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. 2025 Jul 29:15:1604377.
doi: 10.3389/fonc.2025.1604377. eCollection 2025.

RNLS promotes ovarian cancer growth and inhibits ferroptosis via mediating STAT3

Liang Lin #  1 , Zuolian Xie #  1 , Peilin Zhong  1 , Jian Chen  1 , Ning Ma  1 , Ling Li  1 , Li Chen  1 , An Lin  1
Affiliations

RNLS promotes ovarian cancer growth and inhibits ferroptosis via mediating STAT3

Liang Lin et al. Front Oncol. .

Abstract

Introduction: Ovarian cancer (OC) is a lethal malignancy for which there are limited therapeutic options. The role of renalase (RNLS) in cancer progression and ferroptosis regulation remains unclear. This study investigates how RNLS mediates STAT3 to promote OC growth and suppress ferroptosis.

Methods: RNLS expression was analyzed in OC cell lines (OVCAR3) and normal ovarian epithelial cells (IOSE80) via qPCR. Stable RNLS knockdown (sh-RNLS) and overexpression (ov-RNLS) OVCAR3 models were established via lentiviral infection. STAT3 siRNA was transfected to explore RNLS-STAT3 interactions. Functional assays (CCK8, wound healing, Transwell, flow cytometry) evaluated proliferation, migration, invasion, apoptosis, and ROS levels. Mitochondrial morphology was assessed by electron microscopy. Subcutaneous tumor models in mice validated in vivo effects. Molecular markers (STAT3, p-PI3K/PI3K, p-AKT/AKT, Ki-67, MDA, GPX4, GSH) were analyzed via Western blot, immunohistochemistry, and ELISA.

Results: RNLS was significantly upregulated in OC cells, particularly OVCAR3. RNLS knockdown suppressed STAT3 expression, cell proliferation, migration, invasion, and tumor growth, while promoting apoptosis, ROS accumulation, and mitochondrial damage. Conversely, RNLS overexpression exerted opposing effects. STAT3 silencing inhibited PI3K/AKT signaling and ferroptosis resistance, which were rescued by RNLS overexpression. In vivo, sh-RNLS reduced tumor volume/weight, as well as RNLS/STAT3, Ki-67, GPX4, and GSH, while increasing MDA. ov-RNLS enhanced tumor growth and reversed these molecular changes.

Conclusion: RNLS drives OC progression by activating STAT3-dependent PI3K/AKT signaling, enhancing proliferation, metastasis, and ferroptosis suppression. Targeting RNLS-STAT3 axis may offer a novel therapeutic strategy against OC.

Keywords: PI3K/AKT; RNLS; STAT3; ovarian cancer; oxidative stress.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
RNLS expression modulation and STAT3 association in OC cells. (A) qPCR analysis of RNLS mRNA levels in IOSE80 normal ovarian epithelial cells and OC cell lines. (B, C) Validation of RNLS knockdown (sh-RNLS) and overexpression (ov-RNLS) in OVCAR3 cells by qPCR. (D) Western blot analysis of RNLS and STAT3 protein levels in modulated cell lines. *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2
Functional effects of RNLS modulation on OC cells. (A) CCK-8 assay was performed to evaluate the effect of RNLS knockdown and overexpression on the viability of OVCAR3 cells. (B) Wound healing assay was conducted to assess the impact of RNLS modulation on the migratory capacity of OVCAR3 cells. (C) Transwell assay was used to determine the effect of RNLS knockdown and overexpression on the invasive potential of OVCAR3 cells. (D) Flow cytometry analysis was carried out to examine the influence of RNLS expression levels on apoptosis in OVCAR3 cells. ***P<0.001.
Figure 3
Figure 3
STAT3 silencing and RNLS overexpression interplay. (A) The inhibition efficiency of three si-STAT3 assays was verified by qPCR in OVCAR3 cells. (B) qPCR analysis of STAT3 expression in si-STAT3, ov-RNLS, and combination groups. (C) IF analysis of STAT3 nuclear localization in si-STAT3, ov-RNLS, and combination groups. (D) Western blot analysis of RNLS, STAT3, p-STAT3 Ser727 and p-STAT3 Tyr705. (E) Western blot analysis of p-PI3K, PI3K, p-AKT, and AKT protein levels in si-STAT3, ov-RNLS, and combination groups. **P<0.01, ***P<0.001.
Figure 4
Figure 4
Rescue effects of RNLS overexpression on STAT3 silencing. (A) CCK-8 assay for viability in si-STAT3, ov-RNLS, and combination groups. (B) Wound healing assessing migration in si-STAT3, ov-RNLS, and combination groups. (C) Transwell assays assessing invasion recovery in si-STAT3, ov-RNLS, and combination groups. (D) Flow cytometry analysis of apoptosis in si-STAT3, ov-RNLS, and combination groups. (E) Flow cytometry analysis of ROS levels in si-STAT3, ov-RNLS, and combination groups. (F) TEM of mitochondrial ultrastructure in si-STAT3, ov-RNLS, and combination groups. ***P<0.001.
Figure 5
Figure 5
The influence of ferroptosis inhibitor Fer-1 on the role of the RNLS-STAT3 axis in oxidative stress. (A) Flow cytometry analysis of ROS levels in OVCAR3 cells following modulation of RNLS and STAT3 expression. (B-D) ELISA quantification of MDA, GPX4, and GSH levels in OVCAR3 cells co-transfected with sh-RNLS or ov-RNLS and si-STAT3. *P<0.05, ***P<0.001.
Figure 6
Figure 6
In vivo tumorigenic role of RNLS. (A) Subcutaneous tumor growth curves of OVCAR3 cells with RNLS knockdown or overexpression in a xenograft mouse model. (B) Representative tumor images. (C) Tumor weights post-excision. (D) qPCR validation of RNLS and STAT3 expression levels in tumor tissues with RNLS knockdown or overexpression. **P<0.01, ***P<0.001.
Figure 7
Figure 7
Biomarker profiling in xenograft tumors. (A) IHC staining of Ki-67, RNLS, and STAT3 in tumor tissues with RNLS knockdown or overexpression. (B-D) ELISA quantification of MDA, GPX4, and GSH levels in tissue homogenates from RNLS-knockdown and RNLS-overexpressing tumors. *P<0.05, **P<0.01, ***P<0.001.
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