FBLN5 is targeted by microRNA‑27a‑3p and suppresses tumorigenesis and progression in high‑grade serous ovarian carcinoma

Abstract

High-grade serous ovarian carcinoma (HGSOC) is one of the most lethal gynecological malignancies; however, the precise molecular mechanisms have not been fully characterized. Fibulin‑5 (FBLN‑5) is an extracellular matrix (ECM) glycoprotein, and plays a crucial role in maintaining the stability of ECM structures, regulating cell proliferation and tumorigenesis. In the present study, the expression of FBLN‑5, as determined by western blot analysis and immunohistochemistry, was significantly increased in normal fallopian tube (FT) samples compared with that in HGSOC samples, and decreased FBLN5 expression was associated with unfavorable prognosis of HGSOC. Functional characterization revealed that FBLN5 overexpression significantly inhibited migration, invasion and proliferation abilities of ovarian cancer cells in vitro. Furthermore, micro (mi)RNA‑27a‑3p (miR‑27a‑3p) was revealed to be increased in HGSOC, and dual‑luciferase reporter assay indicated that miR‑27a‑3p was functioned as a negative regulator of FBLN5 by directly binding with its 3'‑untranslated region. Collectively, FBLN5 expression was associated with prognosis, proliferation, and metastasis in HGSOC. We hypothesized that FBLN5 was targeted by miR‑27a‑3p and may serve as a biomarker and provide a new therapeutic approach for the treatment of HGSOC.

Keywords: microRNA-27a-3p; fibulin-5; high-grade serous ovarian carcinoma; prognosis; proliferation; metastasis.

Figure 1.

FBLN5 is downregulated in HGSOC tissues. (A) Reverse transcription-quantitative PCR analysis of the FBLN5 mRNA expression level in HGSOC tissues (n=14) and normal FT tissues (n=16). (B) FBLN5 mRNA expression level in ovarian cancer and normal tissues from the GEPIA database. (C) FBLN5 protein expression level of FBLN5 in HGSOC (n=9) and normal FT (n=7) tissues using western blot analysis. *P<0.05. FBLN5, fibulin-5; HGSOC, high-grade serous ovarian cancer; T, tumor; FT, fallopian tube.

Figure 2.

Association of FBLN5 expression with clinical information in patients with HGSOC. (A) Representative immunohistochemical staining of FBLN5 in HGSOC. (B) The high and low expression level of FBLN5 in 57 FT and 159 HGSOC tissues. (C) Overall survival rates in patients with HGSOC, with either high or low expression levels of FBLN5. (D) The multivariate analysis for OS. *P<0.05, **P<0.01. FBLN5, fibulin-5; HGSOC, high-grade serous ovarian cancer; FT, fallopian tube.

Figure 3.

FBLN5 inhibits ovarian cancer cell proliferation in vitro and in vivo. The (A) growth curve and (B) colony formation assay were used to assess the effect of FBLN5 on the proliferation of ovarian cancer cells. (C) The HEY cell line with or without FBLN5 overexpression was injected subcutaneously into nude mice (n=5). (D) The tumor weights in each group were assessed. Data are presented as the mean ± standard error of the mean. n=5. (E) p21 and p27 expression was measured using western blot analysis in ovarian cancer cell lines with FBLN5 overexpression or knockdown. Data are presented as the mean ± standard error of the mean. n=3. *P<0.05, **P<0.01, ***P<0.001. FBLN5, fibulin-5; si, small interfering; NC, negative control.

Figure 4.

FBLN5 inhibits the migration and invasion of ovarian cancer cells in vitro. Transwell assays were used to assess the effect of FBLN5 overexpression or knockdown on the (A) migration and (B) invasion of ovarian cancer cells. (C) Western blot analysis of the EMT markers, N-cadherin, E-cadherin and Snail. Data are presented as the mean ± standard error of the mean. n=3. *P<0.05, **P<0.01, ***P<0.001. FBLN5, fibulin-5; EMT, epithelial-mesenchymal transition; si, small interfering; NC, negative control; N-CAD, N-cadherin; E-CAD, E-cadherin.

Figure 5.

FBLN5 is the target gene of miR-27a-3p. (A) The mRNA level of miR-27a-3p was higher in HGSOC tissues compared with that in FT tissues, using RT-qPCR. (B) Correlation analysis of FBLN5 and miR-27a-3p expression levels. (C) Prediction of the binding sites between miR-27a-3p and 3′-UTR of FBLN5. The binding sequence of miR-27a-3p with the FBLN5 3′UTR sequence was deleted in the mutated type. Dual-luciferase reporter assays were used to analyzed the luciferase activity of the WT or MT FBLN5 3′UTR. (D) The mRNA expression level of miR-27a-3p was detected using RT-qPCR following SKOV3 cell transfection with miR-27a-3p mimics or inhibitor. RT-qPCR and western blot analysis revealed an inverse association between FBLN5 and miR-27a-3p expression. Upregulation of miR-27a-3p inhibited FBLN5 expression level and downregulated miR-27a-3p expression level could promote the expression level of FBLN5. Data are presented as the mean ± standard error of the mean. **P<0.01. FBLN5, fibulin-5; miR-27-3p, microRNA-27-3-p; HGSOC, high-grade serous ovarian cancer; FT, fallopian tube; RT-qPCR, reverse transcription-quantitative PCR; UTR, untranslated region; WT, wild-type; MT, mutant; NC, negative control.