5-methylcytosine regulated CCNL2 promotes tumorigenesis and cisplatin resistance of ovarian cancer with therapeutic implications

Abstract

Backgroud

Ovarian cancer (OC) is the most lethal gynecological tumor, primarily due to resistance to chemotherapy. Cyclin L2 (CCNL2) is a novel member of the cyclin family and mainly localized in nucleus. It regulates transcription and alternative splicing by interacting with cyclin-dependent kinases. However, its role in OC chemoresistance remains unknown.

Results

Here, we demonstrated that the expression level of CCNL2 was higher in OC tissues as well as in various other tumor types. Furthermore, elevated expression of CCNL2 indicated a poor prognosis in ovarian cancer. Functionally, CCNL2 promoted OC cell proliferation and xenograft growth. Depletion of CCNL2 enhanced chemotherapy sensitivity in OC cells. Mechanistically, YBX1 directly bound to CCNL2 mRNA, and its depletion reduced CCNL2 mRNA stability and protein expression. MeRIP assays revealed that YBX1 regulated CCNL2 via 5-methylcytosine (m⁵C) modification. Mutation of the key residue of YBX1 required for m⁵C function led to decreased CCNL2 expression. Further investigation of the YBX1 regulatory network identified a direct interaction between YBX1 and MATR3, which cooperatively modulated downstream targets. Notably, MATR3 knockdown reversed the YBX1-induced upregulation of CCNL2. Virtual screening identified YB-B1 as a YBX1 inhibitor that effectively downregulated both YBX1 and CCNL2 expression. In vitro, YB-B1 suppressed ovarian cancer cell proliferation and enhanced cisplatin cytotoxicity. Furthermore, patient-derived tumor xenograft (PDX) model also confirmed its chemosensitizing effect.

Conclusions

In summary, we demonstrated that CCNL2 promoted OC cell proliferation and chemoresistance, with its expression regulated by YBX1 via m⁵C methylation. The small molecule inhibitor YB-B1 was identified as a promising solution to overcome chemotherapy resistance.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01753-9.

Background

OC has the highest mortality rate among all female cancers and poses a serious threat to women’s health [1]. According to the 2022 global cancer statistics, ovarian cancer ranks among the top gynecological tumors in terms of both incidence (3.4%) and mortality (4.8%) [2]. The standard treatment for OC patients is surgical debulking followed by platinum-based chemotherapy. Some patients may also receive combined targeted therapy to further improve treatment outcomes [3]. OC has a high recurrence rate and poor prognosis, predominantly due to chemotherapy resistance. Platinum resistance is typically defined as tumor recurrence or progression within 6 months after surgery and adjuvant chemotherapy. The emergence of platinum resistance often indicates a very poor prognosis, with an overall survival of only 12 to 14 months from the establishment of resistance to diagnosis [4], severely affecting the treatment efficacy and survival of patients. Despite advances in the treatment of ovarian cancer, the 5-year survival rate after diagnosis is only 46% [5]. Therefore, it is urgent to explore the mechanism underlying OC chemoresistance.

CCNL2 is a new member of the cyclin family, containing a C-terminal RS domain and an N-terminal cyclin box domain. The C-terminal RS domain interacts with CDK11 and plays a key role in regulating pre-mRNA splicing [6, 7]. The CCNL2/CDK11 complex has been reported to be involved in the gene expression of the HIV. It can be recruited by the viral DNA and mediates the cleavage and polyadenylation of transcripts, thereby regulating subsequent transcription [8]. Furthurmore, CCNL2 can participate in HIV replication independently of CDK11. It interacts with DCAF1 to support HIV-1 replication and CCNL2 knockdown attenuates HIV-1 replication in macrophages [9]. However, there are only several studies on CCNL2 in tumors. The alternative splicing of CCNL2 is influenced by SRSF5, thereby enhancing the proliferation of pancreatic cancer [10]. Furthermore, CCNL1/CCNL2 form synthetic lethal pairs as shown by a double-knockout CRISPR-Cas9 approach. The concurrent loss of both genes significantly suppressed tumor cell growth [11]. However, the role of CCNL2 in OC pathogenesis remains unexplored.

5-methylcytosine (m⁵C) was discovered recently and plays a critical role in RNA metabolism [12]. Similar to m⁶A modification, m⁵C methylation is regulated by ‘Reader’, ‘Writer’, and ‘Eraser’ [13]. Generally, mRNAs are methylated by m⁵C writers such as NSUN2 and NSUN6. Readers including Y-box‐binding protein 1 (YBX1) and ALYREF can bind to m⁵C mRNA to regulate mRNA stability, export, and translation [14, 15]. Recent research has highlighted the significant role of m⁵C methylation in cancer progression. For example, YBX1 recruits ELAVL1 in the cytoplasm and binds the m⁵C methylation sites of HDGF, thereby modulating urothelial carcinoma’s pathogenesis [16]. Recent studies have also found that m⁵C modification may be associated with drug resistance in malignant tumors. In tumor immunotherapy, glucose can act as a signaling molecule, directly binding to and activating the m⁵C RNA methyltransferase NSUN2, which maintains the m⁵C methylation and mRNA stability of TREX2. This further inhibits the cGAS/STING pathway, thereby suppressing the effect of IFN and leading to resistance to immunotherapy [17]. m⁵C methylation also plays a critical role in chemotherapy resistance [18]. By inhibiting post-transcriptional m⁵C modification, it can reduce protein synthesis in stress response pathways, thereby decreasing the sensitivity of tumor stem cells to chemotherapy drugs [19]. Therefore, analyzing chemoresistance in ovarian cancer from the perspective of m⁵C modification holds great potential.

The discovery of new chemoresistance-related targets in ovarian cancer can promote drug development. Virtual screening, as a technique that uses computer simulation and molecular docking to assist in the discovery and optimization of drug molecules [20], has become increasingly widespread in both academia and the pharmaceutical industry [21]. Virtual screening of the binding site between Smad1 and p300 identified the small molecule cpd.618, which effectively disrupts their interaction, inhibiting tumor cell proliferation and enhancing chemosensitivity [22]. Therefore, selecting appropriate chemoresistance targets and utilizing virtual screening to identify small molecule drugs is an effective strategy for overcoming chemotherapy resistance.

