Bioworld Technology Inc, Minneapolis, Minnesota, USA) at 37°C for 30 min. After washing with phosphate buffered saline (PBS, 0.01M, pH7.4), the specimens were boiled in 0.01M citric acid buffer solution at 95°C for 30 min and cooled to the room temperature, washed with PBS (0.01M, PH7.4), and sealed in normal goat serum at 37°C for 10 min. The diluted mouse anti-human Notch1 were then added (ab8925, 1 : 500; Abcam, Inc., MA, USA) for reaction at 4°C 12 h. After

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additional PBS washing, biotin-labeled goat anti-mouse secondary antibody (A21020, Abbkine Inc., California, USA) was added allowing for a reaction to occur over a 10 min period at room

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temperature. After full washing, horseradish peroxidase (HRP)-labeled streptavidin-working

solution (DF7852, Shanghai Yao Yun Biological Technology Co., Ltd, Shanghai, China) was added for a 10 min reaction process at room temperature. The specimens were subsequently stained with

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Diaminobenzidine (DAB) and stored in a dark environment for 8 min at room temperature. The

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specimens were then washed with running water, stained with hematoxylin, dehydrated, cleared,

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mounted and viewed under an optical microscope. The positive cells were counted using a Japanese

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Nikon image analysis software (Eclipse 80i). Five non-repetitive fields (× 200) with equal area were selected from each section to calculate the positive staining area and their rates of total area,

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regarded to be positive (+) if the rates between positive tumor cells and all tumor cells were over

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10%.

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2.5 Cell culture and transfection

Glioma cell line TJ905 and normal glial cell HEB were purchased from the Cell Bank of the

Chinese Academy of Sciences (Shanghai, China). The cells were conventionally cultured in

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dulbecco's modified eagle medium (DMEM) complete culture medium containing 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 for subculture, with the culture medium was replaced every 3~5 days. Glioma cells confirmed to be at the third generation were assigned into the normal group (HEB), the blank group (TJ905, without any transfection), the negative control group (NC, TJ905, transfected with random sequence of miR-449a NC), the miR-449a mimic group (TJ905, 7

transfected with miR-449a mimic), the miR-449a inhibitor group (TJ905, transfected with miR449a inhibitor), the siRNA-Notch1 group (TJ905, transfected with siRNA-Notch1), the miR-449a inhibitor + siRNA-Notch1 group (TJ905, transfected with miR-449a inhibitor and siRNA-Notch1), the SB230580 group (TJ905, incubated with 1uM SB230580, the inhibitor of the MAPK signaling pathway) and miR-449a inhibitor + SB230580 group (TJ905, incubated with SB230580 and

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transfected with miR-449a inhibitor). Glioma cells in the logarithmic growth phase were seeded into a 6-well plate. In the event that the cell density had grown to between 30~50%, cell

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transfection was conducted in accordance with the instructions of lipofectamine 2000 reagent

(Invitrogen, Co., Ltd, California, USA). Next, 100 pmol of cells in miR-449a mimic, miR-449a inhibitor, siRNA-Notch1, miR-449a inhibitor + siRNA-Notch1 and NC groups were diluted with

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250 μL serum-free culture medium Opti-MEM (Gibco Inc., California, USA), making a final

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concentration of 50 nM, gently blended and incubated for 5 min at room temperature. Then, 5 μL

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lipofectamin 2000 was diluted with 250 μL serum-free medium Opti-MEM, gently blended and

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incubated for 5 min at room temperature. The two prepared solutions were mixed and incubated for 20 min at room temperature, then placed into wells for cell culture at 37°C with 5% CO2. And 6~8

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h later, the medium was replaced by a complete medium, and the cells were then cultured for 24~48

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h for subsequent experiments.

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2.6 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from both the glioma and adjacent tissues using RNA extraction kit

(Invitrogen, Co., Ltd, California, USA). The primers of miR-449a, Notch1, extracellular regulated

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protein kinases (ERK1/2), p38MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (bax), CyclinD1, U6 and β-actin were designed and synthetized by Takara Holdings Inc. (Kyoto, Japan) (Table 1). RNA was then reversely transcribed into cDNA by the PrimeScript RT kit (Guangzhou Bo Li Biotechnology Co., Ltd., Guangdong, China), and the reverse transcription system was set with 10 μL. The reaction conditions were set based on the instructions at 37°C, 15 min 3 times (reverse transcription), 85°C for 5 s (reverse transcriptase inactivation). The reaction 8

solution was then subjected to fluorescence quantitative PCR and fluorescence quantitative PCR was performed using the SYBR® Premix Ex TaqTM II Kit (Dalian Biotech Co., Ltd., Dalian, Liaoning, China). The reaction system was set with 50 μL, including 25 μL of SYBR® Premix Ex TaqTM II (2 ×), 2 μL of PCR upstream primer, 2 μL of PCR downstream primer, l μL of ROX Reference Dye (50 ×), 4 μL of DNA template and 16 μL of ddH2O. Fluorescence quantitative PCR

