Chapter I. Introduction
6. Rationales
The tumorigenesis of NB could be a divergence of the embryonic development of sympathetic nervous system. ER chaperones including CRT and GRP78 are found to participate during embryonic development. Studies in CRT knockout mice which demonstrated the essential role of CRT for embryogenesis have suggested the important role of CRT in nervous system development as well as the biology of NB.
As we mentioned above, calreticulin (CRT) has been previously correlated with the differentiation of NB tumors, implying a favorable prognostic factor. However, how CRT affects the neuronal differentiation and of NB remains unclear.
VEGF-A and VEGF-A-driven angiogenesis have been reported to participate in the behavior of NB. However, the roles of VEGF-A in NB progression and prognosis are complicated and controversial. Furthermore, recent studies have found a
correlation between CRT and VEGF-A in gastric cancers. Nevertheless, the
relationship between CRT and VEGF-A in NB has never been studied. Thus, in this
study, we proposed to investigate the association of CRT and VEGF-A in
regulating NB tumorigenesis focusing on angiogenesis and neuronal
differentiation in vitro and in vivo.
Chapter II.
Materials and Methods
Ethics statement
All zebrafish and mouse experiments were performed after approval from the Institutional Animal Care and Use Committee at National Taiwan University. The clinical evaluation and use of tumor tissues for this study were approved by the Institutional Review Board of National Taiwan University Hospital. Written informed consent was obtained from the patients before sample were collected.
Fish breeding and embryo collection
Breeding fish will be maintained at 28.5 °C on a 14-h light/10-h dark cycle in a certified fish facility. Embryos will be collected by natural spawning, raised in 0.3X Danieau’s buffer (by diluting 1X Danieau’s buffer: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, and 5.0 mM HEPES (pH 7.6), with double distilled water) until observation or fixation. Embryos will be staged according to Kimmel et al.
[92], and stages are given as hours post-fertilization.
Sequence analysis of calreticulin
The homologous calreticulin genes of human, mouse and zebrafish were identified from the NCBI database. The genomic sequence alignment and phylogenetic tree were carried out using Ensembl and MEGA4.3, respectively.
Total RNA isolation and RT-PCR in zebrafish
A reverse-transcription polymerase chain reaction (RT-PCR) will be performed on total RNA extracted from embryos at the designated times using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA will be subsequently treated with DNase I (Invitrogen), and first-strand cDNA will be synthesized. cDNA will be generated from total RNA of zebrafish larvae using the SMART kit (Clontech, Mountain View, CA) according to the manufacturer’s
instructions.
Morpholino oligonucleotide (MO) microinjections
Antisense MOs will be purchased or custom made by Gene Tools, LLC (Philomath, OR). A standard control MO (stdMO,
5’-CCTCTTACCTCAGTTACAATTTATA-3’) with no sequence homology to any known zebrafish sequences will be used. To knock down the target genes activity, we used two different MOs targeting the translation initiation site and the splicing donor site, respectively. The MOs will be dissolved in sterile double-distilled water to 1 mM and stored at -20 °C.MOs will be diluted to the desired working concentrations in 1X Danieau’s buffer with 0.5% phenol red and stored at 4 °C before being used.
Thin-wall (1 mm (o.d.) × 0.75 mm (i.d.)) glass capillaries with filaments (A-M Systems, Carlsborg, WA) will be pulled using a horizontal puller (P-97, Sutter Instrument, Navato, CA). Embryos at desired stages will be immobilized at an injection trough on a 100-mm 2% agar plate. MOs will be prepared as described at designated concentrations. An injection pipette will be forced into the chorion and the yolk cells to reach the junction between the yolk cells and blastodisc where the solution will be ejected by using a pressure injector (IM-300, Narishige, Japan). After injection, embryos will be recovered from the injection troughs and cultured in 0.3X Danieau’s buffer at 28.5 °C until being examined.
Cell culture
The NB cell lines SH-SY5Y (ATCCH CRL-2266TM) and SK-N-DZ (ATCCH CRL-2149TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), while the stNB-V1 was kindly provided by Dr. Yung-Feng Liao of Academia Sinica. These cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/High glucose medium (Biowest) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). The cells were grown in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. Using a
hemocytometer and trypan blue, cells were sub-cultured in 10 cm plates at a density
of 106 cells per plate.
