Long non-coding RNA RAD51-AS1 promotes the tumorigenesis of ovarian cancer by elevating EIF5A2 expression

Purpose The present study aims to determine the molecular mechanism mediated by RAD51 antisense RNA 1 (RAD51-AS1) in ovarian cancer (OvCA). Methods The data associated with RAD51-AS1 in OvCA were obtained from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. Relative expression of RAD51-AS1 was detected. Determination of cell proliferation, metastasis, and invasion was performed by cell counting, colony formation, would-healing, and transwell invasion assays. Protein levels were detected by western blotting. The molecular mechanism mediated by RAD51-AS1 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. Subcutaneous tumorigenesis models were used to confirm the function of RAD51-AS1 in vivo. Results Data from TCGA and GEO showed that RAD51-AS1 was associated with poor prognosis in OvCA patients and DNA repair, cell cycle, focal adhesion, and apoptosis in SKOV3.ip cells. High levels of RAD51-AS1 were detected in OvCA cells. Overexpressing RAD51-AS1 enhanced the proliferative, invading, and migratory capabilities of OvCA cells in vitro while silencing RAD51-AS1 exhibited the opposite effects. Mechanically, RAD51-AS1 elevated eukaryotic initiation factor 5A2 (EIF5A2) expression as a sponge for microRNA (miR)-140-3p. Finally, the role of RAD51-AS1 was verified by subcutaneous tumorigenesis models. Conclusion RAD51-AS1 promoted OvCA progression by the regulation of the miR-140-3p/EIF5A2 axis, which illustrated the potential therapeutic target for OvCA. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-024-05671-z.


Introduction
Ovarian cancer (OvCA) ranks eleventh and fifth among common cancers and related deaths in female patients, respectively (Morand et al. 2021).Approximately 140,000 women die from OvCA worldwide every year (Penny 2020).The main treatment method for patients with OvCA is to remove all macroscopic diseases through cytoreductive surgery (Feng et al. 2018;Ayhan and Akilli 2021).Approximately 80% of OvCA patients receive treatment regimens of cytoreductive surgery combined with adjuvant chemotherapy (Baci et al. 2020).However, about 70% of patients who adopt this treatment regimen relapse again, and recurrent patients typically have resistance to standard platinum chemotherapy (Nowak and Klink 2020).For patients in advanced stages, only about 29% of patients have a 5-year survival time (Siegel et al. 2019).More molecular changes in the population of OvCA patients have been identified, and exploring these abnormal molecules will have significant implications for exploring new diagnostic and therapeutic targets for OvCA patients (Guan and Lu 2018;Norouzi-Barough et al. 2018).
The largest group of non-coding RNAs (ncRNAs) produced in the genome is long non-coding RNAs (lncRNAs) (Robinson et al. 2020).The key feature of these transcripts is that they can not encode proteins and exceed 200 nucleotides in length (Bridges et al. 2021).Mechanically, lncRNAs exert their regulatory effects on various cancer-related cellular processes through communication with proteins, ncR-NAs, mRNAs, and DNA by serving as different functional molecules, such as decoys and scaffolds (Ørom et al. 2010;Winkle et al. 2021).For instance, tumorigenic lncRNA on chromosome 1p13 could lead to the downregulation of some tumor suppressors by interacting with TPR in liver cancer (Yuan et al. 2022).LncRNA KCNQ1 opposite strand/antisense transcript 1 exerted a tumor-promoting function in OvCA by decreasing eukaryotic translation initiation factor 2B (EIF2B5) expression by recruiting DNA methyltransferases into EIF2B5 promoter (He et al. 2022a, b).In colorectal cancer, NOTCH1-associated lncRNA in T cell acute lymphoblastic leukemia 1 strengthened the proliferative and migrating capacities of the tumor cells by sponging micro-RNA (miR)-574-5p (Ye et al. 2022).LncRNA RAD51 antisense RNA 1 (RAD51-AS1) located on 15q15.1 has been reported to improve the effectiveness of chemo/radiotherapy for cancer cells in liver cancer (Chen et al. 2018a, b) and repress cancer cell growth in colorectal cancer (Chen et al. 2018a, b;Li et al. 2021a, b).On the contrary, RAD51-AS1 could promote the growth of tumor cells in breast cancer (Gazy et al. 2015) and OvCA (Zhang et al. 2017), but to date still little is known about molecular mechanisms by which RAD51-AS modulates OvCA progression.
The eukaryotic initiation factor 5A2 (EIF5A2) gene located on chromosome 3q26 was first discovered in a primary OvCA cell line (Guan et al. 2001).Significant upregulation of EIF5A2 was verified in prostate cancer, and interference with EIF5A2 in PC-3 M IE8 cells decreased cell growth and lung metastasis in mouse models.The ubiquitination of EIF5A2 mediated by HERC3 repressed tumor cell invasion and migration in colorectal cancer (Zhang et al. 2022).Inhibition of EIF5A2 lowered hypoxia-induced cisplatin resistance by repressing hypoxia-induced autophagy in lung cancer (Xu et al. 2022).More importantly, the oncogenic role of EIF5A2 was also confirmed in OvCA (Wang et al. 2021;Zhao et al. 2021).A previous study uncovered the downregulation of EIF5A2 in RAD51-AS1inhibiting SKOV3.ip cells (Zhang et al. 2017).However, whether RAD51-AS can regulate OvCA progression through EIF5A2 remain unknown.
Here, we aimed to investigate the molecular mechanisms related to RAD51-AS1 and EIF5A2 in OvCA.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used for the isolation of total RNA from cells.For the detection of RAD51-AS1 and EIF5A2, the complementary (c) DNA was obtained using the First Strand cDNA Synthesis Kit (Takara, Japan).For miR-140-3p detection, total RNA underwent reverse transcription for cDNA production in the light of the instructions provided by the miRNA First Strand cDNA Synthesis kit (Sangon, Shanghai, China).The reaction was about qPCR being run using real-time fluorescence quantitative PCR instruments (ABI 7500, Applied Biosystems, Foster City, CA, USA) with SYBR Green Master Mix (Takara).All primers were provided in Table 1.Relative expression of RAD51-AS1, EIF5A2 and miR-140-3p was calculated using the 2 −ΔΔCt method by normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 expression.

