RNAi-mediated SYT14 knockdown inhibits the growth of human glioma cell line U87MG

Accepted Manuscript Title: RNAi-mediated SYT14 knockdown inhibits the growth of human glioma cell line U87MG Authors: Bin Sheng, Yuxin Jiang, Degang Wu, Niansheng Lai, Zhennan Ye, Bingbing Zhang, Xinggen Fang, Shanshui Xu PII: DOI: Reference:

S0361-9230(17)30556-7 https://doi.org/10.1016/j.brainresbull.2018.04.002 BRB 9408

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

18-9-2017 4-12-2017 4-4-2018

Please cite this article as: Bin Sheng, Yuxin Jiang, Degang Wu, Niansheng Lai, Zhennan Ye, Bingbing Zhang, Xinggen Fang, Shanshui Xu, RNAi-mediated SYT14 knockdown inhibits the growth of human glioma cell line U87MG, Brain Research Bulletin https://doi.org/10.1016/j.brainresbull.2018.04.002 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.

RNAi-mediated SYT14 knockdown inhibits the growth of human glioma cell line U87MG Bin Shenga,1, Yuxin Jiangb,1, Degang Wua, Niansheng Laia, Zhennan Yec Bingbing Zhanga, Xinggen Fanga,*, Shanshui Xua,* a

Department of Neurosurgery, Yijishan Hospital of Wannan Medical College,

b

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Wuhu City, Anhui, 241001, China Department of Physiology, School of Basic Medicine, Wannan Medical

College, Wuhu City, Anhui, 241000, China c

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Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou

Medical University, Guangzhou City, Guangdong, 510000, China *

Corresponding author at: 2 Zheshan Road, Wuhu City, Anhui, 241001,

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China

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Corresponding authors.

E-mail adress: [email protected] (S.S. Xu),

These two authors contributed equally to this work.

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[email protected] (X.G. Fang)

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Highlight:

Lentivirus-mediated small hairpin RNAs could silence the SYT14 gene.

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Knockdown of SYT14 promotes U87MG cell apoptosis

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Knockdown of SYT14 inhibits U87MG cell proliferation and colony

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formation.

Abstract: SYT14 (Synaptotagmin 14) participates in pathomechanical neurodegeneration and contributes to abnormal neurodevelopment. However, the functional mechanism of SYT14 in human glioma tumorigenesis remains unclear. In the present study, we measured the expression levels of SYT14 mRNA in human glioma cell lines, U373MG, U178, and U87MG and neural

stem cells (NSC) cell line by RT-PCR, and used lentivirus-mediated small hairpin RNAs (shRNAs) to knock down SYT14 expression in U87MG cells. Changes in SYT14 expression were determined by real-time PCR. Cell proliferation and colony formation assays were used to analyze the role of SYT14 in U87MG cell proliferation, and cell apoptosis was assessed by flow cytometry. SYT14 mRNA expression was detected in the three glioma cell lines, and was highest in the U87MG cell line. The RNAi-mediated knockdown

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of SYT14 significantly decreased cell proliferation and colony formation in U87MG cells, and caused a moderate increase in apoptosis. Fewer S phase

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cells and more G2/M phase cells were observed. These data indicate that SYT14 is highly expressed in glioma cells, and may participate in glioma cell proliferation, apoptosis, and colony formation.

Key word: Glioma, Synaptotagmin 14, Lentivirus infection, small hairpin RNA

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1. Instruction

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Gliomas, originating from neural mesenchymal and parenchymal cells, are the most common aggressive primary brain tumors [1]. Generally, gliomas

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have high incidence, recurrence, mortality, and migratory and proliferative abilities, as well as a low cure rate[2,3]. Although surgery combined with

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radiation and chemotherapy is the preferred treatment, it does not significantly prolong survival (the median survival time is less than 14 months), and the

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prognosis is poor [4,5]. Therefore, it is imperative to explore the molecular mechanisms of malignant progression and study the invasive growth of

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gliomas to identify new targets for glioma treatment.

