N6-methyladenosine modification of PLOD2 causes spermatocyte damage in rats with varicocele
Cellular & Molecular Biology Letters volume 28, Article number: 72 (2023)
In recent years, N6-methyladenosine (m6A) methylation modification of mRNA has been studied extensively. It has been reported that m6A determines mRNA fate and participates in many cellular functions and reactions, including oxidative stress. The PLOD2 gene encodes a protein that plays a key role in tissue remodeling and fibrotic processes.
The m6A methylation and expression levels of PLOD2 were determined by m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative polymerase chain reaction (qPCR) in the testes of varicocele rats compared with control. To determine whether IGF2BP2 had a targeted effect on the PLOD2 mRNA, RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed. CRISPR/dCas13b-ALKBH5 could downregulate m6A methylation level of PLOD2, which plays an important role in PLOD2-mediated cell proliferation and apoptosis in GC-2 cells.
PLOD2 was frequently exhibited with high m6A methylation and expression level in the testes of varicocele rats compared with control. In addition, we found that IGF2BP2 binds to the m6A-modified 3′ untranslated region (3′-UTR) of PLOD2 mRNA, thereby positively regulating its mRNA stability. Targeted specific demethylation of PLOD2 m6A by CRISPR/dCas13b-ALKBH5 system can significantly decrease the m6A and expression level of PLOD2. Furthermore, demethylation of PLOD2 mRNA dramatically promote GC-2 cell proliferation and inhibit cell apoptosis under oxidative stress.
As a result, we found that varicocele-induced oxidative stress promoted PLOD2 expression level via m6A methylation modification. In addition, targeting m6A demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 system can regulate GC-2 cell proliferation and apoptosis under oxidative stress. Taken together, our study has acquired a better understanding of the mechanisms underlying male infertility associated with oxidative stress, as well as a novel therapeutic target for male infertility.
The incidence of male infertility has gradually increased in recent years, which is affecting families around the world. Worldwide, male reproductive health is recognized as an important issue. Varicocele (VC) is a pathological occurrence characterized by elongation, expansion, and tortuousness of male spermatic veins. The incidence rate of varicocele in young male urology is about 15%, and it is mainly determined by its physiological and anatomical factors . With a clear diagnosis of VC, infertility can be as high as 35%, which is a major cause of male infertility . The relationship between VC and male infertility has been studied extensively in recent years, resulting in a wide range of theories . VC may induce male infertility through the effects of testicular microcirculation, vasoactive substances, oxidative stress, nitric oxide, hypoxia, immunity, and apoptosis.
VC is caused by blocked spermatic vein returns, which decrease blood flow to the testes and makes the testes hypoxic, ultimately resulting in a decrease in sperm production. Based on the studies of Kilinc et al., VC is associated with tissue hypoxia and related pathophysiological processes, thereby affecting spermatogenesis . Furthermore, Ozturk et al. found that rats in the VC model group had significantly higher testis tissue hypoxia indices . As a result of an anoxic environment, the body adjusts and changes. During tissue hypoxia, hypoxic inducible factor (HIF) is produced, and its level is proportional to the degree of hypoxia. Hypoxia-induced HIF-1 can rapidly enter the nucleus and trigger transcription of VEGF, enabling cells to adapt to hypoxia. A study by Wang et al. found that VC on one side of testis could lead to hypoxia on the other side of testis, eventually causing spermatogenic cells to die . The results of this study suggest that hypoxia promotes cell apoptosis, which can also be used to predict apoptosis of testicular germ cells, and is associated with increased cell apoptosis rate .
RNA modifications play an essential role in regulating gene expression posttranscriptionally. Eukaryotes have the most abundant internal modification, m6A, which constitutes 0.1%–0.3% of total adenosine residues [8, 9]. There is high conservation between humans and mice for m6A methylation modification. It is located in the non-coding 3′ term, near the stop codon and long internal exons, and is related to RNA stability, splicing, intracellular distribution, and translation [10, 11]. The cellular m6A state is controlled by a group of genes called “writers” (WTAP, METTL3 and METTLL4), “erasers” (FTO and ALKBH5), and “readers” (YTHDF1/2/3, IGF2BP2/3, YTHDC1 and YTHDC2) [12,13,14,15,16,17,18]. A multisubunit methyltransferase complex can increase m6A levels, while an m6A demethylase complex can reverse this process [8, 11, 19, 20]. N6-methyladenosine RNA modifications on the sixth nitrogen atom of adenine, one of the most important nitrogen atoms in RNA, have become one of the hottest topics in various human diseases, including hypertension , cardiac hypertrophy , viral infection , diabetes , and cancers [25, 26]. In another study, m6A modification was found to promote PLOD2 protein translation after YTHDF1 recognition, and then promote renal cancer development . Yet, the expression patterns of RNA m6A methylation modification and their underlying mechanisms remain largely unknown in VC.
In the present study, we observed an increase in PLOD2 m6A methylation and expression levels in the testes of VC rats compared with the control group. Preliminary experimental results showed that methylating the 3′UTR of PLOD2 can regulate mRNA stability by recruiting m6A reader proteins. Furthermore, PLOD2 demethylation promotes GC-2 cells apoptosis under oxidative stress.