Transfection
The cells were seeded in 3.5 cm plates at density of 3*105 cells and were transfected with construct plasmids using Lipofectamine 2000 (Invitrogen). Every plate was transfected with 4 μg of the plasmid and 10 μL of the Lipofectamine 2000 in serum-free DMEM. After 8 hours of transfection, the medium was changed into DMEM with 10% FBS. The cells were harvested after 48 h.
Cells transfected with a vector, without the insert gene, were used as vehicle control and cells treated with ddH2O were used as negative control. The construction of CRT expression vector pEGFP-C1-CRT and CRT shRNA vector
pCR3.1-CRTshRNA was as previously described [28].
Construction of stable cell lines
Tetracycline-inducible CRT expression vector pLKO_AS3.1-p5CRT-HY was constructed. pLKO_AS3.1 was the control vector of the inducible vector. CRT shRNA expression vector was purchased from the National RNAi Core Facility Platform, Academia Sinica (Taipei, Taiwan). The shRNA target sequence was: 5’-
CCAGTATCTATGCCTATGATA-3’ (shCRTa, TRCN0000019989), 5’-
CGTCTACTTCAAGGAGCAGTT-3’ (shCRTb, TRCN0000019991). pLKO.1 was the control vector of the shRNA plasmid.
Lentiviral stocks were produced by calcium phosphate transfection. Around 30-40% confluent 293T cells in T25 flasks were prepared and transfected with a DNA mixture containing 7.5 g packaged plasmids and 7.5 g lentivector of the target gene for 16 h. The transfected condition medium was replaced with 12 ml fresh DMEM containing 10 mM sodium butyrate. After 24 h, all conditioned medium were harvested and treated with the stNB-V1 cells for infection. Cells were selected by 1 μg/μL puromycin (InvivoGen, USA).
RNA isolation and reverse-transcription (RT)
Total cellular RNA was extracted using the TRIzol reagent (Invitrogen).
Complementary DNA was synthesized with 1 µg total RNA using a Toyobo RT-polymerase chain reaction (PCR) kit (Toyobo, Osaka, Japan).
Quantitative real-time PCR
The real-time PCR with the mixture reagent KAPA SYBR-Green as the fluorescent dye (Bio-Rad) was conducted on a Mini-Opticon real-time detection system (Bio-Rad, Hercules, CA, USA). Gene-specific primers were used and the
specificity was confirmed by single melting-curve after real-time PCR. Cycling
conditions were 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s.
For quantification, the target gene was normalized to the GAPDH to act as an internal control for SH-SY5Y and SK-N-DZ cells and heat shock protein 60 (HSP 60) for stNB-V1. P53, Bcl-2 and Bax were used as apoptotic markers [93]. Primers for the real-time PCR were: GAPDH (F-5’-AAG GTG AAG GTC GGA GTC-3’ and
R-5’-TGT AGT TGA GGT CAA TGA AGG-3’); HSP60 (F-5’- CA CCG T AA GCC TTT GGT CAT-3’ and R-5’- CTT GAC TGC CAC AAC CTG AA-3’); CRT (F-5’-CC TCC TCT TTG CGT TTC TTG-3’ and R-5’-CAG ACT CCA AGC CTG AGG AC);
HIF-1α (F-5’-CAT AAT GTG AGT TCG CAT CT-3’ and R-5’-ATA TCC AAA TCA CCA GCA TC); VEGF-A (F-5’-GGC ACA CAG GAT GGC TTG AAG-3’ and R-5’-GGC ACA CAG GAT GGC TTG AAG-3’); p53 (F-5′-TGA CTG TAC CAC CAT CCA CTA-3′ and R-5′-AAA CAC GCA CCT CAA AGC-3′); Bax ((F-5′
-TGC TTC AGG GTT TCA TCC AG-3′ and R-5′-GGC GGC AAT CAT CCT CTG-3′); Bcl-2 (F-5′-AGG AAG TGA ACA TTT CGG TGA C-3′ and R-5′- GCT CAG TTC CAG GAC CAG GC-3′); GAP43 (F-5′-TCC GTC GAC ACA TAA CAA -3′ and R-5′-CAG TAG TGG TGC CTT CTC C-3′); neuron-specific enolase (NSE) (F-5′-TGT CTG CTG CTC AAG GTC AA-3′ and R-5′-CGA
TGA CTC ACC ATG ACC C-3′); neurofilimant-H (NFH) (F-5′-CCG ACA TTG CCT CCT ACC-3′ and R-5′-GAG CCA TCT TGA CAT TGA GCA-3′); TrkA (F-5′-TTG GCA TGA GCA GGG ATA TCT-3′ and R-5′-ACG GTA CAG GAT GCT CTC GG-3′).