Synaptotagmins (SYTs) are a large family of transmembrane proteins with

a single transmembrane domain (TM) close to the N terminus and containing

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tandem cytoplasmic domains, C2A and C2B [6,7]. SYTs are anchored to secretory vesicles via the TM and are associated with exocytosis [8,9]. SYTs function as sensors that link changes in calcium levels to a variety of biological

processes,

including

neurotransmission

and

hormone-

responsiveness [10]. A member of the SYT family, SYT14 is located on the 1q32 breakpoint [11] and is conserved across many organisms [10,12,13]. A recent study demonstrated that SYT14 specifically localizes to Purkinje cells

in the cerebellum in humans and mice [14]. Similar studies showed that aberrant SYT14 was associated with psychomotor retardation [14] and neurodevelopmental abnormality [10]. Duan et al. [15] found that a G allele at rs2485893 (10 kb 3' of SYT14) was associated with nonsyndromic cleft palate only (NSCPO) by the transmission disequilibrium test (TDT) method. Genotypic TDT for epistatic interactions for NSCPO also identified an

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interaction of rs4844913 (37 kb 3' of the digestive organ expansion factor homolog) and rs11119388 (SYT14).

The function of SYT14 in human cancers, particularly in gliomas, remains

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unclear. In the present study, we focused on the human NSCs line HB1.F3,

which are immortalized, clonal, non-tumorigenic and minimally immunogenic cells[16]. We confirmed that SYT14 is highly expressed in glioma cell lines, with higher levels in U87MG cells compared to HB1.F3 cells. Additionally, we

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employed the lentivirus-delivered small hairpin RNA (shRNA) technique to

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2. Materials and methods

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examine the effect of SYT14 knockdown on human glioma cell growth in vitro.

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2.1 Cell lines and culture conditions

Human glioma cell lines U373MG, U178, and U87MG and human renal

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epithelial 293T cells were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The human Neural Stem Cell (NSC)

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line HB1.F3 was derived from 15-week human fetal telencephalon and immortalized by v-myc expression[17,18]. All cell lines were cultivated in RPMI-1640 medium, and supplemented with 10% fetal bovine serum (FBS; Gibco®, Shanghai, China), 100 U/ml penicillin, and 0.1 mg/ml streptomycin

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(Sangon Biotech, Shanghai, China) at 37˚C with 5% CO2 .

2.2 Quantitative RT-PCR

Total RNA from the three cell lines, U373MG, U178, and U87MG, was extracted using the Trizol reagent (Invitrogen, Shanghai, China), according to

the manufacturer's protocols. The first strand cDNA was compounded using the miRcute miRNA Frist-Strand cDNA Synthesis Kit (Tiangen, Beijing, China). PCR amplifications were performed with the miRcute miRNA qPCR Detection Kit (SYBR Green) (Tiangen, Beijing, China) in a CFX-96 Bio-Rad Real-Time PCR instrument (Bio-Rad Laboratories, lnc. Hercules, CA, USA). Quantitative RT-PCR was performed with an initial denaturation at 95˚C for 15 sec, then 45 cycles at 95˚C for 15 sec, and 60˚C for 30 sec. GAPDH was used as an

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internal control. The relative gene expression levels were calculated by 2-△△CT

analysis. The primers used were as follows: for SYT14, F: 5'-GGT GGA GAG

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AGA ACC TGT GG-3', R: 5'- ATC TGG AAA CCC GCC AAC AT 3'; for GAPDH, F: 5'-TGA CTT CAA CAG CGA CAC CCA-3', R: 5'-CAC CCT GTT GCT GTA GCC AAA-3'. All reactions were done in triplicate.

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2.3 Recombinant lentiviral vector production and cell infection

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The complementary DNA sequence (ATG TTG GCG GGT TTC CAG AT) of SYT14 was designed from the full-length SYT14 sequence (GenBank no.

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NM_001146261.2), the control shRNA's sequence(TTC TCC GAA CGT GTC ACG T) and synthesized by GeneChem Co. Ltd. (Shanghai, China). After the

knockdown

efficiencies,

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testing

stem-loop

oligonucleotides

were

synthesized and inserted into the lentivirus-based pGV115-GFP (GeneChem)

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with AgeI/EcoRI sites. For lentivirus infection, U87MG cells were cultured into 6-well plates and then the SYT14-shRNA-lentivirus or the shRNA-control

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(shCtrl) lentivirus was added at the appropriate multiplicity of infection (MOI). After 72 h of infection, the cells were observed under a fluorescence microscope (MicroPublisher 3.3RTV; Olympus, Tokyo, Japan), Real-time quantitative PCR was performed to determine the knockdown efficiency of the

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harvested cells 120 h post infection.