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was performed by the ABI PRISM® 7300 system (Shanghai Kunke Instruments Co., Ltd., Shanghai, China). The reaction conditions were comprised of pre-denaturation at 95°C for 30 s,

denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, and for 40 cycles. U6 was

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considered to be the internal reference regarding the relative expression of miR-449a, and β-actin for Notch1, ERK1/2, p38MAPK, bax, Bcl-2 and CyclinD1. The relative quantification 2-△△Ct

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method was applied to calculate the relative mRNA expression of the target genes (miR-449a,

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Notch1, ERK1/2, p38MAPK, bax, Bcl-2 and CyclinD1) based on the following formula: △△Ct =

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△Ct (tumor group) - △Ct (normal group), △Ct = Ct (target gene) - Ct (internal reference). The

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relative expressions of target genes were 2-△△Ct (Ayuk et al. 2016). Total RNA was collected after a 48 h period of incubation of total RNA of glioma cells from each group. The mRNA expression of

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the transfected cells was detected by means of RT-qPCR. The operation process employed was

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identical with the aforementioned method.

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2.7 In situ hybridization

The following experiment consisted of a sequence labeled with digoxin at the 5’-end

connected between the oligonucleotide probe sequence and nonsense probe sequence. Paraffin

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sections were dewaxed, fixed 4% polyoxymethylene at room temperature. After incubation with enzyme, prehybridization solution was added, incubated at 40°C for 180 min, followed by the addition of 0.5 g/L miR-449a probe fluid (Shanghai GenePharma Co., Ltd., Shanghai, China) and then incubated at 40°C overnight. Next, the sections were washed with saline-sodium citrate (SSC) buffer (pH = 7.0) and incubated with the addition of mice anti-digoxin monoclonal antibody (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA) at room temperature for 120 9

min. After washed with PBS 4 times, 5 min per time, the sections were treated with DAB with the color intensity controlled using a microscope. The reaction was then halted with tap water. The sections were then counterstained with hematoxylin, washed for 5~10 min, dried and dehydrated with alcohol, cleared with xylene and sealed with neutral gum. 2.8 Western blot analysis

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Glioma and adjacent tissues were collected and added with liquid nitrogen and grinded until the tissues were confirmed to be uniform fine powder. Next, 1 ml cell lysis buffer, including 50

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mmol/l Tris, 150 mmol/l NaCl, 5 mmol/l ethylene diamine tetraacetic acid (EDTA), 0.1% sodium

dodecyl sulfate (SDS), 1% NP-40, 5 μg/ml Aprotinin, and 2 mmol/l phenylmethylsulfonyl fluoride

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(PMSF) was added, grinded into homogenate on the ice bath, added with protein lysis buffer, and

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centrifuged at 12000 r/min at 4°C for 20 min followed by collection of the supernatant. The protein

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concentration of each specimen was detected using a bicinchoninic acid (BCA) detection kit, with

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deionized water used to adjust the loading buffer quantity. Next, 10% SDS separation gel and concentrated gel were prepared, with the specimens mixed with the loading buffer, boiled at 100°C

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for 5 min, followed by an ice bath. The specimens were then centrifuged and then added to each lane using a microtiter for electrophoresis purposes. The proteins on the gel were subsequently

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transferred to the nitrocellulose membranes, which were then sealed with 5% skimmed milk powder

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overnight at 4°C. The following diluted primary antibodies, including Notch1 antibody (1/500, ab8925, Abcam Inc, MA, USA), ERK1/2 antibody (1/1000, ab17942), P38MAPK antibody (1/750, ab31828), bax antibody (1/750, ab53154), Bcl-2 antibody (1/1000, ab32124), CyclinD1 antibody

