miR-34a-FOXP1 Loop in Ovarian Cancer

Abstract

Ovarian cancer (OC) is the main cause of gynecological cancer mortality in most developed countries. microRNA (miR) expression dysregulation has been highlighted in human cancers, and miR-34a is found to be downregulated and associated with inhibition of tumor growth and invasion in several malignancies, including OC. The winged helix transcription factor forkhead box P1 (FOXP1) is reported as either an oncogene or tumor suppressor in various cancers. This study aimed to elucidate potential clinical and biological associations of miR-34a and transcription factor FOXP1 in OC. We investigated nine OC patients' blood samples and two OC cell lines (SKOV-3 and OVCAR-3) using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to determine both miR-34a and FOXP1 expressions. We have found that miR-34a and FOXP1 are reversely correlated in both in vitro and in vivo. Inhibition of miR-34a transiently led to upregulation of FOXP1 mRNA expression and increased cellular invasion in vitro. Our data indicate that miR-34a could be a potential biomarker for improving the diagnostic efficiency of OC, and miR-34a overexpression may reduce OC pathogenesis by targeting FOXP1.

Figure 1

miR-34a and FOXP1 expression levels were analyzed by qRT–PCR in OC patient samples. Results were grouped in each subtypes. The black round dots represent miR-34a expressions; red squares represent FOXP1 mRNA levels. Data normalized according to RNU6 expression levels for miR-34a and RNA polymerase II (RPII) expression level for FOXP1 (n = 3; p < 0.005 for all). The heatmap was created by using GraphPad Prism (v. 9.3.1).

Figure 2

Expression levels of miR-34a and FOXP1 in SKOV-3 and OVCAR-3 cells. (A) RT-qPCR results show 143-fold lower miR-34a expression in SKOV-3 cells compared to that in OVCAR-3 cells. (B) FOXP1 mRNA level was found to be 25-fold more in SKOV-3 cells than that in OVCAR-3 cells. (C) Transiently inhibiting miR-34a in OVCAR-3 cells compared to nontransfected cells reduced miR34a expression 25-fold. (D) FOXP1 expression increased 18-fold in response to miR-34a repression in OVCAR-3 cells. The data are the mean ± SD of three technical repeats evaluated by one-way ANOVA and Bonferroni’s multiple comparison test. Exact p-values are indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; and ****p ≤ 0.0001; error bars indicate SD.

Figure 3

miR-34a inhibition increased cellular invasion and proliferation in OC cells. Figure (A) represents cellular proliferation of OC cells, while (B) cellular invasion of OC cells. The data are the mean ± SD of three technical repeats evaluated by one-way ANOVA and Bonferroni’s multiple comparison test. Exact p-values are indicated as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; and ****p ≤ 0.0001; error bars indicate SD.

Figure 4

Disease pathway map for OC showing different oncogenic and tumor suppressor miRs and their associated target genes and pathways during OC development. Image taken from KEGG PATHWAY database (Kyoto, Tokyo). Accession number: map05206.