In the present study, we demonstrated that CCNL2 was upregulated in OC and indicated a poor prognosis. CCNL2 depletion promoted OC cells proliferation and xenograft tumor growth. Moreover, CCNL2 contributed to chemoresistance of ovarian cancer. Additionally, we demonstrated that YBX1 collaborated with MATR3 to regulate CCNL2 mRNA stability via an m⁵C manner, thereby enriching the regulatory pathway of CCNL2. By virtual screening, we found a new inhibitor, YB-B1, which suppressed the expression of YBX1 and CCNL2, and enhanced chemotherapy-induced cytotoxicity in ovarian cancer cells and the PDX model. As a result, the YBX1-CCNL2 axis may be a key target for overcoming chemotherapy resistance in ovarian cancer, and the small molecule drugs identified through virtual screening offer new possibilities for treatment.

Materials and methods

Patient tissues collection

Seven ovarian cancer tissues and six fallopian tube samples were used for western blot analysis. A total of 130 ovarian cancer and 50 normal fallopian tube tissues between 2005 and 2013 were used to make tissue microarrays. These microarrays will be used for subsequent IHC analysis. This study was approved by the ethics committee of Qilu Hospital, Shandong University (KYLL-202210-052-1).

Cell lines

Human ovarian cancer cell lines such as HEY, OV90 were purchased from the Stem Cell Bank at the Chinese Academy of Sciences. HEK293T cells were obtained from the Beihua Kong lab. All cell lines used in the study underwent validation through STR profiling. Mycoplasma detection and elimination were performed regularly. HEY, OV90 and HEK293T cells were cultured in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (LONSERA, Uruguay) and 1% penicillin/streptomycin. The same cell culture conditions (5% CO2, 37 °C) in a humidified incubator were employed.

HEK293T cells were employed for lentivirus packaging. Ovarian cancer cells were infected with lentivirus for 24 h and puromycin (2 µg/ml) was used for selection for more than 7 days. The siRNA transfections were performed using lipofectamine 2000 (Invitrogen, 11668030). The siRNAs targeting CCNL2, YBX1, and MATR3 were synthesized by Sangon Biotech. The target sequences of siRNAs are listed in Table S1.

Bioinformatics analyses

The UALCAN database was used to obtain CCNL2 protein expression in ovary and ovarian cancer samples (https://ualcan.path.uab.edu/). The progression-free survival and overall survival were analyzed by Kaplan-Meier Plotter (http://kmplot.com/). The correlation between CCNL2 and m⁵C signature was assessed using GEPIA2 (http://gepia2.cancer-pku.cn/). m⁵C signature was listed in Table S2.

Plasmids

The lentivirus vector-encoding pLVX-CCNL2 and pLVX-YBX1 plasmids were purchased from Tsingke Biotechnology (Beijing, China). The doxycycline-inducible pZIP-TRE3G-shCCNL2 plasmid and pZIP-TRE3G-shYBX1 plasmids were purchased from BoShang (Jinan, China). pcDNA3.1-CCNL2-WT, pcDNA3.1-YBX1-WT, pcDNA3.1-MATR3-WT, pcDNA3.1-YBX1-W65A, pGL3-CCNL2-3’UTR-WT, pGL3-CCNL2-3’UTR-MUT, pGL3-CCNL2-Promoter were purchased from Tsingke Biotechnology (Beijing, China).

Cell proliferation assays

CCK-8 assay (Cell Counting Kit-8, Meilunbio) was used to determine cell viability and proliferation. In brief, 1000–2000 cells were seeded in 96-cell plates after intervention. After reaching the observation time, CCK8 was mixed with cell culture medium at a ratio of 1:9 and added to a 96-well plate. Cells were incubated in a humidified incubator for two hours, and the absorbance was measured at 450 nm. The entire cycle lasted for five consecutive days and conditions remained constant.

Cell-Light EdU Apollo In Vitro Kit (RIBOBIO, China) was employed according to the manufacturer’s instructions. Briefly, 2000–4000 cells were seeded in 96-cell plates and incubated in the culture medium with EdU for 2 h. Then cells were fixed and stained with Apollo and Hoechst 33,342.

For colony formation assay, 500–800 cells were plated in 6-cell plates after intervention and continued to grow in a humidified incubator for 10–14 days. Colonies were treated with methanol for 15 min once reaching sufficient numbers. Subsequently, the clones were stained with Crystal Violet Solution (Beyotime), washed with PBS for several times, dried, and photographed. The number of colonies was quantified by ImageJ.

Transwell assays

In the transwell assays of migration and invasion, 24-well cell culture inserts were used. For the invasion assay, the upper surface of the membrane was pre-coated with Matrigel. 4 × 10⁴ cells in serum-free medium were seeded into the transwell chambers (Corning, 3422). The chambers were plated in a 24-well plate and 600 µl medium with 10% FBS was added to each well. After the incubation in a humidified incubator, the cells attached to the membranes of the chambers were fixed and stained.

Apoptosis assay

In the apoptosis assay, Annexin V-FITC/PI Apoptosis Kit (Elabscience) was used to detect cell apoptosis following the manufacturer’s protocol. Cells were digested by trypsin without EDTA, washed with PBS, and resuspended with Annexin V binding buffer. Then Annexin V-FITC and PI were added into cells. Cells were incubated for 15 min in the dark and analyzed by flow cytometry.

RNA isolation and PCR analysis

Total RNA was extracted from cultured cells with RNA Rapid Extraction Kits (SparkJade) and RNA was transcribed into cDNA with All-in-one RT SuperMix (SparkJade). The qPCR was performed using 2×SYBR Green qPCR Mix (SparkJade). The sequences of primers used for qPCR are listed in Table S3.