Wound-healing assay
In short, transfected OvCA cells (1 × 10 5 /wells) were seeded in 6-well dishes.After reaching about 80-90% confluence, cells were scratched from the bottom of the wells with a sterile 200 uL pipette tip.Capturing images and measuring were performed at 0 and 24 h after the wound was created.

Transwell invasion assay
The serum-free medium (500 μL) containing 1 × 10 5 OvCA cells was added into the upper chamber (8.0 μm, Costar, Cambridge, MA, USA) pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA).The DMEM medium containing 10% FBS was added into the bottom chamber.After 24 h of incubation, the cells in the bottom chamber were stained with 0.1% crystal violet (Beyotime).Counting of the number of invading cells was performed under a light microscope (Olympus).

Dual-luciferase reporter assay
For the evaluation of the relationship between RAD51-AS1 and EIF5A2, the EIF5A2 promoter region was inserted into a pGL3-basic luciferase vector (Promega).The fragments containing the binding sites for miR-140-3p in RAD51-AS1 or 3'UTR of EIF5A2 were cloned into pmirGLO vector separately and named RAD51-AS1 WT and EIF5A2 WT.Generation of the mutated plasmids (RAD51-AS1 MUT and EIF5A2 MUT) was achieved by mutating the putative binding sites in RAD51-AS1 or 3'UTR of EIF5A2.OvCA cells were co-transfected with miR-140-3p inhibitor or NC inhibitor and a luciferase reporter.Forty-eight hours posttransfection, all luciferase activities were then assessed by the Dual-Luciferase Reporter Assay System (Promega).

Subcutaneous tumorigenesis model
The in vivo experiments were approved and manipulated based on protocols approved by the Animal Care Committee of the Second Affiliated Hospital of Nanchang University.Eighteen female BALB/c nude mice (Animal Experimental Science Center of Nanchang University, Nanchang, China) were randomly divided into three groups: control (n = 6), sh-NC (n = 6), and sh2-RAD51-AS1 (n = 6).All mice were fed at 25 ± 2 °C (45-50% constant humidity).SKOV3 cells or SKOV3 cells with sh-NC or sh2-RAD51-AS1 (1 × 10 7 ) were subcutaneously injected into the right hindlimb of each mouse.Tumor volume was measured once a week by measuring their length (l) and width (w) and calculating the volume (V) as follows: V = l × w 2 /2.Four weeks post-injection, all mice were euthanized, followed by measurement and weighing of excised tumors.Paraffin-embedded tumor samples were subjected to immunohistochemistry (IHC) staining to detect EIF5A2-positive cells.

Statistical analysis
All experiments in vitro were performed at least triplicate biological replicates.Data analysis was implemented by GraphPad Prism 8 (GraphPad Software, USA) and presented as the mean ± SEM (standard error of the mean).
For the comparison between the two groups, the difference between normal distribution variables was calculated using an independent Student's t-test, and the difference between non-normal distribution variables was calculated using Mann-Whitney U-test.Comparison of multiple groups was made by analysis of variance with Tukey's correction.Significance was defined as any statistical result that resulted in a p-value less than 0.05.

