Reduced Levels of miR-145-3p Drive Cell Cycle Progression in Advanced High-Grade Serous Ovarian Cancer

Abstract

High-grade serous ovarian cancer (HGSOC) is the most lethal form of gynecologic cancer, with limited treatment options and a poor prognosis. Epigenetic factors, such as microRNAs (miRNAs) and DNA methylation, play pivotal roles in cancer progression, yet their specific contributions to HGSOC remain insufficiently understood. In this study, we performed comprehensive high-throughput analyses to identify dysregulated miRNAs in HGSOC and investigate their epigenetic regulation. Analysis of tissue samples from advanced-stage HGSOC patients revealed 20 differentially expressed miRNAs, 11 of which were corroborated via RT-qPCR in patient samples and cancer cell lines. Among these, miR-145-3p was consistently downregulated post-neoadjuvant therapy and was able to distinguish tumoural from control tissues. Further investigation confirmed that DNA methylation controls MIR145 expression. Functional assays showed that overexpression of miR-145-3p significantly reduced cell migration and induced G0/G1 cell cycle arrest by modulating the cyclin D1-CDK4/6 pathway. These findings suggest that miR-145-3p downregulation enhances cell proliferation and motility in HGSOC, implicating its restoration as a potential therapeutic target focused on G1/S phase regulation in the treatment of HGSOC.

Keywords: DNA methylation; cell cycle proliferation; cyclin D1-CDK4/6 pathway; high-grade serous ovarian cancer; miR-145-3p; miRNAs.

Figure 1

Integrated analysis of miRNA expression profiles in HGSOC: insights from PCA, correlation, and treatment stratification. (A) Principal component analysis of miRNA sequencing results assessing the expression of miRNAs between HGSOC and PCOT groups. (B) miRNA expression results confirm miRNA sequencing results. RT-qPCR validation of miRNA sequencing results of down- and up-regulated miRNAs in HGSOC tissues (n = 20) compared to PCOT (n = 20). miRNA expression levels were normalized to the expression levels of their corresponding control. * p < 0.05; ** p < 0.01; *** p < 0.001; and Mann–Whitney U test. (C) Correlation between expression levels of miR-145-5p and miR-145-3p, miR-143-5p and miR-145-3p, and miR-143-5p and miR-145-5p. Spearman’s rank correlation. (D) Differential expression analysis between patients who received either neoadjuvant treatment (w/NT) or no treatment before surgery (w/o NT) for up-regulated miRNAs and down-regulated miRNAs. NT, neoadjuvant treatment. * p < 0.05; ** p < 0.01; and Mann–Whitney U test.

Figure 2

MIR145 is modulated by DNA methylation. (A) Methylation status of the proximal (200 bp upstream of the transcription starting site) and distal (1500 bp upstream of the transcription starting site) promoter of MIR145. * p < 0.05; ** p < 0.01; and Mann–Whitney U test. (B) Influence of 5-Aza treatment on the relative expression levels of the miR-145-5p and the miR-145-3p in three OC cell lines. ns: not significant; * p < 0.05; *** p < 0.001; and Student t-test.

Figure 3

(A–C) Differential expression analysis of miR-145-3p in (A) Caov-3, (B) SK-OV-3, and (C) SW-626 cells transfected with the miR-145-3p mimic or with a scramble control. * p < 0.05; ** p < 0.01; and Student t-test. (D–G) Caov-3 (D) and SK-OV-3 (E) cells were transfected with the miR-145-3p mimic or with a scramble control; 48 h after transfection, cells were scratch-wounded and were incubated in their appropriate complete medium for 72 h, and pictures were captured every 2 h post-scratching. Yellow lines indicate the wound borders. For Caov-3 (F) and SK-OV-3 (G): wound confluence (%) represents the fractional area of the wound that is occupied by cells; wound width represents the area of the wound that is not occupied by cells; and the area represents the cell-covered area of the well. * p < 0.05; ** p < 0.01; *** p < 0.001; and Mann–Whitney U test.

Figure 4

Effects of miR-145-3p on cell cycle and cyclin D1-CDK4/6 pathway regulation in OC cell lines. (A,B) Cell cycle analysis of: (A) Caov-3 and (B) SK-OV-3 cells without miR-145-3p expression and cells overexpressing miR-145-3p. * p < 0.05; and Student t-test. (C,D) Differential expression analysis of CCND1, CDK4, and CDK6 genes between: (C) Caov-3 and (D) SK-OV-3 cells without miR-145-3p expression and cells overexpressing miR-145-3p. ** p < 0.01; and Student t-test. (E,F) Western blot analysis of CCND1, CDK4, and CDK6 genes between (E) Caov-3 and (F) SK-OV-3 cells without miR-145-3p expression and cells overexpressing miR-145-3p.