Reactive oxygen species‐induced SIAH1 promotes granulosa cells' senescence in premature ovarian failure

Abstract Reactive oxygen species (ROS) exposure triggers granulosa cells' (GCs) senescence, which is an important causal factor for premature ovarian failure (POF). However, underlying mechanism in this process remains unknown. In our study, we observed increased ROS levels in POF ovarian tissues, POF patient follicular GCs and cyclophosphamide (CTX) pretreated GCs. Correspondingly, increased SIAH1, reduced TRF2 and GC senescence were also found in these cases. Silencing of SIAH1 rescued ROS‐induced TRF2 reduction and cell senescence in GCs. Moreover, SIAH1 co‐localized with TRF2 in the cytoplasm, facilitating its ubiquitination degradation, further leading to telomere abnormalities in GCs. In conclusion, our findings indicate that ROS induces telomere abnormalities by augmenting SIAH1‐mediated TRF2 degradation, leading to cell senescence in GCs in POF processing.

prognosis of assisted reproduction treatment (ART). 8 Therefore, to elucidate the underlying mechanism of oxidative cell senescencedriven pathological process in GCs beside oocytes is necessary for comprehensively understanding POF pathogenesis.
Ubiquitination plays a central role in selective degrading damaged proteins in various forms. 9 The ubiquitin-proteasome pathway is also an important defence machinery against oxidative damage. 10 SIAH1, one of the seven members in absentia homologue family, is a critical E3 ubiquitin ligase which prominently controls a variety of biological processes, including transcription, mitosis and cell migration. 11,12 SIAH1 also participates in ubiquitination degradation of proteins involved in tumorigenesis (Bcl-2, JNK and EHMT2), [13][14][15] DNA repair (HIPK2, beta-catenin) [16][17][18] and hypoxia response (PHD3, FIH). 19,20 It is reported that SIAH1 expression could be promoted by E2F1, which commonly induced by ROS. 21 However, it is remained elusive whether SIAH1 is a target of ROS in GC senescence, and whether this process promotes GC senescence in POF initiation.
Telomeres are composed of TTAGGG repeats as the natural ends of chromosomes. 22,23 Its dysfunction is the origin of replicative cell senescence in ageing. 24 TRF2 is a telomere-binding protein, playing a vital role in t-loop generation, protecting chromosomes ends from being mis-recognized as double-strand DNA breaks by DNA damage repair system. 25 Early studies found TRF2 was associated with the normality of double-stranded telomeric DNA structure, telomere morphology and function. 26 TRF2 knockdown led to t-loop breakdown and initiated a rapid dsDNA break response, p53-p21 driven cell cycle arrest and cell senescence. 27 Fu et al. reported a proteolytic regulation of TRF2 by SIAH1 in human fibroblasts. 28 These facts support that SIAH1 may be involved in ROS-mediated GC senescence, and this regulatory path may be developed as novel therapeutic targets for rescuing ovarian function in POF patients. Ovarian GCs were isolated from follicular fluid, centrifuged at 280 g for 10 min and re-suspended in phosphate-buffered saline containing 0.1% hyaluronidase (#H1136-1AMP; Sigma) at 37°C for 30 min.

| Patients and GC isolation, and serum protein extraction
Serum protein was extracted from patient serum with RAPI lysis and sulfhydryl reducing agent. Supernatant was collected after 12,000 g centrifugation for 15 min, denatured and then frozen for further examination.

| In vitro ubiquitination and coimmunoprecipitation
293T cells were transfected with plasmids. 36 h later, cells were pretreated with 15 μM MG132 (#HY-13259; MedChemExpress) for 6 h and then lysed with IP lysis buffer. Whole-cell lysates were incubated with indicated antibody overnight at 4°C and then immunoprecipitated with Protein A/G beads (#CS203178; Millipore).
Immunoprecipitates were washed five times, and then, protein was eluted for Western blotting analysis.

| Immunohistochemical
Isolated ovarian tissues were fixed with 4% paraformaldehyde, embedded in paraffin and then sliced in 4 μm thickness. After dehydration with gradient alcohol and xylene, sections were incubated with anti-SIAH1(#ab2237; Abcam) antibody at 4℃ in humid and then incubated with the secondary antibody at room temperature for 2 h.

| Immunofluorescence
Cells were rinsed with PBS and fixed with 4% PFA at room temperature for 20 min. Then, cells were permeabilized with 0.1% Triton X-100 and blocked with 1% bovine serum albumin in TBS for 1 h. Next, cells were incubated with specific primary antibody at 4℃ overnight and with secondary antibodies for 1 h at room temperature in dark.
Nucleus was stained with DAPI (Beyotime) for 10 min at room temperature before imaging.

| Electron microscopy
At Day 21 post-BMSC transplantation, two rats were randomly taken from negative control, model control and BMSC transplantation group. Then, rats were sacrificed for dissection. Indicated tissue was fixed with 2.5% glutaraldehyde phosphate buffer +1% osmic acid fixative. After dehydration and embedding, tissue blocks were subjected to ultra-thin sectioning. Sections were stained with lead citrate and washed with ultra-pure water for image capture and analysis by transmission electron microscopy.