2.4 Western blot analysis

The cells were collected, washed twice with cold phosphate-buffered saline, and then lysed on ice in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl,

1% SDS, 1 mM EDTA, 1% NP-40) containing 1 mM protein inhibitor and 1 mM PMSF, for 30 min on ice. The lysates were centrifuged at 12,000 x g at 4˚C for 10 min and the supernatants were collected. The BCA protein assay (HyClone-Pierce, Rockford, IL, USA) was used to measure protein concentration. Equal amounts of protein from each treatment were electrophoresed on 10 % acrylamide gels. After SDS-PAGE, the proteins were transferred onto PVDF membranes. The membranes were incubated

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with mouse anti-SYT14 or anti-GAPDH antibodies (Santa Cruz Biotechnology,

Santa Cruz, CA, USA). The protein bands were visualized using an enhanced

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chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

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2.5 Cell growth assay

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Cell growth was measured via multiparametric high-content screening

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(HCS) . Briefly, after being transfected with either the shCtrl lentivirus or shSYT14 lentivirus, logarithmic phase U87MG cells were seeded at 2,000

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cells per well into 96-well plates, and then incubated at 37°C with 5% CO2 for 5 days. The cells in the plates were scanned daily with the Cellomics

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ArrayScan™ VT1 HCS automated reader (Cellomics, Inc., Pittsburgh, PA, USA). For each well, at least 800 cells were analyzed.

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Cell viability was measured by MTT assay with MTT Cell Proliferation and Cytotoxicity Assay Kit (Sangon Biotech, Shanghai, China). After infection with either shCtrl lentivirus or shSYT14 lentivirus, U87MG cells were seeded

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at 2,000 cells per well in 96-well plates, then incubated at 37°C with 5% CO2 for 5 days. Then 5 wells each group were added in MTT and incubated for 4 h at 37°C with 5% CO2. Then standard procedure was conducted as described

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by the manufacturer and OD value was tested at a wavelength of 490 nm (Tecan infinite).

2.6 Analysis of cell cycle distribution and apoptosis Cell cycle distribution or apoptosis was determined by flow cytometry (FCM) analysis. Briefly, U87MG cells were transfected with shSYT14 or shCtrl plasmids and incubated at 37˚C for 1, 2, 3, 4, or 5 days. At the indicated time

point, adherent cells were collected, washed twice with ice-cold phosphatebuffered saline (PBS), fixed with 0.5 ml of ice-cold 70% ethanol at 4˚C for 1 h, and stained with propidium iodide (PI; 50 µg/ml, Sigma-Aldrich® Co. LLC., St. Louis, USA) in the presence of RNase A (100 µg/ml; Fermentas ®, Shanghai, China). The suspension was filtered through a 300-mesh filter, and the cell cycle phase of the stained nuclei were determined by a BD FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA).

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Cell apoptosis was assayed by staining with Annexin V-APC (eBioscience, San Diego, CA, USA) and detected by FCM. To do this, U87MG cells were

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cultured into 6-well plates. After 48 h of transfection with shSYT14 or shCtrl plasmids, the cells were collected and washed twice with ice-cold PBS. The cell concentrations were adjusted to 1x106/ml with 1X staining buffer. Next, 100 ml of cell suspension was stained with 5 µl Annexin V-APC at room

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temperature in the dark for 15 min. Cells were analyzed using FCM within 1 h.

2.7 Analysis of cell cycle colony formation

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Cells were seeded into 6-well plates (200 cells/well) and cultured at 37oC

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in 5% CO2. After 14 days, the cells were fixed with paraformaldehyde for 30 min and then stained with GIEMSA for 10 min. The cells were washed three

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times with ddH2O to obtain a clean background. The number of colonies was

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then determined by counting under a light microscope.

2.8 Statistical analysis

One-way ANOVA and the Student's t-test were used for raw data analysis.

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Statistical analysis was performed using the software SPSS20.0 (SPSS, Inc., USA). The statistical data for each group were determined as the mean value ± the standard deviation (SD). A value of p<0.05 was considered statistically

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significant. 3. Results

3.1 Expression of SYT14 in three glioma cell lines In order to examine whether there is a significant difference in the expression of SYT14 mRNA in glioma cells compared to HB1.F3 cells, we performed real-time quantitative PCR. The results showed that the SYT14

mRNA was expressed in all three glioma cell lines and HB1.F3 cell, and with the highest level in the U87MG cell line, as shown in Fig.1A.

3.2 Knockdown efficiency determined by western blot analysis Human embryonic kidney 293T cells were infected with shSYT14 or shCtrl lentivirus. SYT14 protein expression was detected by western blotting in these cells. The SYT14 protein level was greatly reduced in the shSYT14-

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infected cultures, indicating effective knockdown of the target sequence, as

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shown in Fig.1B.

