tRNA-derived fragment tRF-03357 promotes cell proliferation, migration and invasion in high-grade serous ovarian cancer

Abstract

Background: High-grade serous ovarian cancer (HGSOC) is one of the most common ovarian epithelial malignancies. tRNA-derived fragments (tRFs) have been identified as novel potential biomarkers and targets for cancer therapy. Nevertheless, the influence of tRFs on HGSOC remains unknown. This study aimed to identify HGSOC-associated tRFs and to investigate the function and mechanism of key tRFs in SK-OV-3 ovarian cancer cells.

Methods: The tRF profiles in HGSOC patients and controls were investigated using small RNA sequencing. Differentially expressed tRFs were verified by real-time PCR, and a key tRF was evaluated in a function study.

Results: A total of 27 tRFs were differentially expressed between HGSOC patients and controls. Differentially expressed tRFs were mainly involved in the functions of protein phosphorylation, transcription and cell migration and the pathway of cancer, and the MAPK and Wnt signaling pathways. Real-time PCR verified that tRF-03357 and tRF-03358 were significantly increased in the HGSOC serum samples and SK-OV-3 cells compared to their expression levels in the controls. Importantly, tRF-03357 promoted SK-OV-3 cell proliferation, migration and invasion. Moreover, tRF-03357 was predictively targeted, and significantly downregulated HMBOX1.

Conclusion: This study suggests that tRF-03357 might promote cell proliferation, migration and invasion, partly by modulating HMBOX1 in HGSOC.

Keywords: cell growth; high-grade serous ovarian cancer; invasion; migration; tRNA-derived fragments.

Figure 1

Differentially expressed tRFs between high-grade serous ovarian cancer (HGSOC) patients and three healthy subjects. (A) Volcano plot showing the differentially expressed tRFs between the ovarian cancer patients and healthy controls. The abscissa represents the fold change value, and the ordinate represents the FDR. Red dots indicate upregulated tRFs, and blue dots indicate down regulated tRFs. Gray dots represents not significant. Significantly different expression was identified based on a∣log2-fold change∣>1 and FDR<0.05. (B) Cluster analysis showing the differentially expressed tRFs between the ovarian cancer patients and healthy controls. The T4, T6, and T9 groups belong to the tumor serum samples, and the N1, N3, and N5 groups belong to the normal serum samples. The enrichment factor increases from green to red.

Figure 2

Function and pathway analysis of target genes of differently expressed tRFs between high-grade serous ovarian cancer (HGSOC) and three healthy subjects. (A) The top 20 enriched gene ontology (GO) terms of the differentially expressed tRF target genes. (B) The top 20 enriched KEGG pathways of the differentially expressed tRF target genes. Rich factor included the gene numbers and P-values.

Figure 3

Verification of differentially expressed tRFs. (A) Candidate tRF expression (tRF-07650, tRF-03357, and tRF-03358) was measured by real-time PCR in serum samples from 20 high-grade serous ovarian cancer (HGSOC) patients and 15 healthy controls; t-test. (B) tRF-03357 and tRF-03358 expression in ovarian cancer cells (NO8901 and SK-OV) and normal human ovarian epithelial cells (HOSEPIC) was measured by real-time PCR; one-way ANOVA followed by Turkey’s post hoc test. (C) tRF-03357 expression in HOSEPIC cells transfected with the tRF-03357 mimics or SK-OV-3 cells transfected with the tRF-03357 inhibitor was measured by real-time PCR; t-test. *P<0.05, **P<0.01.

Figure 4

The effects of tRF-03357 on ovarian cancer cells. The effects of the tRF-03357 mimics (A) and inhibitor (B) on proliferation were detected using the CCK-8 assay. (C) The effects of the tRF-03357 mimics on HOSEPIC cell migration and invasion were evaluated using the Transwell assay. (D) The effects of the tRF-03357 inhibitor on SK-OV-3 cell migration and invasion were evaluated using the Transwell assay. Note: *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Target gene analysis for tRF-03357. (A) The expression of five predicted target genes of tRF-03357 were measured by real-time PCR in SK-OV-3 cells transfected with the tRF-03357 inhibitor and inhibitor NC. (B) HMBOX1 expression was measured by real-time PCR in SK-OV-3 cells transfected with the tRF-03357 mimics and NC. (C) The HMBOX1 protein level was measured by Western blotting in SK-OV-3 cells transfected with the tRF-03357 inhibitor or mimics.t-test,*P<0.05.