Western blot analysis
Total proteins were extracted from cells using lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, and 10% glycerol) with 10% protease inhibitor cocktail. The cells were lysed for 15 min on ice and then spun at 4°C and 13,000 rpm for 15 min. The supernatant was then collected for Western blotting. A Bio-Rad protein assay kit was used to measure protein concentration.
Concentration-normalized lysates were boiled at 100°C in an SDS sample buffer for 5 min. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) (80 volts for 30 min in stacking gel and 120 volts for 1.5 h in running gel) and transferred to nitrocellulose membranes (100 Volts for 60 min).
The membranes were blocked with 5% BSA in TBS-T (0.1% Tween 20 in TBS), followed by overnight incubation at 4°C with appropriate dilutions of primary
antibody in 1% TBS-T. After three washes with TBS-T (5 min each), the membranes were incubated with the appropriate secondary antibody coupled with horseradish
peroxidase. Immuno-complexes were visualized using an enhanced
chemi-luminescence (ECL) kit according to the manufacturer’s instructions.
The antibodies used were as follows: rabbit polyclonal anti-CRT antibody (Upstate Biotechnology, Lake Placid, NY), rabbit monoclonal anti-VEGF-A antibody (Santa Cruz, CA, USA), goat polyclonal anti-β-actin (Santa Cruz, CA, USA), and goat monoclonal anti-GAPDH antibody (Genetex, USA).
Enzyme-linked immuno-sorbent assay (ELISA) for VEGF-A secreted proteins in
the conditioned medium
The cells were seeded in six-well plates at 3x105/well. The conditioned medium was collected after 48 h of transfection and analyzed by ELISA specific for human VEGF (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. The conditional medium was previously centrifuged to remove cells and other unnecessary particles. Samples and VEGF-A standards were added into
micro-plates that were pre-coated with anti-human VEGF-A capture antibody. The micro-plates were incubated at room temperature on a horizontal orbital shaker for 2 hours. After incubation, samples and standards were discarded and washed with wash buffer 4 times.
After aspiration of conjugates and a further wash, the substrate solution was
added to each well, which were incubated for 30 min. After the reaction was completed, stop solution was added to end the reaction. The optical density of each well was measured with an ELISA plate reader set to a wavelength of 450nm.
Cell Proliferation Assay
Cells were seeded in the 96-well plate at density of 103/100 μL, 5x103/100 μL and 104/100 μL respectively. Cells cultured in serum-free medium were used as
negative control. After tetracycline induction for 48 hours, MTT reagent (Sigma) was added to each well to a final concentration of 0.05% for reaction. After incubation at 37℃for 4 hours, MTT-containing medium were removed and 50 μL dimethyl sulfoxide (DMSO) were added for 20 minutes at 37℃ to dissolve formazan.
Reactions were monitored by 96-well ELISA plate reader at 595nm.
Apoptosis Detection Assay
106 cells were seeded in 10 cm plate. Cells treated with 1 μg/μL
actinomycin-D (Sigma, St.Louis, MO) were used as positive control. After
tetracycline induction for 24 hours, stNB-V1 cells were harvested and washed by cold PBS twice. Cell apoptosis rate was detected by using fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit (BD, Pharmingen, San Diego, CA). 105
cells were suspended in 100 μL of 1 x binding buffer. Harvested cells were then stained by 10 μL of FITC-conjugated Annexin V antibody and propidium iodide for
15 minutes. The stained cells were analyzed by BD FACSCanto2 cell flow cytometry.
VEGFR-1 Blockade
2x105 stNB-V1 cells were cultured in 6-well plate. Cells were treated with 1μg/mL goat polyclonal anti-human VEGF R1 antibody to block the VEGF-A signaling. Normal goat IgG was used as the negative control.
Patients and sample Preparation
A cohort of histologically proven NB patients with complete follow-up were enrolled in this study. Tumor samples were obtained during surgery and imeditely frozen in liquid nitrogen. The categorization of tumor histology was based on the International Neuroblastoma Pathology Classification scheme [94].