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(1/500, ab6152), p-ERK1/2 antibody (1/10000, ab17526), and p-p38MAPK antibody (1/2500, ab170099) were added and incubated overnight. All the aforementioned antibodies were purchased from Abcam Inc (Cambridge, MA, USA). The membranes were then washed 3 times with PBS at room temperature (5 min each time), added with (HRP)-labeled immunoglobulin G (IgG) (1 : 1000, Wuhan Boster Company, Hubei, China) secondary antibody and incubated at 37°C for 1 h, followed by an additional 3 PBS washes at room temperature (5 min each time). The membrane 10

was then immersed in an electrochemiluminescence (ECL) reaction mixture (Pierce Inc, Rockford, IL USA) at room temperature for 1 min. The specimens were completely covered with food wraps after the liquid was then removed and placed back in a dark environment. Images were then acquired after the developing and fixation processes respectively. The relative protein expression was representative of the ratio of gray value of the related protein bands to the β-actin protein band,

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with β-actin serving as the internal reference. 2.9 Dual-luciferase reporter gene assay

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According to an online biology prediction website available at (http://www. microRNA.org), Notch1 was predicted to be the target gene of miR-499a. The artificially synthesized Notch1 3'UTR

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gene fragment was introduced into pMIR-reporter (Promega, Madison, WI, USA) using the

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endonuclease sites SpeI and Hind III, while the complementary sequence mutation site of the seed

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sequence was designed on the Notch1 wild-type (WT). After digestion by restrictive endonuclease,

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the target fragments were inserted into the pMIR-reporter plasmid by using T4 DNA ligase. The recombinant luciferase reporter plasmids WT and mutation type (MUT) were then co-transfected

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into the HEK-293T cells (Shanghai BeiNuo Biotechnology Co., Ltd. Shanghai, China) with miR449a respectively. After a 48 h transfection process, the cells were lysed and collected, with the

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luciferase activity subsequently measured using a dual-luciferase reporter gene assay kit.

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2.10 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay Well grown cells in the logarithmic growth phase were inoculated into a 96-well culture plate

at 1 × 104 cells/well, and the volume of the medium was 100 μL per well. After cell adhesion for 24

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h, the cells were incubated at 37°C with 5% CO2 for 24 h, 48 h and 72 h, respectively, followed by the addition of 20 μL of MTT solution (5 mg/mL) to each well for incubation at 37°C for 4 h. After the supernatant has been removed, 100 μL dimethylsulfoxide (DMSO) was added to each well followed by continuous shaking for 10 min. The optical density (OD) value at the wavelength of

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490 nm was determined by using microplate reader. The experiment was repeated 3 times in order to obtain mean results. 2.11 5’bromo-2’deoxy-uridine (BrdU) labeling Cells were seeded into 24-well plates, transfected and cultured after solution replacement. Upon reaching 80% confluence, cells were added with 10 μmol/L BrdU and incubated at 37°C for 4

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h. The cells were washed 3 times (5 min per washing) after cell culture solution was discarded. Following the addition of 70% absolute ethanol and fixation at 4°C for 10 min, the cells were

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washed 3 times with PBS (5 min per washing) with the absolute ethanol discarded. Then, mol/L

hydrochloric acid (HCl) solution was added to the cells and kept at 37°C for 40 min allowing for

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denaturation of the DNA of the cells. After HCl removal, the cells were treated by PBS washing

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(3x10 min per wash). Next, 1.0% bovine serum albumin (BSA) was added to the cells and kept at room temperature for 1 h for sealing, and washed 3 times with PBS (5 min per washing). After PBS

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removal, cells were added with BrdU monoclonal antibody (1 : 300, 100 μL/well) for incubation at 4°C overnight. On the following day, fluorescein isothiocyanate (FITC)-labeled goat anti-rat

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secondary antibody was added into cells for 2-h incubation at room temperature. The nucleus of all cells was counterstained with propidium iodide (PI), and then observed under a fluorescence

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microscope. Finally, 10 non-overlapping visual fields were randomly selected (× 100) to calculate

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the positive number of BrdU, with the average value obtained. 2.12 Flow cytometry

The cells were collected after 48 h of transfection, diluted and formulated as single cell

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suspension, washed twice with PBS, fixed with pre-cooled 70% ethanol overnight at 4°C, and rinsed an additional two times with PBS. The cells were then filtered through a 600 mesh filter. The cells were treated with 200 μL of RNase A solution, incubated for 30 min, followed by 500 μL PI staining for 30 min in a dark environment. Finally, the cell cycle was detected at an excitation

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wavelength of 488 nm by flow cytometry (BD FACSCALIBUR flow cytometer, Shanghai Shucheng Medical Technology Development Co., Ltd., Shanghai, China). The cells were collected after transfecting for 48 h, diluted and formulated into a single-cell suspension, followed by two PBS washes. Next, 5 μL FITC and 5 μL PI were added for cell labeling respectively, which were incubated at room temperature under conditions void of light for

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15 min. The cells were then re-suspended with 200 μL 1 × binding buffer and the apoptotic rate of each group was detected by flow cytometry at an excitation wavelength of 488 nm.