Western blot

Cells were placed on ice and lysed using RIPA lysis buffer supplemented with 1% PMSF for 30 min, followed by sonication. The supernatant was collected by centrifugation and the concentration of protein was determined by a BCA assay kit. Protein was separated by SDS-PAGE gels and then transferred onto the PVDF membrane. The membrane was blocked with 5% milk or BSA at room temperature for 1 h. Protein on the membrane was captured by incubation of primary antibodies and secondary antibodies. Finally, specific bands were detected by ECL reagents. All antibody information is listed in Table S4.

Immunohistochemistry (IHC)

Tissue samples were fixed, embedded in paraffin wax, and cut into 4 μm slices. Then tissue slides were deparaffinized and rehydrated following the instruction. Heat-induced epitope retrieval was conducted using an antigen retrieval solution with appropriate pH. Tissues were then blocked and incubated overnight at 4℃ with primary antibody against Ki-67 (1:100, Origene, TA500265) and CCNL2 (1:150, Elabscience, E-AB-40247). The slides were incubated with the secondary antibody on the second day at room temperature for 1 h and DAB reagents were used for visualization. The scores of IHC staining were evaluated based on both the proportion of positive cells and the staining intensity, resulting in a total score ranging from 0 to 12. A score greater than or equal to 6 indicated high expression.

Dual-luciferase reporter assay

Ovarian cancer cells were plated in 24-well plates and transferred with 500ng pGL3 reporter plasmids and 20ng renilla reporter plasmids. Firefly luciferase activity and Renilla luciferase activity were detected according to the instructions of the Dual-Luciferase Reporter Assay kit (Promega, E1910).

RNA stability assay

Cells were seeded overnight in 6-well plates and then treated with Actinomycin D (5 µg/ml, GLPBIO, GC16866) for various time intervals. Total RNA was extracted and analyzed by RT-qPCR. The relative abundance of CCNL2 mRNA was normalized to the t = 0 h time point.

RNA pull-down assay

RNA-Protein pull-down Kit (GENESEED, China) was employed following the manufacturer’s instructions. Biotin-labeled RNA was transcribed with the T7 biotin-labeled transcription Kit (RiboBio, China). In brief, ovarian cancer cells were lysed with capture buffer on ice for 10 min. One-tenth proportion of cell lysate was used as input. Biotin-labeled probe and negative RNA probe were cocultured with magnetic beads individually for 30 min. Then probe-beads complex was cocultured with lysate for 1 h at 4 ℃. Protein was eluted and analyzed by western blot and mass spectrometry.

RNA immunoprecipitation (RIP) assay

RNA-Binding Protein Immunoprecipitation Kit (Millipore) was employed according to the instructions. In brief, HEY cells were washed twice with ice-cold PBS and lysed with RIP buffer on ice for 5 min. One-tenth proportion of cell lysate was used as input. The rest was immunoprecipitated with anti-YBX1 antibody (Proteintech, 20339-1-AP) and magnetic beads. The immunoprecipitate was incubated with proteinase K buffer and the RNA pulled by protein was extracted. RT-qPCR and RT-PCR were used to detect the content of specific RNA. All instruments, plastic ware, and reagents used in the experiment were DNase-free and RNase-free.

Methylated RNA immunoprecipitation (MeRIP) assay

m⁵C Methylated RNA Immunoprecipitation Kit (BersinBio) was used following the instructions. Cells were collected and washed with cold PBS. Total RNA was extracted using Trizol and then was fragmented into around 300nt fragments by Fragmentation Buffer. One-tenth of RNA fragments were used as input. The rest was immunoprecipitated with m⁵C antibody (Abcam, ab10805) or IgG, along with magnetic beads. The immunoprecipitate was incubated with proteinase K buffer and the RNA was extracted. RT-qPCR was used to detect the m⁵C methylation of CCNL2 mRNA. The primers for MeRIP-qPCR were designed based on the CCNL2 m⁵C modification sites in the m⁵C-Atlas (www.xjtlu.edu.cn/biologicalsciences/m5c-atlas) or predicted using the RNAm5Cfinder platform (www.rnanut.net/rnam5cfinder/). Fold enrichment was calculated by the ΔΔCT method.

Co-immunoprecipitation (CO-IP) assay

For CO-IP assay, HEY cells were lysed with IP lysis buffer (Beyotime, P0013) supplemented with PMSF. One-tenth proportion of lysate was used as input. The rest of the lysate was immunoprecipitated with anti-YBX1 antibody (Proteintech, 20339-1-AP) and Protein A/G PLUS-Agarose (Santa Cruz, sc2003). The beads were boiled for 10 min and analyzed by western blot.

Animal experiments

Female BALB/c-nude mice (4 ~ 6 weeks) were purchased from Gem Pharmatech and divided into indicated groups randomly. CCNL2 overexpression or doxycycline-induced knockdown ovarian cancer cells were injected subcutaneously. The experiment groups received Doxycycline (1.2 g/L), while 5% sucrose was given to the control groups. When tumors grew to a sufficient size, they were removed and weighed.

For animal experiments requiring cisplatin injection, cisplatin (2 mg/kg) or saline was injected intraperitoneally every three days once the tumor volume grew to 50-100mm³. When tumors grew to a sufficient size, they were removed for subsequent experiments. Tumor volumes were measured with the formula: 1/2×length×width^². The power analysis was used to determine the sample size with G*power(version 3.1). A two-tailed t-test or one-way ANOVA was applied to achieve 80% statistical power at a significance level of 0.05. The animal studies were executed utilizing a single-blind experimental design. All animal experiments were conducted with approval from the Animal Care and Use Committee of Qilu Hospital, Shandong University (Dwll-2023-120). The maximal tumor diameter did not exceed the requirements.

PDX model establishment and therapy

Female NOD-scid mice (4 ~ 6 weeks) were purchased from Gem Pharmatech. Mice were housed in an SPF animal facility for one week to acclimate to the environment. Fresh ovarian cancer tissues were collected, washed with PBS buffer, and necrotic regions were removed. The tissues were then sectioned into small fragments and subcutaneously implanted into mice to observe tumor formation. Once tumors developed, they were surgically removed and transplanted into new recipient mice. When the tumor volume reached an appropriate size, the mice were randomly divided into two groups: the Cisplatin group (2 mg/kg) and the Cisplatin + YB-B1 group (cisplatin 2 mg/kg + YB-B1 3 mg/kg). Treatment was administered every three days. After the treatment period, the mice were euthanized, and tumors were collected and recorded.

