With the effective utilization of chemotherapy, choriocarcinoma (CC) has become a curable human malignancy. However, 30–40% of high-risk or ultra-high-risk CC patients will develop to chemoresistance after initial remission and require treatment with salvage chemotherapy(2, 23). Therefore, an increased understanding of the molecular and functional mechanisms of CC development and identification of novel biomarkers are needed. MiRNAs are involved in various physiological and pathological activities, play indidpensable roles in the regulation of various transcription factors, cytokines or signalling pathways, and are important in tumour progression (24–27). Evidence has shown that aberrant expression of miRNAs such as miR-21, miR-371-5p, miR-196b, and miR-199b, is involved in development and tumorigenesis of GTN (20, 22, 28, 29). However, the expression of miRNAs in chemotherapy-sensitive or drug-resistant tissues has not been studied.
In the present study, miRNA expression in normal chorionic villi tissues and chemotherapy-sensitive CC tissues was analyzed by GeneChip hybridization of FFPE tissues and differential expression analysis. In total, 148 differentially expressed miRNAs were identified by GeneChip hybridization and comparison of expression levels between samples of normal villi taken during early pregnancy and sample of chemosensitive CC tissues; these 148 differential miRNAs included 42 overexpressed and 106 underexpressed miRNAs. miRNAs regulate approximately one-third of human genes, and this significant regulatory effect on gene expression is an independent feature that distinguishes miRNAs from other RNA types and potential therapeutic targets (30). Therefore, miRNAs may be important regulators in chemosensitivity in CC choriocarcinoma and may effectively identify chemosensitive CC patients.
Fifty and twelve potential target genes were predicted for the top 3 most overexpressed and underexpressed miRNAs, respectively. All of these target genes showed enrichment of the terms regulation of transcription from RNA polymerase II promoter, DNA-templated transcription, negative regulation of transcription from RNA polymerase II promoter, response to drug, negative regulation of cell proliferation, cellular response to hypoxia, negative regulation of apoptotic process, microRNAs in cancer (hsa05206), FoxO signalling pathway (hsa04068), pathways in cancer (hsa05200), proteoglycans in cancer (hsa05205) and HTLV-I infection (hsa05166). These findings suggest that these six miRNAs may represent potential therapeutic targets for chemosensitive CC.
Moreover, we constructed PPI networks and identified the top 18 hub genes with the highest degree of connectivity with the top 3 most overexpressed and underexpressed miRNAs. The hub genes of the overexpressed miRNAs were mainly enriched in the terms miRNAs in cancer, while the hub genes of the underexpressed miRNAs were mainly enriched in the terms miRNAs in cancer, PI3K-Akt signalling pathway and pathways in cancer. All these terms have been reported to be associated with tumour growth, progression, invasion and metastasis of various tumours (though not CC) by previous studies (25, 26, 31–35). Therefore, miRNAs play different roles in chemotherapy-sensitive CC, but more studies are needed to explore the functions of miRNAs in CC patients, especially in chemotherapy-resistant patients.
In the present study, we identified hub genes that were potentially modulated by miR‑144-3p and miR‑519c-3p by constructing an miRNA-hub gene network, suggesting that these two miRNAs may be the most important regulators of CC. Recent studies have demonstrated that miR‑144-3p acts as a tumour suppressor in glioblastoma(36), osteosarcoma(37, 38), cervical cancer(39) and oral squamous carcinoma(40). Unsurprisingly, some studies report that miR‑144-3p acts as a promoter of renal carcinoma(41) and papillary thyroid carcinoma(42). However, the roles of miR‑144-3p in chemosensitive CC, especially for the chemotherapy-resistant CC, have not been studied. Furthermore, a few studies have reported that miR‑519c-3p has a suppressive effect on tumour cell proliferation, migration, and invasion in breast cancer (43). According to our results, we speculate that upregulation of miR‑519c-3p suppresses aggressiveness and progression, while downregulation of miR‑144-3p may have the same role in the chemosensitive CC. Therefore, it will be extremely meaningful to authenticate the functions of miR-144-3p and miR‑519c-3p and elucidate the mechanisms in chemosensitive CC in future studies.
Among the 18 hub genes, PTEN was simultaneously found to have the highest interaction degree with the top 3 most overexpressed and underexpressed miRNAs, suggesting that PTEN may participate in transcription from the RNA polymerase II promoter and negative regulation of apoptotic processes to lead to cell proliferation and apoptosis. Moreover, a previous study stated that miR-144-3p can target PTEN to promote lymphovascular invasion, proliferation, and cardiac fibrosis in rectal neuroendocrine tumours, thyroid tumours, and myocardial infarction (44–46). As a cancer suppressor, PTEN, which is a dual protein/lipid phosphatase that can inhibit the PIP3/PI3K/AKT signalling pathway, plays important roles in carcinogenesis(47), and mutations in this gene may be utilized as targets of treatment of chemosensitive CC. These findings reveal a potential target for the diagnosis and/or treatment of aggressive CC in the future.
There were some limitations and weaknesses in this study. Too many differential miRNAs were identified, the relative target gene analysis was performed stimultaneously, and the workload was too large. Therefore, we only selected the top 3 most highly overexpressed and underexpressed miRNAs as potential biomarkers for chemosensitive CC. Confirmation of their diagnostic value and targetability via PCR experiments and prospective clinical research in studies with many clinical samples is needed. In addition, we preliminarily found that miRNAs may function by regulating their target genes in chemosensitive tissues, but cell experiments to verify the mechanisms are needed. Furthemore, the regulatory relationships between miR-519c-3p and the common target gene PTEN has not been demonstrated in other tumours andas such should be validated by dual luciferase reporter assay, overexpression and knockout experiments.
In summary, we successfully identified two crucial miRNAs, miR‑144-3p and miR‑519c-3p based on GeneChip hybridization. Moreover, we also found that PTEN may play an important role in the development of chemosensitive CC by bioinformatics analysis. These findings may provide potential candidate biomarkers for the early diagnosis and the treatment of chemosensitive CC.