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2.13 Statistical analysis

All data were processed by SPSS21.0 software (IBM Corp., Armonk, NY, USA).

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Measurement data were expressed as mean ± standard deviation. Comparisons between two groups

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were compared by t-test and comparisons among multiple groups by one-way analysis of variance

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(ANOVA). p < 0.05 was considered to reflect statistical difference

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3. Results

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3.1 Pathological changes of glioma and adjacent tissues post HE staining HE staining was performed in order to observe the pathological changes of glioma tissues and

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adjacent tissues. The results demonstrated that the cells in glioma tissues had spindle-shaped tumors

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with large deeply-stained nucleus, frequent observations of nuclear division, active growth, densely and disorderly-arranged structure; in comparison with the glioma tissues at stage I-II, that at stage III-IV exhibited more nuclear division and cavitation; while cells in adjacent tissues were normally

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and uniformly distributed (Figure 1). 3.2 Notch-1 is expressed at a higher level in glioma tissues than in adjacent tissues Next, in order to determine the expression of Notch-1 in the glioma and adjacent tissues, we IHC was performed, the results of which illustrated the positive staining of Notch-1 protein was located in the cytoplasm and cytomembrane, exhibited by brown or tan particles. The positive rate 13

of Notch-1 expression was 15.63% in adjacent tissues, and was 72.22% in glioma tissues at a low level (I-II) (p < 0.05); the positive rate of Notch-1 expression was 16.22% in adjacent tissues, and 82.50% in glioma tissues of a high level (III-IV (p < 0.05). The results obtained indicated that Notch-1 expression might be up-regulated in glioma (Figure 2). 3.3 Glioma tissues have decreased expressions of miR-449a, p38MAPK, p-p38MAPK and Bax

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and increased expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 Subsequently, we assessed the expression of miR-449a and the MAPK signaling pathway- and

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apoptosis-related genes by using RT-qPCR, in situ hybridization and western blot analysis. RT-

qPCR results revealed that when compared with the adjacent tissues, the glioma tissues exhibited

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decreased expressions of miR-449a, the mRNA expressions of p38MAPK and Bax (all p < 0.05),

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and increased mRNA expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 (all p < 0.05). The

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changes observed among the glioma tissues at stage III-IV were greater than that in the glioma

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tissues at stage I-II (Figure 3A).

In the experiment of in situ hybridization, the positive staining of miR-449 was represented by

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bluish violet granules in cytoplasm. The in situ hybridization results showed that there were 15 cases with the positive expression of miR-449 in 69 adjacent tissues, with a positive rate of 21.74%,

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while there were 28 cases with a positive expression of miR-449 in 36 glioma tissues at stage I-II.

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The positive rate of 77.78%; there were 37 cases with a positive expression of miR-449 in 40 glioma tissues at stage III-IV, with a positive rate of 92.50%. The positive expression of miR-449 in glioma tissues was significantly higher than that in the adjacent tissues (Figure 3B).

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Western blot analysis results showed that compared with adjacent tissues, the protein levels of

p38MAPK, p-p38MAPK and Bax were decreased, while the protein levels of Notch1, Bcl-2, CyclinD1, p-ERK1/2 and ERK1/2 were increased in glioma tissues (all p < 0.05). The changes in glioma tissues at stage III-IV were greater than that in glioma tissues at stage I-II (Figure 3C~D).

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The above findings demonstrated that miR-449a, p38MAPK, p-p38MAPK and Bax might be expressed at a low level while Notch1, Bcl-2, CyclinD1 and ERK1/2 at a high level in glioma tissues. 3.4 Notch1 is a target gene of miR-499a Next, in order to verify the relationship between miR-499a and Notch1, we adopted an online

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biology prediction website available at (microRNA.org) and dual-luciferase reporter gene assay.

The online biology prediction website results indicated Notch1 as the target of miR-499a. The dual-

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luciferase reporter gene assay results suggested that compared with the NC group, the luciferase

activity in the Wt-miR-449a/Notch1 co-transfected group was significantly decreased (all p < 0.05)

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in the miR-449a mimic group while that in the Mut-miR-449a/Notch1 plasmid group did not differ

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significantly (all p > 0.05) (Figure 4). The results identified that miR-449a could specifically bind to

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Notch1 and negatively regulat it, demonstrating that Notch1 was a target gene of miR-449a.