Virtual screening

Schrödinger Maestro 12.8 was used for virtual screening. The 3D structure of human YBX1 (PDB ID: 6KUG) was downloaded from the RCSB PDB database. The protein Preparation Wizard module was used to refine the raw YBX1 crystal structure for enhanced accuracy. The MCE 50 K diversity library and MCE bioactive compound library Plus, containing 71,495 compounds, were processed by the LigPrep Module. Using the Virtual Screening Workflow module, the ligand and receptor were docked by HTVS, SP, and XP mode. Screening results were exported with docking scores. The higher the absolute value of the docking score, the stronger the binding force between the small molecules and the protein.

Surface plasmon resonance (SPR)

Biacore T200 was used for pH scouting, immobilization, and interaction analysis. The CM5 chip (GE Life, 29104988) was used for immobilization by amine coupling. Commercial YBX1 protein (CUSABIO, EP026247HU) was diluted in buffers with different pH between 4.0 and 5.5. Then the YBX1 protein solution was injected and the response in resonance units (RU) was measured to determine the optimal immobilization pH. The CM5 chip was activated by NHS and EDC. YBX1 protein at pH 4.0 was repeatedly injected to immobilize a sufficient amount on the chip surface. Then ethanolamine was passed over to deactivate the remaining active sites on the chip. The compound YB-B1 was diluted from 3.125 µM to 100 µM and injected to evaluate interaction strength. The results were analyzed by Biacore T200 Evaluation software.

Statistical analyses

All data were analyzed using GraphPad Prism 9. Student’s t-test was used to evaluate the quantitative data. The chi-square test was used to analyze the relation between categorical variables. The log-rank test was used to test the survival differences between high and low expression groups. Results were shown as the mean ± SD of three independent experiments. p < 0.05 was considered significant results (*P < 0.05, **P < 0.01, ***P < 0.001).

Results

CCNL2 is upregulated and associated with poor prognosis in ovarian cancer

We first investigated the expression level of CCNL2 by analyzing the GEO dataset (GSE69428) and the UALCAN database. Our findings revealed that CCNL2 was upregulated at both RNA and protein levels in ovarian cancer compared to normal tissues (Fig. 1A, C). Further analysis revealed that CCNL2 expression was elevated in both grade 2 and grade 3 ovarian cancer tissues compared to normal ovarian tissues (Fig. 1D). Pan-cancer analyses showed that CCNL2 was upregulated at both RNA and protein levels across various cancer types (Fig. 1E, S1A). We also validated the above conclusions using clinical tissue samples. Western blot analysis revealed that CCNL2 expression was higher in serous ovarian cancer tissues (SOCs) compared to fallopian tube tissues (FTs)(Fig. 1B). Then, we performed immunohistochemistry (IHC) on tissue microarrays, containing 130 OC tissues and 50 FT tissues. The results indicated that the proportion of high-expression tissues was greater in serous ovarian cancer compared to normal tissues. The proportion of low expression in normal tissues is 80%, while in tumor tissues it is 62% (Fig. 1F-H). Kaplan-Meier analyses based on the data from tissue microarrays showed that high expression levels of CCNL2 correlated with poor progression-free survival (PFS) and overall survival (OS) in patients with SOCs (Fig. 1I, J). This result was validated using the Kaplan-Meier database(Fig. 1K, L). Taken together, these results indicate that the expression of CCNL2 is upregulated in OC and predicts poor prognosis.

Fig. 1.

CCNL2 is upregulated in OC and associated with poor prognosis. A) CCNL2 expression level in OC tissues (n = 10) and fallopian tube tissues (n = 10) from the GEO dataset, GSE69428. B) Western blot analysis of CCNL2 protein expression levels in serous ovarian cancer tissues (SOCs) (n = 7) and fallopian tube tissues (FTs) (n = 6). C) CCNL2 protein expression was assessed in OC (n = 100) and normal tissues (n = 25) in the CPTAC database. D) CCNL2 protein expression level was analyzed across different OC grades in the CPTAC database. E) Pan-cancer analysis of CCNL2 at the protein level based on the CPTAC database. F) Statistical analysis was conducted on CCNL2 expression levels assessed via IHC staining of tissue microarrays. The proportion of tissues with high CCNL2 expression was higher in SOCs (n = 130) compared to FTs (n = 50). G, H) Representative images with high and low CCNL2 expression levels from tissue microarrays via IHC staining. I, J) Kaplan-Meier analyses of the Qilu cohort demonstrated the effect of CCNL2 on the PFS (I) and OS (J) in OC patients. K, L) Kaplan-Meier analyses demonstrated how CCNL2 affects the PFS (L) and OS (K) of OC patients based on the Kaplan-Meier cohort. The p-value was obtained using two-tailed unpaired Student’s t-test (for A, C, D), Chi-square test (for F), and log-rank test (for I, J, K, L). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

CCNL2 promotes the proliferation of ovarian cancer

To further investigate the role of CCNL2, we designed specific siRNAs targeting CCNL2 and transfected them into ovarian cancer cell lines. Additionally, we established both overexpression and knockdown models of CCNL2 in OC cells to evaluate its functional impact (Fig. 2A). We then conducted CCK8 and clonogenic assays, which demonstrated that depletion of CCNL2 by siRNA significantly inhibited the proliferation of HEY and OV90 cells (Fig. 2B, C). Furthermore, EdU assays performed on OV90 cells revealed a decreased percentage of EdU-positive cells following CCNL2 knockdown, further confirming its role in regulating cell proliferation (Fig. 2D). Transwell assays showed that CCNL2 knockdown decreased the migration and invasion abilities of OC cells (Fig. 2E, F). These results collectively suggest that silencing CCNL2 can effectively suppress the proliferation and metastasis of OC cells in vitro.

Fig. 2.