| Measurement of ROS
Redox-sensitive, cell-permeable fluorophore DHE (#KGAF019; KeyGen biotech) was used to measure superoxide production in situ. DHE (10 mM) was incubated with unfixed frozen sections or cultured cells in a light-protected humidified chamber at 37°C for 30-60 min. Incubated samples were washed with PBS for three times, mounted with fluorescent mounting medium (DAKO) and then stained with 10 µM DCFH-DA (#D6470; Solarbio). Images in similar exposure conditions were picked for fluorescence semiquantification. The grayscale was detected as the signal intensity, and the average in group was compared. For each group, 10 randomly picked sights were included for statistical analysis.

| Senescence-associated βgalactosidase activity
Cell senescence in KGN and GCs were measured by staining with

| Statistical analysis
All experiments have repeated at least three times. Statistical analysis was performed with SPSS 13.0 (IBM) and GraphPad Prism 6 (GraphPad Software). Unless otherwise noted, difference was analysed by the two-tailed Student's t-test or one-way ANOVA test with Tukey's post hoc analysis (for comparing more than two groups).
Differences were defined as significant when p < 0.05. Data were shown as mean ± SD.

| ROS accumulation accelerates GC senescence during ovarian ageing
To elucidate the connection between ROS accumulation and oocyte quality decline in ovarian ageing, we first measured ROS in POF in vitro and in vivo. POF rat model was established as previously described. 29 As shown, we observed dysregulated oestrus cycle, increased FSH and LH levels, and decreased AMH values, corresponding with more active apoptosis and decreased ovarian reserve ( Figure S1). In addition, pathologic phenotypes in GCs including dissolved nucleus, nuclear membranes loss, pyknotic chromatin, edge aggregation, apoptotic bodies and vacuolated mitochondria were all observed. We also detected excessive ROS accumulation in POF rat ovaries, along with downregulation of antioxidant genes (such as SOD2, CAT and GLUT1) ( Figure S3). We previously reported poten-

| ROS promotes GC senescence via upregulating SIAH1
In order to clarify whether SIAH1 was involved in POF development, we measured SIAH1 expression level in POF in vivo and in vitro. As shown in Figure 1A,B, SIAH1 mRNA and protein levels were elevated in POF rat ovaries and CTX-treated GCs, whereas back to normal upon BMSC treatment ( Figure 1C,D). Upon H 2 O 2 inducing ROS in the GC line KGN, SIAH1 and P53 protein level considerably increased, accompanied with more senescent cells ( Figure 1E).
In contrast, silencing of SIAH1 with shRNA effectively protected cells from ROS-induced senescence, suggesting SIAH1 may be a key mediator in regulating this process ( Figure 1F). In CTX-treated GCs, again we confirmed that SAβ-gal signal was significantly reduced by NAC, but notably, upregulating SIAH1 re-induced SAβ-gal signal in NAC-treated cells ( Figure 1H), implying a vital role SIAH1 played in GC senescence.

| TRF2-mediated SIAH1 function on regulating ROS-induced GC senescence
SIAH1 was previously reported promoting TRF2 degradation in human fibroblasts, 28 while TRF2 has a crucial function on telomere protection. 30 Therefore, we extended our study to assessed whether TRF2 and telomere dysfunction were involved in SIAH1regulated GC senescence upon ROS treatment. As showed, TRF2 protein level was decreased in both of POF rats and CTX-GCs model ( Figure 2A,B). Furthermore, SIAH1 expression elevated by increasing H 2 O 2 concentration, whereas TRF2 protein stability decreased ( Figure 2C). As shown in Figure 2D, overexpression of SIAH1 dramatically promoted cell senescence while co-expression with TRF2 had the opposite function. Moreover, in H 2 O 2 -pretreated cells, TRF2 also restrained SIAH1-induced senescence ( Figure 2E). By telomeric PNA-FISH with GCs in metaphase, we analysed the impact of SIAH1/TRF2 to telomeric DNA. As showed, SIAH1 significantly induced telomere fusions and other chromosomal abnormalities in GCs ( Figure 2F), which could be reversed by TRF2 overexpression.
In summary, TRF2-mediated telomere protection might be involved in the process of SIAH1 inducing GC senescence.