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3.5 MiR-449a lowers the expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 yet elevates those

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of p38MAPK and Bax

In order to investigate the effects of miR-449a on the expressions of Notch1, Bcl-2, CyclinD1,

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ERK1/2, p38MAPK and Bax, RT-qPCR and western blot analysis methods were conducted. Compared with the normal group, all the other groups in the experiment exhibited decreased miR-

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449a expression and decreased mRNA expressions of p38MAPK and Bax (all p < 0.05), and increased mRNA expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 (all p < 0.05). Compared with the blank group and the NC group, the miR-449a expression in the miR-449a mimic group was

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increased (p < 0.05), while no significant difference was observed in the siRNA-Notch1 group (p > 0.05); the mRNA expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 decreased remarkably (all p < 0.05), and those of p38MAPK, while Bax was distinctly increased in the miR-449a mimic and siRNA-Notch1 groups (all p < 0.05); the miR-449a inhibitor group had declined miR-449a expression (p < 0.05), increased mRNA expressions of Notch1, Bcl-2, CyclinD1, ERK1/2 (all p < 15

0.05) and decreased p38MAPK and Bax (both p < 0.05); the miR-449a inhibitor + siRNA-Notch1 group had decreased miR-449a expression (p < 0.05), while no difference among mRNA expressions of Notch1, Bcl-2, CyclinD1, ERK1/2, p38MAPK and Bax was detected (Figure 5). Compared with the normal group, the other groups in the experiment had increased protein levels of ERK1/2, Notch1, Bcl-2, CyclinD1 and p-ERK1/2, but decreased protein levels of

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p38MAPK, Bax and p-p38MAPK (all p < 0.05). Compared with the blank group and the NC group, the protein levels of Notch1, Bcl-2, CyclinD1 and ERK1/2 were markedly decreased, while those of

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p38MAPK and Bax were significantly elevated in the miR-449a mimic and siRNA-Notch1 groups (all p < 0.05); the protein levels of Notch1, Bcl-2, CyclinD1 and ERK1/2 were notably increased with p38MAPK and Bax significantly decreased in the miR-449a inhibitor group (all p < 0.05). No

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significant difference among the aforementioned indexes was observed in the miR-449a inhibitor +

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siRNA-Notch1 group (Figure 6). Collectively, the data obtained revealed that miR-449a could

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down-regulate the expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 yet up-regulate that of

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p38MAPK and Bax.

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3.6 MiR-449a inhibits glioma cell proliferation and contributes to glioma cell apoptosis In order to investigate the effects of miR-449a on cell proliferation, MTT and BrdU assays

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were conducted, the results of the MTT assay (Figure 7) indicated that compared with the normal

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group, cell proliferation in the other groups all exhibited significantly increased (all p < 0.05). Compared with the blank group and the NC group, cell proliferation was significantly decreased in the miR-449a mimic, siRNA-Notch1 and SB230580 groups (all p < 0.05), while significant

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increases were recorded in the miR-449a inhibitor group (p < 0.05). No remarkable difference in the cell proliferation in the miR-449a inhibitor + siRNA-Notch1 and miR-449a inhibitor + SB230580 groups was detected (p > 0.05). The above results showed that miR-449a could prevent glioma cell proliferation.

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The effects of miR-449a on cell apoptosis, was investigated by means of flow cytometry. PI staining results indicated that when compared with the normal group, the cell ratio in the other groups was significantly increased in the G0/G1 phase and significantly decreased in S the phase (all p < 0.05). Compared with the blank group and the NC group, the cell ratio in the miR-449a mimic, siRNA-Notch1 and SB230580 groups was decreased in the G0/G1 phase and increased in

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the S phase (all p < 0.05), while the cell ratio in the miR-449a inhibitor group exhibited an increase in the G0/G1 phase and decreased in the S phase (all p < 0.05). No significant difference in the

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miR-449a inhibitor + siRNA-Notch1 and miR-449a inhibitor + SB230580 groups (p > 0.05). Taken together, the results revealed that the overexpressed miR-499a and siRNA-Notch1 could inhibit the MAPK signaling pathway, thus impeding glioma cell proliferation (Figure 8A~B).

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Annexin V-FITC/PI staining results showed that compared with the normal group, the cell

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apoptosis in the other groups were significantly decreased (p < 0.05). Cell apoptosis was

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significantly increased in the miR-449a mimic group, siRNA-Notch1 and SB230580 groups (p <

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0.05), but significantly decreased in the miR-449a inhibitor group (p < 0.05). There was no

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remarkable difference observed in the miR-449a inhibitor + siRNA-Notch1 and miR-449a inhibitor + SB230580 groups (p > 0.05). Taken together, the results showed that overexpression of miR-499a

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4. Discussion

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and siRNA-Notch1 could promote glioma cell apoptosis (Figure 8C~D).