20 adult male Wistar rats (180–200 g) were purchased from the Guangdong Provincial Center for Disease Control and Prevention. A 12 h/12 h light/dark cycle was maintained for the animals and they were fed standard food pellets and water as needed. The Institutional Animal Care and Use Committee approved all animal experiments in strict accordance with its guidelines.
Establishment of a rat varicocele model
In an experimental study, twenty male Wistar rats were split into two distinct groups: the sham control group and the varicocele group. Anesthesia was induced with a solution containing 10 g of tribromoethanol and 10 mL of tert-pentyl alcohol, both from Sinopharm Chemical Reagent Co. (Shanghai, China), diluted to a 2% concentration in distilled water. A 2-cm surgical cut was initiated in the lower abdomen of each rat. The intersection of the left renal vein and the inferior vena cava was carefully isolated. A channel was created via blunt dissection between the inner side of the left renal vein and the exterior of the inferior vena cava. Subsequently, a 4-0 silk suture was employed to constrict the left renal vein to half its initial diameter. For the sham control group, an identical surgical procedure was followed, barring the vein constriction. The success of the modeling was gauged by two criteria: (i) a spermatic vein diameter greater than 1 mm, and (ii) the absence of any size disparity between the left and right kidneys. Eight weeks postoperation, the rats were euthanized for further analysis.
RNA m6A and mRNA sequencing
As described previously, MeRIP-seq and RNA-seq were performed by Novogene (Beijing, China). Trizol (Thermo Fisher Scientific) was used to isolate total RNA from 5 pairs of tumors and adjacent tissues. Then total RNA was broken into almost 100 nt fragmentation and incubated with anti-m6A antibody (Synaptic Systems, 202003, Goettingen, Germany) for 2 h at 4 °C. The beads (Thermo Fisher Scientific) were then prepared and incubated with total RNA for 2 h at 4 °C. The final step is to wash the mixture and purify the m6A-bound RNA with TE buffer. Following purification, the samples can be used to construct the library using the NEBNext UltraTM RNA Library Prep Kit (New England Biolabs, MA, USA) on the Illumina HiSeq sequencer (Illumina, CA, USA). In the NCBI database, raw RNA-seq and m6A-seq data have been uploaded.
Sequencing data analysis
To obtain the sequence data for IP and control samples, we should preprocess the read segment data (for example, filtering out the poor-quality segments), and a reference genome is used to process and analyze all the read segment sequence mapping of the two samples. As a result, read segments captured by methylation sites in the IP samples formed a region or peak near the methylation sites in the reference genome. It is therefore known as the peak-calling algorithm to identify points of methylation enrichment. We identified m6A methylated peaks among transcripts using Model-based Analysis of ChIP-Seq (MACS), and we investigated metagene m6A distribution using MetaPlotR. The DMGs were identified by diffReps. An analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) was conducted on DMGs and DEGs from MeRIP-seq and RNA-seq.
RNA-binding protein immunoprecipitation (RIP)
In accordance with the manufacturer’s instructions, RIP assays were performed with the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore). A protease inhibitor cocktail and RNase inhibitor were included in the complete radioimmunoprecipitation assay buffer for lysing the cells. Protein A/G magnetic beads were prebound to antibodies (5 g) for 2 h before being incubated overnight with 100 L of cell lysate at 4 °C. By incubating the beads in 400 μL of elution buffer for 2 h, eluting with ethanol, and dissolving in RNase-free water, the RNA was eluted from the beads. Real-time PCR (qPCR) was used to determine fragment enrichment.
RT-qPCR, RIP-qPCR and MeRIP-qPCR
Following the instructions of Vazyme Biotech, Nanjing, China, total RNA was extracted from tissues and cell lines using the RNA-easy Isolation Reagent. Anti-m6A antibody-coupled beads were used to incubate fragmented RNA. IGF2BP2 was used to immunoprecipitate the m6A-containing RNA and then it was eluted from the beads. The RT-qPCR was performed with gene-specific primers on both input control and m6A-IP samples. A HiScript III RT SuperMix for qPCR was used to synthesize the cDNA (Vazyme Biotech, Nanjing, China). With universal SYBR Green qPCR Master Mix (Vazyme Biotech, Nanjing, China) and spectrophotometry (ABI Prism 7500TM instrument, Applied Biosystems), qRT-PCR was performed.
Protein isolation and western blot
KeyGEN Bio TECH protein extraction kit (KGP1100) was used to extract protein from cells and separate it on 10% SDS–PAGE and transfer it to nitrocellulose membrane. According to previous instructions, blots were then immunostained with primary antibodies and secondary antibodies. The antibodies were as follows: PLOD2 (1:1000; Abcam, United States), p-AKT (1:1000; Abcam, United States), AKT (1:1000; Abcam, United States), Bax (1:1,000; Invitrogen, United States), Bcl-2 (1:1000; Invitrogen, United States), IGF2BP2 (1:1000; Invitrogen, United States), and GAPDH (1:10000; Proteintech, United States).
