Screening DE-miRNAs between ovarian cancer primary tumours and ascites-derived tumour cells
The microRNA array GSE65819 contains 114 samples collected from 92 high-grade serous ovarian cancer (HGSOC) patients at different time points. The sample types included primary tumour tissue, ascites-derived tumour cells and metastasis tumour tissue. We selected the microRNA array results of the primary tumour tissue and ascites-derived tumour cells harvested from 3 HGSOC patients at the first time they were diagnosed with HGSOC (Fig.1A). Using GEO2R to analyse the DE-miRNAs from the 3 pairs of primary tumour tissue and ascites-derived tumour cells, we identified 69 DE-miRNAs, among which 68 were downregulated and only miR-1245a was upregulated in ascites-derived tumour cells. MiR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p, miR-126-3p and miR-508-5p were the top 6 most downregulated miRNA in ascites-derived tumour cells compared with the primary tumour tissues(Fig.1B,C).
Prediction of target genes of DE-miRNAs and pathway enrichment analysis
We used miRTarBase to predict the target downstream genes of miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p, miR-126-3p and miR-508-5p, resulting in the identification of 1273 probable target genes. After KEGG pathway analysis, we observed that the target genes of DE-miRNAs were primarily enriched in “pancreatic cancer”, “chronic myeloid leukaemia” and “focal adhesion”. Regarding the GO analysis, “positive regulation of cell migration”, “positive regulation of cellular component movement” and “vasculature development” were the most significant terms in biological process (BP); “transferase complex, transferring phosphorus containing groups”, “nuclear transcription factor complex” and “transcription factor complex” were the most significant terms in cellular component (CC); and “protein kinase activity”, phosphotransferase activity, alcohol group as acceptor” and “kinase activity” were the most significant terms in molecular function (MF)(Fig.2B-E).
Identification of hub target genes of DE-miRNAs
To identify the hub target genes of DE-miRNAs, a PPI network was evaluated by using the STRING database and visualized with Cytoscape. Considering the degree of the target genes, AKT1, MYC, EGFR, VEGFA, CDH1, MAPK1, KRAS, ESR1, MAPK8, UBC, IGF1, ERBB2, MTOR. SIRT1, ACTB, CD44, FGF2, SMAD4, MAPK14 and SMAD2 were top 20 genes with the highest degree values and were identified as hub genes of DE-miRNAs. From the PPI network shown in Fig.2A, it was obvious that miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p regulate the expression of multiple hub genes and can also collectively regulate the expression of a single gene. However, miR-508-5p can only regulate the expression of UBC and is not associated with the other identified DE-miRNAs. Therefore, we chose miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p for further study.
Identification of potential upstream transcription factors of DE-miRNAs
miRNAs regulate the expression of their target genes, but the expression of miRNAs themselves can also be regulated by transcription factors (TFs). miR-199a-1, miR-199a-2, miR-199b, miR-145 and miR-126 are the pre-miRNAs of miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p. We used ALGGEN PROMO and TransmiR v2.0 to predict the probable transcription factors that regulate the 5 pre-miRNAs mentioned above. Twenty-six and 6 TFs were identified as potential regulators of all 5 pre-miRNAs using ALGGEN PROMO and TransmiR v2.0, respectively (Fig.3A, B). After taking the intersection, we observed that TFAP2A was the common TF that may regulate all 5 pre-miRNAs mentioned above and further affect the expression of miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p (Fig.3C).
Verification of DE-miRNA expression differences between primary tumours and ascites-derived tumour cells using ovarian cancer patient samples and ovarian cell lines
We collected primary tumours and ascites-derived tumour cells from 12 ovarian cancer patients from 2017 to 2019. After evaluating the expression levels of miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p, we observed that the expression of miR-199a-3p, miR-199b-3p, miR-199a-5p and miR-145-5p was lower in ascites-derived tumour cells than in the primary tumour (p<0.01). The ascites-derived tumour cells from 11 of 12 patients had a lower miR-199b-3p expression level than that of the primary tumour in (p<0.01)(Fig.4A).
To investigate whether this phenomenon could also be observed using ovarian cancer cell line models, we use 2D and 3D cultures to mimic the growth states of primary tumours ascites-derived tumour cells, respectively. The results showed that compared with the 2D culture, miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miR-126-3p were decreased in the 3D-cultured HEY cell line. The expression of miR-199a-3p, miR-199b-3p, miR-145-5p and miR-126-3p but not miR-199a-5p was decreased in the 3D-cultured A2780 cell line. For 3D-cultured OVCA433, SKOV3 and OVCAR3 cells, most of 5 assayed DE-miRNAs were not downregulated compared with the 2D cultures, and some of the DE-miRNAs were even upregulated under 3D culture conditions (Fig.4B).
Effects of miR-199a-3p, miR-199b-3p, miR-199a-5p, miR-145-5p and miRNA-126-3p on invasion and angiogenesis
The pathway analysis results revealed that the dominant functions of the identified DE-miRNAs were migration and vasculature development. Therefore, we used Transwell invasion and angiogenesis assays to investigate the effects of the 3D-cultured HEY cell line. After overexpressing miR-199a-3p, miR-199b-3p and miR-199a-5p, the invasion ability of 3D-cultured HEY cells were inhibited. The angiogenesis of HUVECs was abrogated when miR-145-5p was overexpressed in 3D-cultured HEY cells. In summary, miR-199a-3p, miR-199b-3p and miR-199a-5p can inhibit spheroid ovarian cancer cell invasion, while miR-145-5p can inhibit spheroid ovarian cancer cell angiogenesis.(Fig.5A, B).