Immunohistochemical staining
A total of 69 tumor specimens collected before chemotherapy were fixed and embedded in paraffin. The expression of CRT, VEGF-A and CD34 was assayed using an avidin–biotin complex immunoperoxidase staining technique on archival
paraffin-embedded tissue specimens obtained before chemotherapy. Tissue sections (5mm) of tumors were deparaffinized and rehydrated in a routine manner. After microwave pretreatment, the CRT, VEGF-A and CD34 antibodies were then applied at a dilution of 1:150 overnight at 48C. The N-Histofine Simple Stain MAXPO (Nichirei, Tokyo, Japan) was then applied for 30 min at room temperature.
Diaminobenzidine was used for visualization and nuclei were counterstained with hematoxylin. One ganglioneuroma tumor with consistent CRT expression by immunohistochemistry was used as a positive control in each staining.
Non-immunized rabbit serum was used as a negative control. Tumors with various differentiating histologies were included in each staining. The immunoreactivity of CRT, VEGF-A and CD34 was assessed by a single pathologist who was blinded to the clinical background of the patients.
Mouse xenograft studies with inducible CRT stNB-V1 cells
5x106 CRT inducible stNB-V1 cells were injected subcutaneously into four-week old female athymic nude mice with matrigel (BD Bioscience). Mice were randomized into two treatment groups. Tumor-bearing mice were treated with doxycycline in their daily drinking water (2g/L) or vehicle alone (sucrose) for 21 days. The growth ability of xenografted tumors on animals was measured according to the metric measurement
of tumor size. Tumor diameters were measured with calipers, and volumes were calculated as LxW2x0.5, where L and W are the tumor length and width in mm, respectively.
Statistical analysis
The correlation between CRT and VEGF-A mRNA expression level were
analyzed using non-parametric Wilcoxon rank-sum test and Spearman’s correlation test. Other data analyses were performed using one-way analysis of variance (ANOVA), followed by Fisher’s protected least-significant difference (LSD) test (StatView; Abacus Concept, Berkeley, CA, USA). Each result was obtained from at least three independent experiments and expressed as mean ± standard deviation.
Statistical significance was set at p<0.05.
Chapter III.
Results
Cloning and gene analysis of zebrafish CRT (zCRT) showed high sequence
homology
To investigate the role of CRT in zebrafish development, we have cloned the full length transcript of zCRT..The zebrafish zCRT are highly homologous to their human and mouse counterparts with protein sequence similarity close to 90% as shown in the calreticulin sequence alignment graph (Fig. 2A). The evolutional distances of zCRT to other species’ CRT are shown in the phylogenetic trees (Fig. 2B). To further
investigate the genetic conservation of CRT in vertebrate including zebrafish, mouse and human, we compared their chromosome location maps from the Ensemble database. The results showed that all CRT genes are located near rad23a. The gene orientation of zCRT is the same as that of mouse CRT but is opposite to that of human CRT (Fig. 2C).
Expression profiles of CRT in embryonic development of zebrafish
To evaluate the temporal and spatial expression analysis of zCRT, the
quantitative RT-PCR (qRT-PCR) was performed. Our studies revealed that zebrafish CRT could be detected very early and reduced gradually during the segmentation and pharyngula stages (Fig. 3). The whole mount in situ hybridization (WISH) analysis of zCRT from ZFIN showed that zCRT was highly expressed before 5 dpf, compatible
with our expression analysis of qRT-PCR. Besides, the spatial analysis from ZIFN revealed that zCRT was expressed mainly in hatching gland, floor plate,
chordo-neural hinge and lateral line in embryos. However, in adult fish (older than three months), the zebrafish CRT were detected in all organs by qRT-PCR analyses (Fig. 4).
Knockdown of zCRT protein expression by morpholinos cause an embryonic
defect and developmental retardation
Microinjection of morpholinos targeting the ATG start site into embryos effectively abolished the expression of zCRT protein. Knockdown of zCRT expression resulted in severe embryonic lethality within 3 dpf. The surviving morphants showed developmental retardation, such as slow growth, reduced brain size and heart edema compared to the wild type fish and standard control-MO fish (Fig. 5A). The severe embryonic defect of the zCRT knockdown prevented us from studying its impact on locomotive movement. Both heart edema and phenotype change are morpholino concentration-dependent (Fig. 5B).