CCNL2 promotes the proliferation of ovarian cancer. A) Western blot analysis of CCNL2 in stably CCNL2-depleted and CCNL2-overexpressed OC cell lines. B) The effect of CCNL2 on cell proliferation was assessed by colony formation assay in HEY and OV90 cells (n = 3). Quantified data are shown on the right. C) CCK8 assay was performed to assess the proliferation ability after CCNL2 knockdown or overexpression in HEY cells (n = 3). D) The EdU assay was performed in OV90 cells with CCNL2 knockdown (n = 3). Scale bar, 100 μm. Quantified data are shown on the right. E, F) Transwell assays were performed in HEY and OV90 cells with CCNL2 knockdown (n = 3). Quantified data are shown on the right. G-I) The image of subcutaneous tumors (G), tumor mass (H), and IHC images of Ki-67 and CCNL2 (I) in control and CCNL2 knockdown groups (n = 5 per group). Scale bar, 100 μm. J-L) The image of subcutaneous tumors (J), tumor mass (K), and IHC images of Ki-67 and CCNL2 (L) in control and CCNL2 overexpression groups (n = 5 per group). Scale bar, 100 μm. All quantification analyses were based on three independent experiments. Data are mean ± SD. The p-value was obtained using two-tailed unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

To assess the effect of CCNL2 on tumor growth in vivo, we utilized tumor xenograft models. HEY cells with either CCNL2 overexpression or knockdown were injected subcutaneously into nude mice. The results revealed that CCNL2 knockdown in HEY cells led to significant reductions in both tumor volumes and weights (Fig. 2G, H). Immunohistochemical analysis showed decreased expression of the proliferative marker Ki-67 in tumors from CCNL2 knockdown groups (Fig. 2I). Conversely, overexpression of CCNL2 in HEY cells resulted in enhanced tumor growth and increased proliferation (Fig. 2J-L). These findings indicate that CCNL2 promotes the proliferation of xenograft tumors, supporting its potential role as a key regulator of tumor growth in ovarian cancer.

Knockdown of CCNL2 enhances the sensitivity of OC cells to cisplatin

To further investigate the role of CCNL2 in chemoresistance, we performed a series of experiments. First, we conducted a CCK8 cytotoxic assay to assess the effect of CCNL2 on cisplatin sensitivity and found that the knockdown of CCNL2 significantly lowered the IC50 of cisplatin in ovarian cancer cells (Fig. 3A), indicating an enhanced sensitivity to the drug. The IC50 of cisplatin in HEY cells was 4.83 µg/mL in the NC group, and was reduced to 2.79 µg/mL after CCNL2 knockdown. Similarly, in OV90 cells, the IC50 was 4.78 µg/mL in NC group and decreased to 2.95 µg/mL in shCCNL2 group. Next, we examined the apoptotic response to cisplatin treatment by flow cytometry and observed that the depletion of CCNL2 led to an increased apoptotic rate in OC cells after cisplatin exposure (Fig. 3B). This suggests that CCNL2 knockdown sensitized the cells to cisplatin-induced cell death. Additionally, we performed clonogenic assays, which demonstrated that the clonogenic ability of OC cells was significantly reduced following CCNL2 knockdown and cisplatin treatment, further supporting the role of CCNL2 in promoting chemoresistance (Fig. 3C).

Fig. 3.

Knockdown of CCNL2 enhances the sensitivity of OC cells to cisplatin. A) CCK8 assay was conducted to assess the effect of the CCNL2 depletion on chemosensitivity. OC cells were treated with various concentrations of cisplatin for 48 h (n = 3). B) Apoptotic cells were analyzed using Annexin V/PI staining in HEY and OV90 cells with cisplatin treatment (5 µg/ml, 48 h) (n = 3). The bar graphs on the right display the percentage of apoptotic cells. C) Clonogenic assay was performed with CCNL2 depletion under cisplatin treatment in OV90 cells. D-G) The image of subcutaneous tumors (D), tumor mass (E), IHC images of Ki-67 (F), and tumor growth curves (G) in CCNL2 knockdown and control groups with cisplatin treatment (4 mg/kg every three days) (n = 5 per group). Scale bar, 100 μm. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value.*P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

In vivo, we used tumor xenograft models to evaluate the impact of CCNL2 on cisplatin sensitivity. HEY cells with CCNL2 knockdown or control cells were treated with cisplatin. The results showed that CCNL2 knockdown significantly enhanced the therapeutic effect of cisplatin, as evidenced by a reduction in tumor growth and lower Ki-67 expression, a marker of cell proliferation (Fig. 3D-G). These findings suggest that CCNL2 plays a crucial role in promoting chemoresistance and that its depletion can increase the efficacy of cisplatin treatment in both in vitro and in vivo models.

YBX1 enhances CCNL2 mRNA stability via an m⁵C-dependent manner

To investigate the regulation mechanism of CCNL2, we performed an RNA pull-down assay followed by mass spectrometry analysis. Interestingly, YBX1, which has been reported in multiple studies to mediate tumor chemoresistance [23], was among the hub proteins pulled down by the full-length biotinylated-CCNL2 probe, as calculated by Cytoscape (Fig. 4A). Given its previously reported role in RNA stability regulation, we hypothesized that YBX1 might modulate CCNL2 expression. Subsequent validation assays demonstrated that knockdown of YBX1 significantly decreased the mRNA expression of CCNL2 in HEY and OV90 (Fig. 4B). Similar results were also observed in protein expression (Fig. 4C, S2A). Importantly, the depletion of YBX1 attenuated the increase of colony number caused by CCNL2 overexpression (Fig. 4D). Therefore, there may be a regulatory relationship between YBX1 and CCNL2, mediating chemotherapy resistance in ovarian cancer.

Fig. 4.