Glioma remains the most common and aggressive type of primary malignant brain tumor

(Yang et al. 2017). Numerous studies have in recent years highlighted miRNAs due to their

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significant regulatory roles in the processes of tumor progression, functioning as tumor suppressors or oncogenes by regulating gene expressions and signaling pathways (Wang et al. 2016). During the present study, an investigation was conducted in order to identify the effects caused by miR-499a on the regulation of cell proliferation and apoptosis through the MAPK signaling pathway by targeting Notch1 in glioma. The results of our study demonstrated that miR-499a could act to

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suppress proliferation and promote the apoptosis of glioma cells through inhibition of the MAPK signaling pathway by targeting Notch1. Initially, the expressions of miR-449a, p38MAPK, Bax and p-p38MAPK were lower whereas the expressions of Notch1, Bcl-2, CyclinD1, ERK1/2 and p-ERK1/2 were higher in glioma tissues than in the adjacent tissues. A previous study revealed that miR-449a was depleted in human

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prostate tumor tissue linked to patient-matched controls, capable of tumor suppressor-like function through the targeting HDAC1 and activating p27 expression (Noonan et al. 2010). Ye et al revealed

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that the expressions of miR-449a were expressed at a low level in type II endometrial cancer tissues compared with the benign endometrial tissue specimens (Ye et al. 2014). Notch1 is a member of the Notch receptor family, a human gene encoding a single-pass transmembrane receptor, which plays a

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crucial role in various diseases (Sharma et al. 2011). A previous study suggested that Notch1

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expression was markedly higher in glioma than in normal control brain tissues (Jiang et al. 2011).

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p38MAPK represents one of the four signaling pathways of the MAPK family that has been

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identified in eukaryotic cells (Zhang et al. 2015). Amantini et al revealed that the expression of

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p38MAPK was lower in glioma tissues than that in normal tissues (Amantini et al. 2007). The best characterized MAPK cascade is comprised of Raf, isoforms MEK1/2, and extracellular signal

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regulated protein kinase (ERK) 1/2, while ERK1/2 has been reported to inhibit glioma cell proliferation (Cuevas et al. 2006). Zhang et al demonstrated that the average expression of ERK1/2

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in sacral chordoma tissue specimens was markedly higher than in normal tissue specimens (Zhang et al. 2015). CyclinD1 acts as an essential regulatory role in the G1 phase, while indicating that the

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gene of CyclinD1 is overexpressed in various cancers including that of stomach cancer and nonsmall cell lung cancer (Cai et al. 2012). Previous studies have revealed there to be higher expressions of Bcl-2 and CyclinD1 in glioma tissues than in that of adjacent tissues (Wang et al. 2012, Qu et al. 2014). Bax plays a pro-apoptotic role in the mitochondrial pathway of apoptosis, which is regulated by the Bcl-2 protein family (Barnes et al. 2017). Studies have previously

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suggested that Bax plays a role in both of the processes of apoptosis and necrosis in human glioma cells (Mitlianga et al. 2002). In this study, cells transfected with miR-449a mimic and siRNA-Notch1 exhibited decreased expressions of Notch1, Bcl-2, CyclinD1, ERK1/2 and p-ERK1/2, while p38MAPK, p-p38MAPK and Bax all displayed increased expression levels. Cells transfected with miR-145 mimics exhibited

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decreased expression of the anti-apoptotic protein Bcl-2, while increased expressions of the proapoptotic proteins Bax, and active caspase-3, as well as an elevated Bax/Bcl-2 ratio (Du et al.

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2017). Jiang et al asserted that the expression of Notch1 was related to tumor progression and upregulated in glioma (Jiang et al. 2011). Zhang et al demonstrated that Notch1 enhances the migration and invasion of glioma cells by activating β-catenin and nuclear factor-κB (NF-κB)

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signaling by means of Protein Kinase B activation (Zhang et al. 2012). Accumulating evidence has

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suggested an association between the ERK1/2 pathway and death receptor-mediated cell

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proliferation in glioma cells (Vilimanovich and Bumbasirevic 2008). Chen et al highlighted the role

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of miR-449a as a tumor suppressor in human bladder cancer through its interaction with pocket

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proteins regulation (Chen et al. 2012). Overexpression of miR-499 has been shown to inhibit the apoptosis of cardiomyocyte in heart failure as a result of volume overload (Chua et al. 2016).

