The human ovarian cancer cell line SKOV3 was purchased from the ATCC and was authenticated by the analysis of a short tandem repeat (STR). The cell was cultured in RPMI-1640 medium (Hyclone, SH30809.01) with 10% fetal bovine serum (Tianhang, 11012-8611).
Knockdown of CD44
The lentiviruses were constructed according to the target sequence of CD44 for RNAi: 5'- TTG CAG TCA ACA GTC GAA GAA -3' and the negative control sequence: 5'- TTC TCC GAA CGT GTC ACG T-3' (vector: GV493); by Shanghai Genechem Co., LTD. (Shanghai, China). The lentiviruses (MOI=20) were added to SKOV3 cells for transfection. Puromycin was used to screen infected cells (2.5 µg/ml). The transfection efficiency was observed by GFP expression, and RT-qPCR and western blot determined the knockdown of CD44.
RNA was extracted using Trizol (Beyotime, Shanghai, China) and reverse transcribed into cDNA. RT-qPCR analysis used the StepOne™ real-time qPCR system (Applied Biosystems, USA). The primer sequences: CD44 forward, 5’-TCCCAGACGAAGACAGTCCCTGGAT-3' and reverse, 5’-CACTGGGGTGGAATGTGTCTTGGTC-3'; β-actin forward, 5’-TGTGGCATCCACGAAACTAC-3' and reverse 5’-GGAGCAATGATCTTGATCTTCA-3'. The 2−ΔΔCT was used to quantified the expression.
RNA extraction, library construction, and sequencing
RNA was extracted. The mRNA was enriched by Oligo(dT) beads, fragmented into short fragments, and reverse transcripted into cDNA. Then the cDNA fragments were purified, end-repaired, poly(A) added, and ligated to Illumina sequencing adapters. The Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China) was used to sequence.
Identification of differentially expressed genes (DEGs)
RNAs differential expression analysis was performed by edgeR between two samples. The gene which a P value of below 0.05 and absolute fold change ≥1.5 was considered differentially expressed genes.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis
DEGs were analyzed by differential RNA between 2 groups. Transcripts which a P value of below 0.05 and absolute fold change ≥1.5 were defined as differentially expressed. The KEGG was used to analyze the pathway enrichment.
String v10 was used to identify the network of proteins interaction, which defined genes as nodes and interaction as lines in a network. Cytoscape (v3.7.1) was used to display gene biological interaction.
Cells were plated on a 15 mm circle microsope cover glass (NEST, China) placed in the 24-well culture plate. Cells were fixed for 15 min and blocked for 30 min. Cells were incubated with antibodies against CD36 (1:200, Proteintech, 18836-1-AP) overnight at 4°C. CY3-Goat Anti-rabbit IgG (BOSTER, Wuhan, China) was used as the secondary antibody. DAPI (BOSTER, Wuhan, China) was used to stain the nucleus. Cells were observed by a Leica TCS SP8 confocal microscope (Leica Microsystems GmbH, Mannheim, Germany).
Western blot analysis
Cells were lysed by RIPA Lysis Buffer (BOSTER, Wuhan, China). The antibodies against CD44 (1:1,000, Proteintech, 60224-1-Ig), CD36 (1: 1,000, Proteintech, 18836-1-AP), and beta-amyloid (1:50, Proteintech, 25524-1-AP) were used as primary antibodies and Actin (1:5,000, Bioss, bs-0061R) used as internal control. Image Lab™ Software 4.1 (Bio-Rad, USA) was used to analyze the expression of proteins.
Use GraphPad Prism 8 to analyze the data. Use unpaired Student's t-test to compare the two groups, and the results showed by mean ± SEM. The in vitro experiments were repeated at least three times. A value of P<0.05 was considered statistically significant. The progress Free Survival (PFS) and overall survival (OS) rates in different cohorts of serous ovarian cancer patients were assessed by Kaplan-Meier plot (http://kmplot.com/), and the databases calculated the hazard ratio (HR) and log-rank P-values. The correlation of CD44 and CD36 expression in ovarian cancer was analyzed by The Gene Expression Profiling Interactive Analysis database (http://gepia2.cancer-pku.cn/), while CD36 mRNA levels in different stages were analyzed. The correlation between different genes was calculated by Pearson's correlation coefficient.