YBX1 enhances CCNL2 mRNA stability via an m⁵C-dependent manner. A) Hub proteins were screened by Cytoscape from proteins pulled by biotinylated-CCNL2 probe. B) Relative CCNL2 mRNA expression after YBX1 knockdown in HEY and OV90 cells (n = 3). C) Relative CCNL2 protein expression was analyzed with YBX1 knockdown in HEY and OV90 cells. D) Colony formation assay was conducted to assess the rescue effect of YBX1 on CCNL2 overexpression (n = 3). E) Correlation analysis of CCNL2 and m⁵C signatures in ovarian cancer tissues based on TCGA database. F) The half-life of CCNL2 mRNA was analyzed by RT-qPCR after YBX1 knockdown with Actinomycin D treatment in HEY and OV90 cells. G) Immunoblotting of YBX1 after RNA pull-down assays with full-length biotinylated-CCNL2 probe and negative RNA probe. H) RIP assays showing the interaction between YBX1 and CCNL2 mRNA (n = 3). The agarose gel electrophoresis results above showed the results of RT-PCR. The NC lane meant negative control without the template. The IgG and Anti-YBX1 lanes used products pulled by IgG or anti-YBX1 antibodies separately. The input lane used input as the template. I) MeRIP assays were conducted after YBX1 knockdown. Primers used in the Negative Control group were designed for non-m⁵C modification sites, while primers used in the other two groups were designed for the m⁵C modification sites (n = 3). J) Relative luciferase activity of CCNL2-WT or CCNL2-mut in YBX1 overexpression or control cells (n = 3). K) Relative luciferase activity of CCNL2-WT was detected after YBX1 overexpression or W65A mutation (n = 3). All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. The relative enrichment of CCNL2 in the RIP assay was normalized by input. The fold enrichment of CCNL2 in the MeRIP assay was normalized by input and IgG

Traditionally, YBX1 has been viewed as a transcription factor regulating tumor drug resistance [23]. However, recent findings have unveiled its new role as an m⁵C methylation reader that maintains mRNA stability [16]. Consistent with this notion, KEGG enrichment analysis showed that CCNL2 RNA-interacting proteins were enriched in RNA degradation (Figure S2B). These results indicate that YBX1 may regulate CCNL2 through modulating mRNA stability rather than transcription. To elucidate how YBX1 regulates CCNL2 expression, we performed a dual-luciferase assay (Figure S2D). The results showed that YBX1 did not directly modulate the transcription of CCNL2. Instead, we found a positive correlation between CCNL2 and m⁵C regulators other than ‘Erasers’, suggesting that m⁵C methylation is involved in the regulation of CCNL2 (Fig. 4E). In addition, YBX1 had the highest mutation rate in ovarian cancer among all m⁵C regulators (Figure S2C). Therefore, we speculate that YBX1 regulates CCNL2 via an m⁵C manner and plays an important role in ovarian cancer.

To directly assess whether YBX1 stabilizes CCNL2 mRNA, we performed an RNA stability assay using actinomycin D. Importantly, the half-life of CCNL2 mRNA decreased after YBX1 depletion in HEY and OV90 (Fig. 4F). Next, we validated the direct interaction between YBX1 and CCNL2 mRNA using RNA pull-down assay followed by western blot analysis, which confirmed that YBX1 specifically binds to CCNL2 mRNA in both cell lines (Fig. 4G). RNA immunoprecipitation assay (RIP) was also conducted to verify that YBX1 is predominantly bound to CCNL2 mRNA (Fig. 4H).

To confirm that YBX1 regulates CCNL2 in an m⁵C manner, the methylated RNA immunoprecipitation assay (MeRIP) was subsequently performed after the knockdown of YBX1. The results confirmed the existence of m⁵C modification sites and that the level of m⁵C methylation decreased after YBX1 depletion (Fig. 4I). Generally, 3’UTR is enriched with m⁵C modifications. So we constructed the firefly luciferase (Fluc) reporter plasmids containing either the wild type CCNL2 3’UTR or 3’UTR with m⁵C sites mutation. The results showed that the luciferase activity of the wild-type CCNL2 reporter increased with YBX1 overexpression. However, the increase in luciferase activity was attenuated by mutations of CCNL2 m⁵C sites (Fig. 4J), indicating that YBX1-mediated regulation of CCNL2 relies on m⁵C modifications.

Previous studies have identified the W65 residue as a key site for YBX1’s m⁵C recognition function [16], To determine whether this residue is crucial for YBX1-mediated CCNL2 stabilization, we constructed a YBX1 mutant (W65A) and assessed its impact on CCNL2 regulation. The result showed that YBX1 overexpression-induced increase in luciferase activity was attenuated by the mutation in the W65 site (Fig. 4K). Collectively, these findings demonstrate that YBX1 stabilizes CCNL2 mRNA through an m⁵C-dependent mechanism, thereby potentially contributing to ovarian cancer chemoresistance.

YBX1 cooperates with MATR3 to maintain the stability of CCNL2 mRNA

It is worth noting that MATR3, like YBX1, is also a hub protein among RNA pull-down products (Fig. 4A). MATR3, an RNA-binding protein, was reported to interact with m⁶A reader proteins to maintain mRNA stability [24]. To explore the potential interaction between MATR3 and YBX1, we conducted a co-immunoprecipitation (CO-IP) assay using anti-MATR3 and anti-YBX1 antibodies. The results confirmed that MATR3 physically interacts with YBX1 in vitro (Fig. 5A), suggesting a functional relationship between these two proteins.

Fig. 5.

YBX1 cooperates with MATR3 to maintain the stability of CCNL2 mRNA. A) CO-IP assay demonstrated the interaction between YBX1 and MATR3. B) CCNL2 protein expression was measured with MATR3 depletion by western blot in HEY and OV90 cells. C) The half-life of CCNL2 mRNA was analyzed by RT-qPCR with MATR3 knockdown in OV90 cells with actinomycin D treatment (n = 3). D) CCNL2 mRNA expression was analyzed in indicated HEY cells (n = 3). E) The half-life of CCNL2 mRNA was analyzed by RT-qPCR in indicate OV90 cells with actinomycin D treatment (n = 3). F, G) CCNL2 protein expression was measured in indicated HEY and OV90 cells. H) Colony formation assay was conducted to investigate the regulation mechanism of YBX1-MATR3-CCNL2 axis. I) CCK8 assay confirmed the regulation mechanism of YBX1-MATR3-CCNL2 axis in HEY cells. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

Given the role of YBX1 in regulating CCNL2 expression, we next investigated whether MATR3 similarly influences CCNL2 levels. Western blot analysis revealed that MATR3 knockdown significantly reduced CCNL2 protein expression in HEY and OV90 cells, mimicking the effect of YBX1 depletion (Fig. 5B). To determine whether this regulation occurs at the post-transcriptional level, we performed an RNA stability assay using actinomycin D. The results demonstrated that the mRNA stability of CCNL2 was markedly decreased upon MATR3 knockdown (Fig. 5C), further supporting its role in stabilizing CCNL2 transcripts.