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Importantly, evidence has been presented, suggesting that miR-449a could directly and negatively regulate CyclinD1 and Bcl-2 (Hu et al. 2014). Previous studies have showed the involvement of

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overexpressed Notch1 in the majority of glioma cell lines and primary human gliomas with different grades (Xu et al. 2010, Zhao et al. 2010), demonstrating that the activation of Notch may

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be one of causes of glioma. Similarly, in our study, greater expressions of Notch were detected in the glioma tissues in comparison with the adjacent tissues. Furthermore, dual-luciferase reporter gene assay in our study provided verification confirming that Notch1 was as a target gene of miR499a. The current study revealed that the overexpression of miR-449a and Notch1 silencing resulted in reductions in the expressions of Notch1, Bcl-2, CyclinD1, ERK1/2 and p-ERK1/2 which was accompanied by increased expressions of p38MAPK, p-p38MAPK and bax in glioma. 19

Our study revealed that miR-449a mimic, SB230580 and siRNA-Notch1 groups had decreased cell proliferation, increased apoptosis, with less cells arrested at the G0/G1 stage and more cells arrested at the S stage. Accrued evidence has suggested that overexpression of miR-499-5p could inhibit non-small-cell lung carcinoma cells proliferation, while acting to promote cell cycle arrest and apoptosis (Li et al. 2016). Noonan et al revealed that the overexpression of miR-449a in

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prostate cancer PC-3 cells leads to cell cycle arrest and cell apoptosis (Noonan et al. 2009). Jundt et al revealed that Notch1 activation could elevate the proliferation of tumor cells and survival in

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Hodgkin and anaplastic large cell lymphoma (Jundt et al. 2002). A previous study indicated that

activated Notch elevated the proliferation of cells in glioma (Zhang et al. 2008). SB230580 is an antagonist of p38-MAPK, has been identified to function as an inhibitor for the urotensin II-

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mediated cardiomyocyte hypertrophy (Onan et al. 2004). The present study identified that the

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overexpression of miR-499a inhibited cell proliferation and promoted cell apoptosis in glioma by

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targeting Notch1 through suppressing the MAPK signaling pathway.

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In conclusion, our current study demonstrated that miR-499a could act as a tumor suppressor

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in glioma via the MAPK signaling pathway, as well as providing evidence indicating that Notch1 is a direct target gene of miR-499a in glioma. Therefore, the identification of miR-494 and MAPK

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signaling pathway may aid in facilitating the existing understanding of the mechanisms of glioma, with potential of serving as a diagnostic marker for the treatment of glioma in the future. However,

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due to the limitation of sample size, further studies are required to fully understand the specific mechanisms of miR-499a combined with MAPK signaling pathway in glioma treatment.

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Competing interests We declare that we have no conflicts of interest.

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Acknowledgement This study was supported by Henan Provincial Medical Science and Technology Planning Project Jointly Established by the Provincial Department (No. 201601016), Henan Province Key Scientific and Technological Projects (No. 152102310136). We would like to give our sincere appreciation to

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the reviewers for their helpful comments on this article.

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Figure 1 Pathological observation of glioma tissues and adjacent tissues (normal tissues) (× 400).

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Figure 2 Immunohistochemistry staining shows Notch-1 is expressed at a high level in glioma (× 400). Panel A, pathological observation of glioma tissues and adjacent tissues; Panel B, the positive

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rate of Notch1 expression in glioma tissues and adjacent tissues; n = 6.

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Figure 3 MiR-449a, p38MAPK, p-p38MAPK and Bax are decreased yet Notch1, Bcl-2, CyclinD1 and ERK1/2 are increased in glioma tissues. Panel A, the mRNA expressions of relative genes in each group detected by RT-qPCR; Panel B, detection for miR-449a using in situ hybridization ( 400); Panel C, the protein levels of relative proteins in each group detected by western blot analysis; Panel D, protein bands of relative proteins in each group; n = 6; Data from at least three

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independent experiments performed in triplicates are presented as the mean ± standard deviation; comparison between two groups was analyzed by t-test; *, p < 0.05 vs. adjacent tissue; #, p < 0.05

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vs. glioma tissue at stage I-II; p38MAPK, p38 mitogen-activated protein kinase; ERK, extracellular

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regulated protein kinases; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X protein.