CRT positively regulated VEGF-A and HIF-1 expressions
To investigate the relationship between CRT and VEGF-A, CRT was
over-expressed using pEGFP-C1-CRT expression vector via Lipofectamine 2000 transfection system in SK-N-DZ and SH-SY5Y cells, which was performed by Kuan-Hung Lin. According to real-time PCR analysis, the expression vector
significantly enhanced CRT mRNA expression in SK-N-DZ and SH-SY5Y to 1800- and 1400-folds higher, respectively, compared to the negative control (none) and vector control (pEGFP-C1) (Fig. 6A). This CRT over-expression at the protein level was also confirmed by western blotting (Fig. 6B).
To elucidate the effects of CRT on VEGF-A and HIF-1, VEGF-A mRNA
expression was analyzed in transiently CRT-over-expressing NB cells. The
over-expression of CRT increased VEGF-A mRNA expression in both SK-N-DZ and SH-SY5Y cells (Fig. 6C&D). The mRNA expression level of HIF-1, a well-known
up-regulator of VEGFs in NB, also positively correlated with CRT level [95].
Elevated HIF-1 expression suggested that HIF-1 might be involved in the
CRT-dependent VEGF-A up-regulation.
To further clarify the relationship between CRT and VEGF-A, CRT was
transiently knocked-down using shRNA in SK-N-DZ and SH-SY5Y cells. According to real-time PCR analysis, the CRT mRNA expression levels were significantly inhibited by the pCR3.1-CRT-shRNA in both SK-N-DZ and SH-SY5Y cells (Fig.
7A). The knockdown of CRT was further confirmed at the protein level by western
blotting (Fig. 7B).
To examine the effect of CRT knockdown on VEGF-A and HIF-1, VEGF-A and HIF-1 mRNA expressions were analyzed in transiently CRT-knocked down NB cells. The VEGF-A and HIF-1 mRNA expression were lower in SK-N-DZ and
SH-SY5Y cells with CRT knockdown (Fig. 7C&D). The studies about the CRT knockdown were also performed by Kuan-Hung Lin. The results further confirmed that CRT could regulate VEGF-A and its up-regulator, HIF-1 expression in NB cells.
Effects of CRT on VEGF-A protein expression and secretion level in NB cells
Whether the CRT expression affected the VEGF-A protein expression in NB
cells was further determined. Results showed that VEGF-A protein expression was up-regulated by CRT over-expression both in SK-N-DZ and SH-SY5Y cells (Fig. 8A).
Moreover, knockdown of CRT decreased the protein expression of VEGF-A (Fig. 8A).
The results demonstrated that CRT positively regulated VEGF-A both at the mRNA and protein level in different NB cells.
The VEGF family members, including VEGF-A, are known to be secreted polypeptides. The VEGF-A secretion level was then examined in the conditioned media. Results showed that the CRT over-expression up-regulated VEGF-A secretion in the conditioned media of SK-N-DZ and SH-SY5Y cells (Fig. 8B). On the other
hand, CRT knockdown suppressed the VEGF-A protein secretion in conditioned media (Fig. 8B). These results further supported the effects of CRT on VEGF
expression and secretion in different NB cells. The results about the VEGF-A protein expression and secretion level were provided by Kuan-Hung Lin.
Establishment of stable cell lines
In order to further clarify the effects of CRT on NB cell lines, CRT
over-expression and knockdown stable cell lines were selected. However, constitutive over-expression of CRT led to NB cell differentiation without proliferation. Thus, kindly provided by Dr. Yung-Feng Liao and Dr. Bo-Jeng Wang, we received an inducible-CRT stNB-V1 cell line by a tetracycline-regulated gene system. To induce CRT expression, cells were treated with tetracycline. After 1 μg/μL tetracycline induction for 24, 48, and 72 h, CRT mRNA expression was elevated by 3-, 6-, and 4-folds higher, respectively, than non-induced cells (Fig. 9A). As such, 48 h induction was chosen as the best condition for subsequent experiments. By western blotting, the protein expression of CRT was also enhanced by tetracycline induction (Fig. 9B).
For knockdown stable cell lines, stNB-V1 NB cells were transfected with a CRT-shRNA plasmid via lentiviral system and then selected by respective antibiotics.
After selection by puromycin, CRT-shRNA cells were generated (19989 and 19991)