Introduction

Ovarian cancer (OC) is one of the most common cancers in the female genital tract, which is the third most common cancer behind cervical cancer and endometrial cancer, and the most deadly, being the 8th most common cause of cancer deaths among women [1]. The overall survival of OC patients has not improved significantly over the past decades due to the lack of typical and easily detectable early clinical signs, the lack of durable and effective treatment, and the elevated risk of chemoresistance and recurrence, with most cases being clinically advanced and with distant metastases at the time of diagnosis [2]. Therefore, in order to provide a theoretical basis for improving clinical outcomes in OC, there is an urgent need to better understand the potential pathogenesis of OC and to significantly improve our understanding of tumor progression and metastasis.

Post-translational modifications (PTMs) are a dynamic and reversible post-translational epigenetic modification of proteins with low metabolic cost and sensitive response [3]. It is an important mechanism for increasing the variety and functional diversity of proteins and plays a key role in regulating metabolism, signal transduction, reproductive development, tumor inflammation and other physiological and pathological conditions. Ubiquitination is an important type of PTMs, plays a crucial role in regulating the “quantity” and “quality” of substrate proteins, thus hel** to ensure homeostasis of the intracellular environment and the smooth running of life processes. Malfunctions of the ubiquitin-proteasome system are responsible for more than 80% of proteins are degraded in cells and have been demonstrated to cause pathologies, particularly malignant tumors [4]. Ubiquitination is a cascading process in which ubiquitin, a universally expressed 76 amino acid protein, first binds to ubiquitin-activating enzymes (E1s) and activates ubiquitin, then transfers the activated ubiquitin molecule to ubiquitin-binding enzymes (E2s). Ubiquitin ligases (E3s) then transfer ubiquitin molecules from the E2s to the underlying protein, ultimately leading to its degradation by the proteasome. Due to their relative specificity in recognizing substrate proteins, E3 ligases play a key role in the overall ubiquitination process.

Smad ubiquitin regulatory factor 2 (SMURF2) is a HECT-type E3 ubiquitin (Ub) ligase that regulates many key functional proteins, including SATB1 [5], RNF20 [6], YY1 [7], and Smad2 [8], which are involved in oncogenic or tumor suppressor functions. Recent studies have shown that SMURF2 interacts with SIRT1 to mediate the degradation of SIRT1, while deletion of SMURF2 expression leads to upregulation of SIRT1, inducing tumor initiation and the invasive metastasis of colorectal cancer in vivo and in vitro [9]. In addition, SMURF2 induces enhanced tumor metastasis in nude mice models of breast cancer [10]. Nevertheless, the role of SMURF2 in the progression of OC has not yet been the subject of in-depth investigation.

The receptor for activated C-kinase 1 (RACK1) is involved in a wide range of cellular signaling pathways, as a member of the tryptophan-aspartate repeat (WD repeat) family of proteins. Identified as a classic scaffolding protein for a variety of kinases and receptors, RACK1 is involved in a number of intracellular signaling pathways and plays a key role in a variety of physiological processes including cell growth, migration and differentiation [11,12,13,Protein expression and purification

For the production of proteins from bacteria, Escherichia coli BL21 (DE3) cells containing the GST, GST-SMURF2 and GST-SMURF2 C716A plasmids were induced for protein expression using 0.5 mM IPTG at 37 °C for 4–6 h. The cells were lysed in buffered saline. Cell lysis was performed with lysis buffer (0.5% Triton X-100, pH 7.5, 1 mM DTT, 50 mM Tris-Cl, 200 mM NaCl, 10% glycerol and 1 mM PMSF and sonicated). Lysates were centrifuged and incubated with glutathione-Sepharose 4B (GE Healthcare) at 4 °C for 4 h or overnight. The resin was washed three times with lysis buffer plus 300 mM NaCl and then washed two more times with PBS. Immobilization on glutathione-Sepharose beads was verified by SDS-PAGE and aliquoted for storage at −80 °C. 6×His-RACK1 was purified using nickel-nitrilotriacetic acid (Ni-NTA) matrices (QIAGEN).

In vivo ubiquitination assay

Cells were transfected with the indicated plasmids, then treated with 20 μM MG132 for 8 h for the in vivo RACK1 ubiquitylation assay. The cells were harvested and lysed in RIPA lysis buffer plus 1% SDS, 20 µM MG132, and protease inhibitors. The lysates were incubated with anti-RACK1 or anti-Myc antibodies for 12 h and with Protein A/G magnetic beads for an additional 12 h at 4 °C. The precipitated protein was boiled in SDS-PAGE loading buffer for 10 min and then treated with IB.

GST pull-down assay

Bacterially expressed GST, GST-SMURF2, or GST-SMURF2 C716A was bound to glutathione-Sepharose 4B beads (GE Healthcare). The complexes were mixed with Myc-RACK1 expressed in HEK293T cells for 2 h at 4 °C. After incubation, the complexes were washed with GST binding buffer for at least 3 times, then eluted with SDS-PAGE loaded buffer by boiling and treated with IB antibody shown.

Statistics and reproducibility

GraphPad Prism (version 8.0) was used for all statistical analyses. All in vitro experiments were carried out in at least three replicates and the data presented are from one representative experiment. The data are expressed as mean ± standard deviation. Double-tailed Student’s t-test or two-factor analysis of variance was used to assess the statistical significance of differences. The correlation between SMURF2 and RACK1 expression in OC patients was calculated by Pearson correlation analysis. Overall survival was assessed by the Kaplan-Meier method and compared by the log-rank test. P < 0.05 was considered statistically significant.