Furthermore, we examined the interplay between YBX1 and MATR3 in regulating CCNL2 expression. MATR3 knockdown exacerbated the reduction in CCNL2 protein levels induced by YBX1 depletion (Fig. 5F). Conversely, when YBX1 was overexpressed, MATR3 knockdown effectively reversed the increase in CCNL2 expression and stability (Fig. 5D, E, G). To further validate the regulatory relationship among YBX1, MATR3, and CCNL2, we conducted rescue experiments using the CCK8 assay and colony formation assay in HEY cells. Overexpression of YBX1 enhanced the proliferation of HEY cells, while MATR3 knockdown decreased. Furthermore, MATR3 was silenced in the context of YBX1 overexpression, and the proliferative effect induced by YBX1 overexpression was greatly diminished. Subsequent re-expression of CCNL2 restored the proliferation capacity of HEY cells under these conditions, indicating that MATR3 is indispensable for YBX1 to maintain CCNL2 expression, thereby promoting the proliferation of OC cells (Fig. 5H, I). These findings indicate that YBX1 and MATR3 function cooperatively to regulate CCNL2 expression, likely through a shared mechanism that enhances CCNL2 mRNA stability.

Identification of a small molecule YB-B1 which enhances chemotherapy sensitivity

Due to the lack of CCNL2 protein structure information and the clear role of YBX1 in chemoresistance, we decided to develop a small molecule inhibitor targeting YBX1. The overall workflow is shown in Fig. 6A. 71,495 compounds were screened with virtual screening based on the YBX1 protein structure. YB-B1’s docking score ranked among the top. The molecular docking showed that YB-B1 formed hydrogen bonds with Glu82 and Ala120 of YBX1 (Fig. 6B).

Fig. 6.

Identification of a small molecule YB-B1 which enhances chemotherapy sensitivity. A) Schematic diagram of virtual screening for YBX1 inhibitors. B) Chemical structure of YB-B1 and its docking with the YBX1 protein. C, D) SPR assay was performed to evaluate the affinity between YB-B1 and YBX1 human protein. RU, resonance unit. E) Western blot analyses were performed to evaluate the effects of YB-B1 treatment over the indicated time in HEY and OV90 cells. F) The line chart illustrating the time-dependent trends of CCNL2 and YBX1 protein expression during YB-B1 treatment. G) Dose-response curve for YB-B1 in HEY and OV90 cells after 48 h of treatment (n = 3). H) Clonogenic assay with YB-B1 treatment in HEY and OV90 cells (n = 3). I) Cell viability was assessed in OV90 cells after 48 h with cisplatin and YB-B1 treatment (n = 3). J, K) Apoptotic cells were analyzed using Annexin V/PI staining in OV90 cells with cisplatin and YB-B1 treatment for 48 h (n = 3). L-O) The PDX model was constructed using fresh ovarian cancer tissues and divided into Cisplatin and Cisplatin + YB-B1 groups for corresponding treatments. The image of subcutaneous tumors (L), tumor mass (M), and IHC images of Ki-67 (N) in Cisplatin and Cisplatin + YB-B1 groups (n = 5 per group). Scale bar, 50 μm. Quantified data are shown on the right. All quantification analyses were based on three independent experiments. Data are mean ± SD. Student’s unpaired t-test was used to calculate the p value. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

To further validate this interaction, we conducted a surface plasmon resonance (SPR) assay, which showed that YB-B1 bound to YBX1 protein with a Dissociation Constant (KD) of 5.67 µM, confirming its direct affinity for YBX1 (Fig. 6C, D). The Western blot results indicated that YB-B1 decreased the expression of CCNL2 and YBX1 over time. However, a slight rebound in expression was observed at 36 h in HEY cells, potentially due to transient drug resistance or compensatory mechanisms (Fig. 6E, F). Notably, the DMSO control group exhibited no significant changes, indicating that the observed protein reduction was specifically induced by YB-B1 rather than natural protein degradation (Figure S3A).

We further assessed the functional impact of YB-B1 on OC cells. CCK8 and colony formation assays revealed that YB-B1 significantly suppressed the proliferation of HEY and OV90 cells (Fig. 6G, H). More importantly, YB-B1 exhibited a synergistic effect with cisplatin, as evidenced by a greater reduction in cell viability (Fig. 6I) and an increased apoptosis rate in the combination treatment group, as determined by flow cytometry (Fig. 6J, K).

To extend our findings in vivo, we established the PDX model using fresh ovarian cancer tissues to explore the chemosensitizing effect of YB-B1. The results demonstrated that tumors in the YB-B1 + Cisplatin group were significantly smaller in both volume and weight compared to the Cisplatin-alone group, indicating that YB-B1 enhances the cytotoxic efficacy of cisplatin (Fig. 6L, M). Consistently, immunohistochemical analysis showed that Ki-67 expression was markedly lower in the YB-B1 + Cisplatin group than in the Cisplatin group (Fig. 6N), further supporting the potent anti-tumor effects of this combination therapy.