RAD51-AS1 was associated with poor prognosis in OvCA patients and DNA repair, cell cycle, focal adhesion, and apoptosis in SKOV3.ip cells
Relative RAD51-AS1 expression in OvCA cell lines was first estimated by RT-qPCR.Higher levels of RAD51-AS1 in SKOV3 and OVCAR3 cell lines were observed in comparison with the HOSEpiC cell line (Fig. 1A).In the TCGA cohort, the area under the curve (AUC) for OvCA diagnosis was 0.975 (CI: 0.962-0.988) in the analysis of the receiver operating characteristics (ROC) curve of RAD51-AS1, suggesting that RAD51-AS1 might serve as an improved diagnostic biomarker for OvCA (Fig. 1B).Furthermore, OvCA patients with low RAD51-AS1 expression survived longer in the TCGA cohort (Fig. 1C).
For the bioinformatics analysis on the GSE89374 dataset, the box plot results showed that the expression levels in different datasets were not on the same horizontal line, so we performed normalization to remove batch effects (Fig. 1D).To distinguish significant differences between normal cells and RAD51-AS1-knockdown cells, principal component analysis was performed to reduce dimensionality and assess the independence of each group.The results showed that there were significant differences between SKOV3.ip cells and RAD51-AS1-knockdown SKOV3.ip cells (Fig. 1E).Moreover, the gene set enrichment analysis (GSEA) indicated that RAD51-AS1 was involved in four biologic processes (DNA repair, regulation of DNA repair, regulation of cell cycle phage transition, and positive regulation of execution phase of APOP1) and 3 pathways (cell cycle, focal adhesion and apoptosis) (Fig. 1F andG).These bioinformatics analyses collectively suggested that high RAD51-AS1 expression was associated with poor prognosis in OvCA patients, and RAD51-AS1 might be involved in DNA repair, cell cycle, focal adhesion, and apoptosis of OvCA cells.

Inhibition of RAD51-AS1 diminished SKOV3 cell proliferation, migration, invasion, and EMT
Next, we established RAD51-AS1-knockdown SKOV3 cells for the assessment of the biological function of RAD51-AS1, and the knockdown efficiency was exhibited in Figure S1B.Contrary to the overexpression of RAD51-AS1, RAD51-AS1 silencing markedly decreased the proliferation, colony formation, migration and invasion of SKOV3 cells (Fig. 3A-D).Consistently, PCNA, cyclin D1, CDK4, N-cadherin, and Vimentin protein levels in SKOV3 cells were downregulated, whereas c-caspase 3 and E-cadherin protein levels were upregulated after knocking down RAD51-AS1 (Fig. 2E and F).These results supported that RAD51-AS1 might be required for SKOV3 cell proliferation, migration, invasion, and EMT.

RAD51-AS1 mediated SKOV3 cell proliferation, migration, invasion, and EMT via regulation of EIF5A2 expression
To explore the molecular mechanism by which RAD51-AS1 promotes OvCA progression, we screened for significant differential expressed genes between OvCA cells with and without RAD51-AS1 knockdown and plotted the results into a volcano plot (p < 0.05, |fold change|> 2) based on GSE89374 dataset.Among them, EIF5A2 with a significant downregulation differential change attracted our attention (Fig. 4A).Western blotting showed that EIF5A2 protein levels were markedly upregulated in RAD51-AS1-overexpressing OVCAR3 cells and downregulated in RAD51-AS1inhibiting SKOV3 cells, implying that RAD51-AS1 positively regulated EIF5A2 expression in OvCA cells (Fig. 4B).Rescue experiments were then conducted in SKOV3 cells to determine whether RAD51-AS1 promotes OvCA progression through EIF5A2.We constructed an EIF5A2 overexpression plasmid in SKOV3 cells and observed that the downregulation of EIF5A2 caused by RAD51-AS1 silencing was weakened following the EIF5A2 introduction (Fig. 4C).Meanwhile, EIF5A2 promoted SKOV3 cell proliferation, colony formation, migration, and invasion, and the introduction of EIF5A2 disminished the inhibitory effects of RAD51-AS1 silencing on the malignant behaviors of SKOV3 cells (Fig. 4D-G).Correspondingly, the changes in protein levels of PCNA, cyclin D1, CDK4, c-caspase 3, E-cadherin N-cadherin, and Vimentin mediated by EIF5A2 knockdown were weakened after overexpression of EIF5A2 (Fig. 4G and H).Together, RAD51-AS1 modulated the malignant behaviors of OvCA cells through EIF5A2.