Fig.1 SYT14 levels in glioma cell lines compared with HB1.F3 cell line and SYT14 protein expression in 293T cells with SYT14 knocked down. (A) The

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expression of SYT14 mRNA in three glioma cell lines and HB1.F3 cell line as

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determined by RT-PCR. (B) Knockdown of SYT14 protein expression was

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analyzed by western blotting in 293T cells. Compared with expression of shCtrl, the level of SYT14 protein decreased after SYT14 expression was

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silenced by RNAi. GAPDH was used as an internal control.

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3.3 Lentivirus-mediated knockdown of SYT14 in glioma cell line U87MG

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To explore the role of SYT14 in glioma cells, we knocked down SYT14 in the U87MG cell line. As shown in Fig. 2A, by day 3 after infection, the proportion of infected cells was over 80% for both the shSYT14 lentivirus and

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shCtrl lentivirus. SYT14 mRNA levels in U87MG cells were assessed by RTPCR at day 5 after infection with either the shSYT14 or shCtrl lentivirus. The shSYT14 lentivirus-infected cultures exhibited significantly lower levels of

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SYT14 mRNA compared to levels in cultures infected with the shCtrl lentivirus (Fig.2B).

Fig.2 Lentivirus-mediated shSYT14 significantly suppressed the expression of SYT14. (A) U87MG cells were infected with shSYT14 or shCtrl lentivirus and examined by fluorescent microscopy and light microscopy after 3 days of

infection. The green fluorescence in cells indicates lentivirus infection. Magnification, x100. (B) U87MG cells were infected with shSYT14 or shCtrl lentivirus and SYT14 mRNA levels were analyzed by RT-PCR after 5 days of infection. As predicted, the SYT14 mRNA level decreased significantly after SYT14 knockdown. **p<0.01.

3.4 Knockdown of SYT14 in U87MG cells inhibits cell proliferation and

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colony formation

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To explore the effect of SYT14 on cell growth, cell growth was measured

via multiparametric high content screening (HCS) at different times after shSYT14 lentivirus infection. As illustrated in Fig. 3B, the proliferation of U87MG cells was significantly inhibited when SYT14 was knocked down. The

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same result was also observed in MTT assay, as shown in Fig. 3C.

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We studied the colony formation capacity of the U87MG cell line with

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lentivirus treatment to explore the effect of SYT14 on cell growth. After infection with lentivirus and fixation with paraformaldehyde and Giemsa

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staining, the growth of cells was observed for the two groups. As shown in Fig. 3D, the number of colonies and analysis results showed decreased growth of

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U87MG cells in the shSYT14 group compared to those in the shCtrl group (shSYT14, 7.00 ± 1.52, vs shCtrl, 103.30 ± 2.60, p <0.01). Obviously, the

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knockdown of SYT14 significantly decreased cell proliferation and colony

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formation of U87MG cells.

Fig.3. Knockdown of SYT14 inhibits proliferation and colony formation of U87MG cells. (A) Images of lentivirus transfection of shCtrl and shSYT14 and

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at different times after transfection. (B, C) Cell growth was measured by multiparametric high content screening (HCS) and by MTT assay. Proliferation of U87MG cells was significantly inhibited when SYT14 was knocked down. (D) Colony formation of cells in the shCtrl and shSYT14 after infection of the U87MG cells with lentivirus. The clone number per well was determined and is shown in the histogram. The results revealed a significant difference in the treatment group compared to the shCtrl group (**p<0.01)

3.5 Knockdown of SYT14 in U87MG cells leads to cell cycle arrest and induces cell apoptosis

Flow cytometry was used to determine whether knockdown of SYT14 is necessary for cell cycle progression in U87MG cells. The results shown in Fig.4A indicated that shSYT14 knockdown in U87MG cells decreased the

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proportion of cells in G0/G1 and S phases, and increased the G2/M phase

population, compared with the shCtrl group. The percentage of G2/M phase

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increased from (1.15 ± 0.63) in the shCtrl groups to (8.16 ± 0.93) in the shSYT14 groups. These results indicated that SYT14 may arrest the cell cycle at the G2/M phase.

