2.1 Differentially expressed genes (DEGs) screening in EOC with platinum resistance
The gene expression profile was obtained from GSE114206 dataset of GEO database. GSE114206 dataset contained 6 low PARP and platinum-resistant EOC samples and 6 high PARP and platinum-sensitive EOC samples. We use the “limma” R package to screen the DEGs between the platinum- resistant and sensitive EOC samples. Adjusted P-value < 0.05 and |log2fold change (FC)| > 1, were chosen as the cut-off threshold. Candidate DE chemokines were visualized by “circos” package (https://www.omicstudio.cn/tool).
2.2 Patients and clinical sample collection
All patients signed informed consent before using clinical specimens, and the use of specimens for this study has been proved by the ethics committee of the First Affiliated Hospital with Nanjing Medical University. All the ovarian cancer tissues and serum samples were obtained from patients diagnosed and underwent surgery in the First Affiliated Hospital with Nanjing Medical University between January 2015 to January 2020. Surgical specimens were immediately at −80°C for nucleic acid and protein extraction. Patient who did not achieve the clinical complete response in the initial therapy or whom with recurrent tumor within 6 months was identified as an primary resistance case[22]. Due to the secondary operation is not recommended in patients with recurrent resistant tumor, collected samples in our study were limited (15 platinum-sensitive and 9 platinum-resistant EOC samples). Meanwhile, serum samples of 6 platinum-resistant and 9 platinum-sensitive EOC patients were also collected.
2.2. Cell culture and chemicals
HO8910 and A2780 are platinum-sensitive EOC cells. By exposure to increasing concentration of cisplatin, the cisplatin-resistant cells A2780-DDP, HO8910-DDP were constructed in our study for further research. FBS (CLRAK Bioscience) and 1% penicillin/streptomycin (Sigma-Aldrich) at 37℃ supplied with 5% CO2.
2.3 Plasmid and siRNA transfection
The plasmid encoding the transcript of CXCL2 (oeCXCL2) and the negative control (oeNC) was synthesized by Tsingke (Nanjing, China). The siRNA targeted CXCL2 (siCXCL2) and the negative control (siNC) were purchased from GemmaPharma (Shanghai, China). Cells were transfected by Lipofectamine 3000 Transfection Reagent (L3000150, Invitrogen) for 24-48 hours. The CXCL2 siRNA sequence was described in the previous study [23].
2.4 Cell viability assay
About 6000-8000 preprocessed EOC cells were plated in 96-well plates, and incubated in a series of cisplatin concentration (0, 5, 10, 15 and 20μM) (Sigma-Aldrich) for 48 hours. Cell viability was qualified by Cell Counting Kit-8 (CCK8) (Vazyme) under the manufacturer's instructions. A microplate reader (TECAN, Infinite M200 PRO) was applied to measure the absorbance at 450 nm, and the cell’ IC50 were analyzed in Graphpad 8.0.
2.5 RNA extraction and quantitative Real-time PCR (qRT-PCR)
According to the manufacturer's instructions, the total RNA of cultured cells and EOC tissues was extracted with Trizol (Invitrogen). cDNA HiScript Q RT SuperMix for qRT-PCR (Vazyme) were used to prepare cDNA. qRT-PCR was performed under the instructions of SYBR Green PCR Kit (Vazyme). The sequences of gene primers used for qRT-PCR were synthesized by Tsingke (Nanjing, China) (Supplementary Table 1). The mRNA expression level was normalized to GAPDH to calculate the relative gene expression.
2.5 Western blot assay
Firstly, cultured cells were lysed in RIPA buffer (Beyotime) with protease inhibitor (Beyotime). Proteins were then extracted and quantified. Western blot assay was performed by the protocol that we previously reported [24]. The reagents and antibodies were shown in Supplementary Table 2
2.6 Enzyme-linked immunosorbent assay (ELISA)
Serum samples of platinum-resistant and platinum-sensitive patients and cell culture supernatants were respectively extracted for cytokine analysis. Human CXCL2 ELISA kits were purchased from Hengyuan Biological Co. Ltd (Shanghai, China), and ELISA assay was performed as the manufacturers’ instructions. Each sample was duplicated.
2.7 Cell apoptosis assay
EOC cells were plated in 6-well plates and treated with cisplatin (5μM/10μM) for 48 hours. Then, 2× 104 cells and the cultural supernatant were collected for apoptosis assay. FITC Annexin V (5 μl) and propidium iodide (5 μl) (BD Biopharmingen) were used to stain the collected cells by suspending in 300 μl of binding buffer for 15 min in the dark. Cell apoptosis was eventually detected by a flow cytometer (FACScan; BD Biosciences) with Cell Quest software (BD Biosciences).
2.8 Statistical analysis
Each sample was analyzed based on repeated results at least three times and analyzed in Graphpad 8.0. A standard Student's t-test determined the statistical significance of differences between the two groups. Differences at p< 0.05 were regarded as statistically significant (*), ones at p < 0.01(**), p < 0.001(***) or p<0.0001(****) was considered higher statistical significances.