RAD51-AS1 modulated EIF5A2 expression by interacting with miR-140-3p
To evaluate whether RAD51-AS1 directly interacts with EIF5A2, we constructed a dual-luciferase reporter plasmid containing the EIF5A2 promoter.As shown in Fig. 5A and  B, either overexpression or knockdown of RAD51-AS1 did not have a significant effect on the fluorescence activity of the plasmid, implying that EIF5A2 expression was indirectly regulated by RAD51-AS1.SKOV3 cells with silenced RAD51-AS1 or OVCAR3 cells with overexpression of RAD51-AS1 were incubated with actinomycin D and then subjected to analyzing the expression of EIF5A2.RT-qPCR results manifested that RAD51-AS1 upregulation prolonged the half-life of EIF5A2 mRNA while RAD51-AS1 silencing reduced the half-life of EIF5A2 mRNA (Fig. 5C  and D).Accumulative evidence has indicated that lncRNAs can function as miRNA sponges.Therefore, we predicted miRNAs bound to RAD51-AS1 or EIF5A2 using ENCORI.Two miRNAs (miR-154-5p and miR-140-3p) that interact with them collectively were obtained through Venn analysis, and miR-140-3p downregulated in OvCA was selected for further analysis (Fig. 5E).Interestingly, miR-140-3p inhibitor markedly elevated the luciferase activity in OVCAR3 cells of the RAD51-AS1 WT and EIF5A2 WT groups, but there were no changes in the RAD51-AS1-MUT and EIF5A2 MUT groups (Fig. 5F and G).We also observed the downregulation of miR-140-3p in RAD51-AS1-overexpressing OVCAR3 cells and the upregulation of miR-140-3p in RAD51-AS1-knockdown SKOV3 cells (Fig. 5H and I).In addition, the miR-140-3p inhibitor increased EIF5A2 expression at mRNA and protein levels in OVCAR3 cells (Fig. 5J).On the contrary, the miR-140-3p mimic resulted in a significant reduction of EIF5A2 in SKOV3 cells (Fig. 5K).All results manifested that RAD51-AS1 functioned as a miR-140-3p sponge and regulated EIF5A2 expression by sponging miR-140-3p.