The effect of SYT14 knockdown in U87MG cells on cell apoptosis was

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determined by Annexin V staining followed by flow cytometry. As shown in

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Fig.4B, the results showed that cell apoptosis was significantly increased in

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the shSYT14 group compared to the shCtrl group (shCtrl, 2.91 ± 0.06%, vs shSYT14, 9.55 ± 0.24%, p<0.001). These results indicate that SYT14

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expression is a determinant of cell apoptosis in U87MG cells.

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Fig.4 SYT14 knockdown leads to cell cycle arrest and induces apoptosis in U87MG cells. (A) Analysis of the cell cycle of U87MG cells by flow cytometry.

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Compared with the shCtrl group, shSYT14 cultures showed a significant increase in the proportion of cells in the G2/M phase (**p<0.01). (B) Cell death was determined by Annexin V staining and flow cytometry. The shSYT14

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cultures showed a significant increase in apoptosis compared with shCtrl cultures (***p<0.001).

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4. Discussion Synaptotagmins (SYTs) are membrane trafficking proteins found in various species from different phyla and can form Ca2+-independent oligomers [12]. SYTs may play a key role in the repair of degenerating neurons and act as Ca2+ sensors for the regulated release of neurotransmitters [19,20]. SYT14, a

SYT, belongs to the C-terminal-type (C-type) tandem C2 protein family, with a single transmembrane (TM) domain at the N-terminus and a putative fattyacylation site just downstream [12]. Waerzeggers et.al [21] showed that synaptotagminⅠfunctions as a special biomarker for pre-clinical development of malignant glioma. Kunapuli et.al [22] demonstrated that the LGI1 gene is involved in synaptic vesicle function in neurons. However, there have been no

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reports analyzing the function of SYT14 in human glioma cells. The purpose of this study was to explore the function of SYT14 in human glioma cells.

This study is the first demonstration of RNAi-mediated knockdown of

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SYT14 in human glioma cells. To investigate the expression of SYT14 in human glioma cells, we constructed three glioma cell lines and compared

them with the NSC cell line HB1.F3. Real-time PCR assays confirmed altered SYT14 expression in these cell lines. Compared with HB1.F3 cells, SYT14

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expression was detected in all three glioma cell lines, with the highest level in

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U87MG cells. Next, we used lentivial-mediated shRNAs to effectively

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knockdown levels of SYT14 in U87MG cells. Compared to the shCtrl group, we found that knockdown of SYT14 resulted in cell cycle arrest at G2/M phase

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and increased cell apoptosis. Additionally, we also found that knockdown of SYT14 decreased the clone number in U87MG cells. Taken together, these

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results indicated that SYT14 promotes the growth of human glioma cells. The mechanism of SYT14 in glioma cells remains unclear, however, a

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growing number of studies have suggested that SYT14 is involved in the function of the brain. Previous studies found that the expression of SYT14

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mRNA is highly restricted to the mouse heart and testis and absent in the brain, suggesting that SYT14 may be involved in membrane trafficking in specific tissues outside the brain. Doi et al. [14] indicated that alteration of the exocytosis membrane-trafficking machinery by mutant SYT14 may represent a

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distinct pathomechanism associated with human neurodegenerative disorders. Another study [10] demonstrated that disruption of SYT14 protein function was associated with neurodevelopmental abnormalities. Similar studies [23,24] showed association of SYT14 with cerebellar ataxia or vestibular disorders. Taken together, these findings indicate that SYT14 contributes to neuronal function. Basing on our trial results, we concluded that SYT14 may play an

indispensable role in the development of pathophysiological mechanisms in glioma. There are some potential limitations of our study. First, this was an in vitro study and the in vivo mechanism is unclear. Therefore, these findings should be verified by the construction of animal models. Second, we have detected that knockdown of SYT14 decreased cell proliferation and colony formation but increased cell apoptosis in the U87MG cell line, however, this is a single

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human glioma cell line, and this finding should be tested in other systems.

Finally, we observed that SYT14 promoted cell growth with a control shRNA,

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but this finding should be verified with another control shRNA. Therefore,

future studies should include the construction of animal models, additional cell lines, and other control shRNA to verify this conclusion.

5. Conclusion

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In summary, these findings demonstrate that RNA-mediated SYT14

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knockdown can inhibit proliferation and colony formation and promote

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apoptosis of glioma cells. These results suggest that SYT14 plays an important role in glioma development and suggests a new therapy for patients

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with glioma. However, the detailed mechanism of SYT14 action remains

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unclear, and further research is needed.

Conflict of interest

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The authors declare that there are no conflicts of interest.

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Acknowledgements This research was supported by the National Natural Science Foundation of

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China (No.81671568).

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