Patients and specimens
All clinical pictures and tissue specimens were taken from the sample library of the Tenth People's Hospital affiliated to Tongji University (2012-2016). The patients were enrolled with informed consent, and the specimens were collected and approved by the hospital ethics committee. All patients were confirmed by pathology. No treatment measures such as radiotherapy and chemotherapy were received before surgery, and other systemic neoplastic diseases were excluded at the same time.
Immunohistochemistry
The Immunohistochemical staining was performed on paraffin-embedded tissues according to the manufactuer's instructions of EnVision kit (MaiXin Biotech Co.,Fuzhou,China). The primary antibody was used rabbit anti-human CD11b monoclonal antibody (1:100, Abcam, Cambridge,UK,ab133357).The immunohistochemical scoring principle was according to the staining intensity (no signal=0,weak=1, moderate=2, high=3), and the percentage of staining cells (0=0%; 1= 1%-25%; 2= 26%-50%; 3= 51%-75%, 4= 76%-100%). The final score of 0-12 was based on multiplying the scores of intensity and percentage. The staining scores of CD11b ≥ 4 was considered as high expression, <4 being regarded as low expression.
Cell culture and plasmid transfection
SKOV3 cells purchased from ATCC and cultivated in McCoys
medium(Biochrom AG, Berlin, Germany) containing 10% fetal bovine serum at 37°C in a 5% CO2 humidified atmosphere. OVCAR3 cells purchased from ATCC (Manassas, Virginia, USA) were cultivated in RPMI medium (Invitrogen) containing 10% fetal bovine serum, .HL60 cells purchased from were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)and cultivated in RPMI medium (Invitrogen) containing 10% fetal bovine serum,which was induced by1.25%DMSO.TGF-b-RNAi lentiviral vectors were purchased from Shanghai GeneChem Company (Shanghai, China). and the shRNA control sequence was 5’- GGACTATCCACCTGCAAGA -3’.
RNA extraction and real-time PCR
Total RNA was extracted using Trizol(Invitrogen, 15596-018). First strand cDNA synthesis was performed using a reverse transcription kit (TransGen Biotech, Beijing, China) and the real-time PCR analyses were conducted on an iQ5 Multicolor Real-Time PCR Detection System(Bio-Rad, Hercules, CA, USA) using the SYBR Green dye (Roche Diagnostics, Mannheim, Germany). The GAPDH gene was used as an internal control, and the data are shown as the fold change. The experiment was performed in triplicate. Primers for MMP-2,MMP-9,E-Cadherin,N-cadherin,Vimentin and GAPDH are shown in Table 1.
Table 1
Gene
|
Forward sequences
|
Reverse sequences
|
MMP-2
|
CCCACTGCGGTTTTCTCGAAT
|
CAAAGGGGTATCCATCGCCAT
|
MMP-9
|
GGGACGCAGACATCGTCATC
|
TCGTCATCGTCGAAATGGGC
|
E-Cadherin
|
CGAGAGCTACACGTTCACGG
|
GGGTGTCGAGGGAAAAATAGG
|
N-cadherin
|
TCAGGCGTCTGTAGAGGCTT
|
ATGCACATCCTTCGATAAGACTG
|
Vimentin
|
AGTCCACTGAGTACCGGAGAC
|
CATTTCACGCATCTGGCGTTC
|
GAPDH
|
GGAGCGAGATCCCTCCAAAAT
|
GGCTGTTGTCATACTTCTCATGG
|
Western blotting
Total protein was extracted in RIPA lysate wit PMSF 1mM (Solarbio, Co. Ltd, Beijing, China), and quantified with BCA method. A total of 30 μ g of protein was separated by 10% sodium dodecy1sulfate-polyacrylaminde gel electrophoresis (SDSPAGE),followed by transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA,USA). The PVDF membranes were incubated with primary antibody: anti-CD11b antibody (1:1000,Abcam, Cambridge,UK,ab133357), CXCR4 (1:1000, Abcam ,ab124824), CXCR1 (1:1000,Abcam ,ab14936), CXCL8 (1:1000, Abcam ,ab18672),CCL5 (1:1000, Abcam , ab108933
), TGF-b (1:1000, Abcam , ab235178),MMP2 (1:1000, Abcam, ab92536), MMP9 (1:1000, Abcam, ab38898), Vimentin (1:1000,Abcam, ab92547),E-cadherin(1:1000, CST,3195T), N-cadherin(1:1000, CST,13116T), , p-Erk (1:1000, CST,4370s) , Erk(1:1000, CST,4695s), p-P38(1:1000, CST,4631s) , P38(1:1000, CST,8690s),p-JNK (1:1000, CST,4668s) , JNK(1:1000, CST, 9252S) , GAPDH(1:1000, CST, 5174) at 4℃ overnight. After the membranes were incubated with horseradish peroxidase (HRP)conjugated secondary antibody Anti-mouse IgG(HRP) (1:5000, CST,7076)and Anti-rabbit IgG(HRP) (1:5000, CST,7074)and visualized bychemiluminescence ECL detection system (Bio-Rad).
Transwell assay
Transwell assay was used to evaluate cell migration and invasion. For migration assay, 4*104 ovarian cancer cells as control groups(SKOV3 and OVCAR3) were seeded in the upper in serum-free culture medium (200μl ), Ovarian cancer cells4*104 and N2 neutrophils2*104co-culture as experimental group.The lower chamber filled with complete medium. The cells were fixed with 4% paraformaldehyde and stained with gemsa 15min,after 24h incubation. The images were acquired under microscope and migrated cells were counted in 5 random fields. The method of invasion assay was similar to migration, but the upper chamber coated with matrigel (BD Bioscience, Bedford, MA, USA).
High-throughput sequencing
Total mRNA of OVCAR3 and OVCAR3-N2 were extracted from cell by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for experiment. The
complementary DNA (cDNA) libraries were prepared using the NEBNext TM Ultra Directional RNA Library Prep Kit, Next PolyA
mRNA Magnetic Isolation Module.Next Multiplex Oligos according to the manufacturer’s instructions. The products were purified and enriched by PCR to create the final cDNA libraries and quantified by Agilent2200. The tagged cDNA libraries were pooled in equal ratio and used for 150 bp paired-end sequencing in a single lane of the Illumina HiSeqXTen.HTseq was used to count gene and RPKM method was used to determine the gene expression. We applied DESeq2 algorithm to filter the differentially expressed genes.
Statistical analysis
Statistical analysis was performed using GraphPad Prism Version 5.0 and SPSS 19.0 were performed to analyze data which were presented as value ± standard deviation.Comparisions between 2 groups were performed by standard Student’s t test. p<0.05 was considered to indicate a significant difference (*p< 0.05, **p≤ 0.01, ***p≤ 0.001).