RAD51-AS1 silencing decreased SKOV3 cell growth in vivo
Subcutaneous tumorigenesis models were then established by subcutaneous injection of SKOV3 cells or SKOV3 cells infected with lentivirus-mediated sh2-RAD51-AS1 or sh-NC.The tumor size, growth rate, and weight of tumors derived from cells with RAD51-AS1 knockdown were significantly lower than those of the control and sh-NC groups (Fig. 6A-C).Tumors derived from cells with RAD51-AS1 knockdown showed lower levels of RAD51-AS1 and EIF5A2 mRNA than those in the control and sh-NC groups, but miR-140-3p expression was higher (Fig. 6D).IHC staining showed that the EIF5A2 index in tumors derived from cells with RAD51-AS1 knockdown was significantly lower than that in the other 2 groups (Fig. 6E).What is more, the diminished protein levels of EIF5A2, Ki67, Bcl-2, cyclin D1, and N-cadherin were observed in tumors of the sh2-RAD51-AS1 group in comparison to the other 2 groups, whereas Bax and E-cadherin protein levels were elevated (Fig. 6E).In summary, RAD51-AS1 contributed to the growth of SKOV3 cells in nude mice.
Previous studies have reported the different functions of RAD51-AS1 in different tumors.The elevation of RAD51-AS1 was uncovered to repress cell invasion, glycolysis process, and migration by mediating NDRG2 expression via sequestering miR-29b/c-3p, leading to the reduction of colorectal cancer progression (Li et al. 2021a, b).Similarly, the upregulation of RAD51-AS1 enhanced cell sensitivity to chemotherapy by binding to RAD51 mRNA and repressing its translation in liver cancer (Chen et al. 2018a, b;Chen et al. 2018a, b).Oppositely, RAD51-AS1 was overexpressed in OvCA samples and correlated with poor survival in epithelial OvCA patients, and p53 was repressed by RAD51-AS1, leading to the promotion of cell proliferation (Zhang et al. 2017).In our study, bioinformatics analysis showed the diagnostic and prognostic value of RAD51-AS1 for OvCA and suggested that RAD51-AS1 was related to DNA repair, cell cycle, focal adhesion, and apoptosis in OvCA cells.Our data showed the overexpression of RAD51-AS1 in OvCA cells, and further function analyses showed the contributory effect of RAD51-AS1 on cell malignancy in OvCA cells in vitro, including proliferative, invasive, migratory, and EMT, as well as tumor growth in mouse models, supporting the oncogenic role of RAD51-AS1 in OvCA.
Based on bioinformatics analysis of differentially expressed genes in OvCA cells caused by RAD51-AS1 knockdown, we discovered that RAD51-AS1 might regulate EIF5A2 expression in OvCA cells.EIF5A2 has been revealed as a new independent predictor of outcomes in OvCA patients (Yang et al. 2009).The contributory effect of EIF5A2 on OvCA cell stemness was caused by its mediation of the E2F1/KLF4 pathway (Wang et al. 2021).In addition, EIF5A2 enhanced tumor growth by facilitating the EMT process via the TGF-β pathway (Zhao et al. 2021).Here, further experimental results confirmed the negative regulation of EIF5A2 in OvCA cells by RAD51-AS1.Cell biological experiments further showed that EIF5A2 overexpression blocked the inhibiting effect of RAD51-AS1 silencing on OvCA cell proliferation, invasion, migration, and EMT, indicating that RAD51-AS1 modulated the malignancy of OvCA cells through EIF5A2.
For exploring the regulatory mechanism between RAD51-AS1 and EIF5A2, the luciferase reporter plasmid with EIF5A2 promoter did not show significant differences in OvCA cells with overexpression or knockdown of RAD51-AS1, suggesting that RAD51-AS1 could not directly bind and interact with EIF5A2.Much evidence suggests that lncRNAs act as competitive endogenous RNAs by competitively sponging and sequestering miRs, thereby eliminating miRs-mediated inhibition of their target mRNAs and indirectly regulating the expression of mRNAs (Tay et al. 2014;Zhou et al. 2022).MiRs are very powerful genetic regulators, as evidenced by the fact that a single miR can interact with a broad spectrum of target genes and direct entire cellular pathways (Diener et al. 2022).Herein, miR-140-3p was predicted to interact with RAD51-AS1 and EIF5A2 jointly by ENCORI.The downregulation and tumor inhibitory effect of miR-140-3p has been confirmed in a broad spectrum of cancers, such as bladder cancer (Yang et al. 2022), prostate cancer (He et al. 2022a, b), lung cancer (Huang et al. 2019), breast cancer (Dou et al. 2021) and gastric cancer (You et al. 2023).In OvCA, lncRNA AC005224.4-mediatedupregulation of SNAI2 elevated cancer cell invasion and migration by sponging miR-140-3p (Xiong et al. 2023).Moreover, linc00852 sequestered miR-140-3p to elevate AGTR1 expression, ultimately contributing to the invading and proliferative capacities of OvCA cells (Qiao et al. 2021).More interestingly, we illustrated that RAD51-AS1 had a negative regulation on miR-140-3p and EIF5A2 was a miR-140-3p target by dual-luciferase reporter assays, RT-qPCR and western blotting.Our research results proposed a regulatory model in which high RAD51-AS1 expression sponged miR-140-3p to elevate EIF5A2 expression.Unfortunately, the risk model construction in the research was mainly based on the TCGA (OvCA cohort), so it is best to obtain sufficient clinical samples and data to verify the diagnostic and prognostic value of RAD51-AS1 in OvCA.What's more, whether RAD51-AS1 plays a role in OvCA through other functions still needs to be explored, which is also worth exploring in future.
In summary, this work identified the oncogenic role of RAD51-AS1 in OvCA.Furthermore, RAD51-AS1 interacted with miR-140-3p, leading to the upregulation of EIF5A2, with the outcome of promoting tumor growth in OvCA.The research might be beneficial to further elucidate OvCA pathogenesis and provide new ideas for clinical treatment.

Fig. 1
Fig. 1 RAD51-AS1 was associated with poor prognosis in OvCA patients and DNA repair, cell cycle, focal adhesion, and apoptosis in SKOV3.ipcells.A Relative expression of RAD51-AS1 in OvCA cell lines detected by RT-qPCR (p = 0.0071 vs. HOSEpiC).B ROvCA curve analysis for the diagnostic performance of RAD51-AS1 in OvCA samples.C Kaplan-Meier survival curves showed the survival differences in OvCA patients with high or low RAD51-AS1

Fig. 2
Fig. 2 RAD51-AS1 markedly elevated OVCAR3 cell proliferation, migration, invasion and EMT.A The growth curve of CCK-8 assay for OVCAR3 cells with overexpression of RAD51-AS1 (p = 0.0003 vs. Vector).B-D Representative images and quantitative results of colony formation, wound-healing and transwell invasion assays for

Table 1
Sequences of PCR primers used in this study