| SIAH1 mediates TRF2 protein ubiquitination degradation
Inspired by opposite trends of SIAH1 and TRF2 expression in POF, we set out to investigate the underlying mechanism of SIAH1 regulating TRF2. SIAH1 participated in ubiquitin-mediated proteolytic cascade, so we tested whether SIAH1 serves as an E3 ubiquitin ligase in TRF2 ubiquitination. It was found that TRF2 protein level was significantly reduced by SIAH1 ( Figure 3A), but this effect could be attenuated by MG132 (a proteasome inhibitor) ( Figure 3B), suggesting that TRF2 may be SIAH1's substrate. To further identify whether SIAH1 is the responsible E3 ligase of TRF2, KGN overexpressed with SIAH1 were treated with combination of cycloheximide (CHX, an inhibitor of protein biosynthesis). Measurement of immunoblots confirmed that SIAH1 significantly reduced TRF2 protein stability ( Figure 3C,D). With suppressing TRF2 degradation, we observed SIAH1 significantly enhanced TRF2 ubiquitination ( Figure 3E,F).
In contrast, silencing of SIAH1 inhibited TRF2 ubiquitination ( Figure 3G). All above results indicate that SIAH1 destabilizes TRF2 proteins in a ubiquitin-proteasome pathway.
Next, we verified protein-protein interaction of endogenous SIAH1 and TRF2 by CO-IP ( Figure 3H). Interestingly, nuclear localization of TRF2 was reduced upon H 2 O 2 while cytoplasmic TRF2 increased ( Figure 3I), which was consistent with the fact that ubiquitinated TRF2 protein would be exported to cytoplasm. 28 In further validation, we showed TRF2 was retained in nucleus upon MG132 treatment by measuring colocalization signals. With withdrawing MG132, TRF2 was exported to cytoplasm and co-localized with SIAH1, and then underwent degradation ( Figure 3J, Figure S4C).
These results indicate that SIAH1 promotes TRF2 ubiquitination degradation in the cytoplasm upon ROS.

| SIAH1 expression elevated in POF patient GCs
To check clinical relevance of the ROS-p53-SIAH1-TRF2 axis in POF, we assessed ROS levels, cell senescence and SIAH1 expression

| DISCUSSION
Our study reported that elevated ROS promoted SIAH1 expression in POF in both in vivo and in vitro cases. We also confirmed this process in clinical human samples. As known, SIAH1 was well-studied in hypoxia, ageing and DNA damage with identification of numerous SIAH1-interacting proteins. Our results indicated that SIAH1 responded to ROS and promoted GC senescence in POF. Our data also demonstrated that SIAH1 directly bound with TRF2 to promote its ubiquitination and degradation, rendering telomere abnormalities and cell senescence in GCs, subsequently contributing to POF development.
Clinically, POF patients have significant early exhaustion of ovarian follicles along with amenorrhea, hypoestrogenism and elevated gonadotropin levels before age 40. 2 Due to premature atresia of follicles and the arrest of spontaneous ovulation, the chances of conceiving are very low in POF patients. 31 However, POF pathogenesis has not been fully interpreted so far. Numerous studies demonstrated that reproductive capacity was very sensitive to redox homeostasis and oxidant enzyme levels. 32 A widely acknowledged theory of follicle senescence emphasized that ROS induced GCs degeneration as a critical causative factor, 33  Reactive oxygen species vitiates the maturation and quality of oocytes through destroying the spindles in chromosome assembly, interrupting mitochondrial function, affecting motility during early meiosis by shortening telomeres. 38,39 TRF2 is responsible for the formation and maintenance of the 't-loop' structure, 25 and TRF2 loss is associated with senescence-related signalling. 40 We found that when SIAH1 overexpression in GCs reduced the telomere protection factor TRF2 by promoting its ubiquitination. Telomeric DNA was continually consuming, resulting in chromosome-end fusions and other types of damage. Consistently, in ROS-driven GC senescence, ascending P53 and SIAH1 restrained TRF2 protein level and led to excessive chromosome end fusion and damage. This outcome was significantly avoided by exogenously expressed TRF2 ( Figure 2G), confirming that TRF2 could protect telomere from fusions or other damage induced by SIAH1. It is reported that TRF2 decrease was associated with telomere dysfunction-driven ageing acceleration.
Supraphysiological amounts of TRF2 are also susceptible to chromosomal instability and impaired normal senescence checkpoint. 41,42 These non-conflicting conclusions from different studies supported the fact that telomere regulation is an extremely elaborated, and both loss and excessive TRF2 lead to telomere dysfunction and chromosomal abnormalities.

| CON CLUS ION
In summary, our study revealed that SIAH1 mediated cytoplasmic TRF2 ubiquitination degradation and then induced telomere abnormalities, F I G U R E 5 Overview of the ROS-p53-SIAH1-TRF2 signalling axis in POF. The diagram shows the hypothetical model of ROS-induced GC senescence in our study. Exogenous H 2 O 2 induced intracellular ROS in GCs. ROS accumulation activated p53. Then, active P53 upregulated SIAH1 which promoted TRF2 ubiquitination degradation, resulting in GC senescence along with telomere shortening, loss, deforming. GCs, granulosa cells; POF, premature ovarian failure; ROS, Reactive oxygen species contributing to GC senescence upon oxidative stress. These findings implied SIAH1 might be developed into a novel therapeutical target to alleviate ROS-induced GC senescence in POF ( Figure 5). However, we also need additional investigation to in-depth elucidate SIAH1 biological function for the goal of improving fecundity in POF in clinical.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.