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Figure 4 Notch1 is confirmed as a target gene of miR-499a. Panel A, predicted binding site of miR449a on Notch1 3'UTR; Panel B, luciferase activity assay; n = 6; Data from at least three independent experiments performed in triplicates are presented as the mean ± standard deviation; comparison between two groups was analyzed by t-test; *, p < 0.05 vs. the NC group; miR-499a,

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microRNA-499a; NC, negative control.

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Figure 5 RT-qPCR results demonstrates that miR-449a down-regulates the mRNA expressions of Notch1, Bcl-2, CyclinD1 and ERK1/2 yet up-regulates those of p38MAPK and Bax. n = 6; Data from at least three independent experiments performed in triplicates are presented as the mean ± standard deviation; comparisons among multiple groups were analyzed by ANOVA; *, p < 0.05 vs. the normal group; #, p < 0.05 vs. the blank and NC groups, miR-499a, microRNA-499a; p38MAPK,

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lymphoma-2; Bax, Bcl-2 associated X protein; NC, negative control.

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p38 mitogen-activated protein kinase; ERK, extracellular regulated protein kinases; Bcl-2, B-cell

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Figure 6 Western blot analysis results shows that miR-449a decreases the protein levels of Notch1, Bcl-2, CyclinD1 and ERK1/2 yet elevates those of p38MAPK and Bax. Panel A, the protein levels of Notch1, p38MAPK, ERK1/2, and p-ERK1/2 in each group; Panel B, the protein levels of Bcl-2, CyclinD1, Bax and p-p38MAPK in each group; Panel C, protein bands of Notch1, p38MAPK, ERK1/2, Bcl-2, CyclinD1, Bax, p-p38MAPK and p-ERK1/2 among the groups; n = 6; Data from at

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least three independent experiments performed in triplicates are presented as the mean ± standard deviation; comparisons among multiple groups were analyzed by ANOVA; *, p < 0.05 vs. the

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normal group; #, p < 0.05 vs. the blank and NC groups; p38MAPK, p38 mitogen-activated protein kinase; ERK, extracellular regulated protein kinases; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2

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associated X protein.

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Figure 7 The findings of MTT and BrdU assays indicate that miR-449a inhibits glioma cell proliferation. Panel A, cell proliferation in each group detected by MTT assay; Panel B, cell proliferation in each group detected by BrdU assay; Panel C, fluorescence image of cell proliferation in each group detected by BrdU assay ( 100); n = 6; Data from at least three independent experiments performed in triplicates are presented as the mean ± standard deviation;

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comparisons among multiple groups were analyzed by ANOVA; *, p < 0.05 vs. the normal group; #,

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p < 0.05 vs. the blank and NC groups; miR-499a, microRNA-499a; NC, negative control.

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Figure 8 Flow cytometry results reveals miR-449a accelerates glioma cell apoptosis. Panel A~B, cell cycle of glioma cells in each group; Panel C~D, cell apoptosis of glioma cells in each group; n = 6; Data from at least three independent experiments performed in triplicates are presented as the mean ± standard deviation; comparisons among multiple groups were analyzed by ANOVA; *, p < 0.05 vs. the normal group; #, p < 0.05 vs. the blank and NC groups; miR-499a, microRNA-499a;

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NC, negative control.

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Table1 Primer sequences for RT-qPCR Premier sequences (5’-3’)

Genes

F: TGCGGTGGCAGTGTATTGTTAGC miR-449a R: CCAGTGCAGGGTCCGAGGT F: GAAGAAGCTCTCCAGACCATTTC P38MAPK R:AACGTCCAACAGACCAATCAC

R:CAAACCCCCGTCTGTTACAC F: TGCACCTGAGCGCCTTCAC Bcl-2 F: CCACCAGCTCTGAACAGTT Bax R:TCAGCCCATCTTCTTCCAG F: CGTGCAGAGTGAGACCGTGGA

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Notch1

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R:TAGCTGATTCGACCATTTGCCTGA

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F: GCCGATTACCAGACAAGCA ERK1/2

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R:TGCGGTCTGTCTGGTTGTGCA F: AGGAGAACAAACAGATCA

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CyclinD1

R:TAGGACAGGAAGTTGTTG

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F: GGGTGCTCGCTTCGGCAGC U6

R:CAGTGCAGGGTCCGAGGT

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F: CGAGAAGATGACCCAGATCA

β-actin

R:GATCTTCATGAGGTAGTCAG

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Note: RT-qPCR, reveres transcription quantitative polymerase chain reaction; F, forward; R, reverse; miR-449a, microRNA-449a; p38MAPK, p38 mitogen-activated protein kinase; ERK, extracellular regulated protein kinases; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated X protein.

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