Discussion

Several studies have explored the association between CCNL2 and HIV replication. A previous study showed that CCNL2 interacted with HIV restriction factor, SAMHD1, and supported HIV replication [9]. However, fewer studies have investigated its relationship with tumors. CCNL2 has been identified as a key player in RNA processing [6], particularly in the regulation of alternative splicing and transcription. So it may have a significant impact on the transcription levels of tumor cells. In our present study, we demonstrated that CCNL2 was significantly upregulated in OC and correlated with a poor prognosis. Furthermore, we observed that CCNL2 expression level increased with higher tumor grades. Subsequent experiments showed that ectopic expression of CCNL2 enhanced the proliferation and reduced the chemosensitivity of OC. These findings suggest that CCNL2 is involved in both tumor growth and chemoresistance in OC.

m⁵C is widespread in mRNA, tRNA, rRNA, and ncRNA [25], playing a critical role in post-transcriptional regulation. Among these, m⁵C methylation on mRNA has been reported to influence multiple aspects of RNA metabolism, including nuclear export [26], stability, and translation efficiency. Abnormal mRNA m⁵C methylation has been shown to contribute to the development of various cancers, such as bladder cancer [16], gastric cancer, and esophageal squamous cell carcinoma. In our study, we revealed a positive correlation between CCNL2 and m⁵C regulators other than ‘Erasers’, suggesting that m⁵C methylation may regulate CCNL2 expression. We further discovered that the m⁵C reader protein YBX1 regulated the RNA stability of CCNL2. Knockdown of YBX1 led to a decrease in m⁵C methylation level of CCNL2. Furthermore, mutation of the key site required for YBX1’s methylation function resulted in reduced CCNL2 expression. These findings suggest that YBX1 regulates CCNL2 expression through m⁵C methylation.

RNA-binding proteins normally regulate downstream networks in the form of complexes. YBX1 frequently recruits other RNA-binding proteins to exert its regulatory functions. Previous studies showed that YBX1 recruited RNA-binding proteins, such as ELAVL1 and PABPC1, to maintain mRNA stability [16, 27]. IGF2BPs, as m6A readers similar to YBX1, also require the assistance of other proteins, such as ELAVL1, MATR3, and PABPC1, to perform its RNA stability function [28]. In our study, RNA pull-down and CO-IP assays identified MATR3 as one of the CCNL2 mRNA-binding proteins that interacts with YBX1. Knockdown of MATR3 led to a decrease in both the mRNA stability and expression level of CCNL2. Furthermore, MATR3 depletion reversed the increase in CCNL2 expression and stability induced by YBX1 overexpression. These findings suggest that MATR3 plays a critical role in modulating CCNL2 stability and expression, potentially in collaboration with YBX1 (Fig. 7).

Fig. 7.

Graphic abstract of the role of CCNL2 in ferroptosis and its regulatory mechanism. YBX1 cooperates with MATR3 to regulate CCNL2, driving OC cells into the ferrotosis resistance state. LIP referes to labile iron pool

Recently, the prognosis of OC patients has improved due to the emergence of targeted therapies, such as PARP inhibitors [29]. However, due to the high heterogeneity of OC and its tendency to develop chemoresistance, further research is still needed to elucidate the mechanism of chemoresistance and to develop new targeted drugs to overcome resistance. In our study, we discovered new chemoresistance pathways, screened small-molecule compounds, and explored new combination strategies of OC with cisplatin. Several studies have investigated the development of small molecule inhibitors for YBX1. Ye et al. found sciadopitysin through virtual screening as an inhibitor of YBX1, which subsequently increased YBX1 expression and attenuated age-related bone loss [30]. Moreover, another study found that HSc025, a YBX1 inhibitor, can alleviate hepatic fibrosis by promoting the nuclear translocation of YB-1 [31]. However, their clinical application has not primarily targeted chemotherapy resistance. In our study, we identified a small molecule inhibitor, YB-B1, through virtual screening targeting YBX1-CCNL2 axis. YB-B1 effectively suppressed OC cell proliferation and, importantly, enhanced the cytotoxicity of cisplatin when used in combination. These results highlight the therapeutic potential of disrupting the YBX1–CCNL2 axis in OC treatment. Nevertheless, the precise mechanisms underlying the chemosensitizing effect of YB-B1 remain to be fully elucidated. In particular, the involvement of CCNL2 and its regulation via m⁵C RNA methylation needs further investigation.

Conclusion

In conclusion, our study found that CCNL2 was upregulated in OC tissues and correlated with poor prognosis. Knockdown of CCNL2 suppressed OC cell proliferation and significantly enhanced cisplatin sensitivity. YBX1 recruited MATR3 to regulate CCNL2 expression and maintain its mRNA stability. This regulation was achieved through an m⁵C-dependent mechanism, where YBX1 directly bound to CCNL2 mRNA and facilitated its m⁵C modification, while MATR3 acted as a co-regulator to stabilize the modified transcript. YB-B1, identified through virtual screening, inhibits OC cell proliferation and enhances cisplatin sensitivity. The PDX model constructed using clinical tissues also demonstrated that the small-molecule drug YB-B1 enhanced the efficacy of cisplatin. The PDX model constructed using clinical OC tissues also demonstrated that the small-molecule drug YB-B1 enhanced the efficacy of cisplatin. These results explain the role of CCNL2 in chemoresistance and propose a novel approach to address chemoresistance in ovarian cancer.

Electronic supplementary material

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Abbreviations

CCNL2

Cyclin L2

CO-IP

Co-immunoprecipitation assay

IHC

Immunohistochemistry

KD

Dissociation constant

MeRIP

Methylated RNA immunoprecipitation assay

m⁵C

5-methylcytosine

OC

Ovarian cancer

OS

Overall survival

PFS

Progression-free survival

RIP

RNA Immunoprecipitation assay

SOC

Serous ovarian cancer

SPR

Surface plasmon resonance

YBX1

Y-Box‐binding protein 1

Author contributions

KZ and XL designed the study and drafted the manuscript; KZ, GC and WJ contributed to the acquisition of experimental data. SY and BK analyzed data and revised the manuscript. All authors have read and agreed to the final version of the manuscript.

Funding

This study was supported by the Postdoctoral Innovation Program of Shandong Province, China (grant number: SDBX2022007) and Natural Science Foundation of Shandong Province (approval number ZR2022QH035).

Data availability

The raw RNA sequencing data are available in the GEO database under accession number GSE279620. Additional data supporting the experimental results are provided in the supplementary materials.

Declarations

Ethics approval and consent to participate

Ethical approval was obtained from the ethics committee of Qilu Hospital, Shandong University (KYLL-202210-052-1). All animal experiments were conducted with approval from the Animal Care and Use Committee of Qilu Hospital, Shandong University (Dwll-2023-120).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.