Expression plasmids, short interfering RNAs, and lentivirus transfection
Addgene provided the CRISPR dCas13b plasmids and Cas13b-gRNA plasmids. A number of designed gRNAs and the dCas13b-ALKBH5 vector have been constructed by Synbio Technologies. An overexpression plasmid was generated using the CDS of ALKBH5 cloned into pcDNA3.1. The vector control used for analysis was pcDNA3.1. IGF2BP2 was knocked down with Sigma-synthesized duplex RNAi oligos.
Cell culture and plasmid transfection
The GC-2 cell line for this study was purchased from the American Type Culture Collection (ATCC, Manassas, VA, United States). DMEM with 10% fetal bovine serum was routinely used for cell culture, supplemented with 5% CO2 in a 37 °C environment (Invitrogen, Carlsbad, CA, United States). Lipo3000 (Invitrogen) was used for transfection of all siRNAs and plasmids as per the manufacturer’s instructions, and 1 μg of plasmids was used in each experiment. There was a 50 nM working concentration of siRNA.
Luciferase reporter assay
To determine the effect of PLOD2 expression, PLOD2’s wild type or mutant 3′UTR was inserted at the end of the firefly luciferase (F-luc) coding sequence. In both wild type and ALKBH5 overexpression cells, the pmirGLO-PLOD2-3′UTR-WT and pmirGLO-PLOD2-3′UTR-MUT were transfected for 24 h. We analyzed firefly luciferase (F-luc) and renilla luciferase (R-luc) by using the Dual-Glo Luciferase Assay system (Promega) according to instructions. The activity of Renilla Luciferase (R-luc) was used to normalize the activity of Firefly Luciferase (F-luc) to evaluate reporter transcription. The experiments were repeated three times and the results were similar each time.
Cells transfected with different plasmids were stabilized with actinomycin D (Act-D, catalog #A9415, Sigma, U.S.A.) at 50 mg/mL during incubation. To conduct real-time PCR, RNA was isolated from the cells at the indicated times. PLOD2 mRNA half-life was calculated by using ln2/slope and normalized using GAPDH.
Cell proliferation and apoptosis assays
Detection of cell proliferation was carried out using the Cell Counting Kit 8 (CCK8) assay (Transgen, China). In 96-well plates, 3 × 103 cells were seeded per well. A total of 24 h, 48 h, 72 h, and 96 h of cell culture was followed by 3 h of incubation with CCK8 at 37 °C. After that, 450 nm absorbance was measured using a microplate reader. Approximately 70%–80% confluence was achieved with vector-transfected cells plated in a 12-well plate (2 × 105 /well). Flow cytometry assays were performed 48 h after cell harvesting.
At least three independent experiments were used to gather data. Data are reported as mean ± standard deviation. A two-tailed unpaired Student’s t-test was used between two groups, along with one-way or two-way ANOVA and Bonferroni testing for multiple comparisons. A two-sided test was used for all statistical analyses, using SPSS 16.0 for Windows. To be considered statistically significant, the p-value must be 0.05 or less, e.g., *p < 0.05, **p < 0.01. “NS” stands for not significant.
Distribution of m6A modification in the testes of VC rat and control group
To understand the mechanisms of m6A methylation modification in the testes of VC rats in greater detail, whole testicular samples were used for mRNA m6A methylation sequencing (MeRIP-seq). First, we analyzed the metagene profiles of transcript peaks in the testes of control and VC rats. We found that the majority of m6A peaks were situated at the end of the 5′UTRs and start of the 3′UTRs in two groups (Fig. 1A and B). Further analysis revealed that peaks located at coding sequence (CDS) were the most frequent, and peaks located at translation start site (TSS) were the least frequent in both groups (Fig. 1C and D). Afterwards, the rat transcriptome was analyzed for their m6A methylation modifications. In VC and control groups, most of the methylated sequences within mRNA contained less than five m6A peaks, while few had more than five (Fig. 1E and F). Similar results were found in previous studies for the distribution of m6A modifications.
Functional analysis of differentially m6A methylated mRNAs between two groups
To determine whether the m6A peaks we identified from MeRIP-seq represented the RRACH motif, we performed the HOMER motif software analysis on the m6A peaks. The motif sequences in the control and VC groups were AAACU and GGACA, respectively (Fig. 2A). Based on |log2FC| > 1 and p-value < 0.05, the differentially methylated m6A peaks were identified between the control and VC groups. As compared with the control group, 2599 hypomethylated peaks and 2357 hypermethylated peaks were found in the VC group (Fig. 2B). As a result of KEGG pathway analysis, differentially m6A methylated peaks within mRNA were predominantly associated with metabolic pathways, PI3K-Akt signaling pathways, and cell apoptosis (Fig. 2C). MeRIP-qPCR assays were performed to confirm our MeRIP-seq results for five hypermethylated genes (PLOD2, CASP3, CASP9, ITGB4, and DDIT4) and five hypomethylated genes (IGF1R, ATG7, SLC22A13, GRB2, and TAP1), which were potentially involved in cell apoptosis and the PI3K-Akt signaling pathway. These genes exhibited almost the same m6A-level changes with sequencing, supporting our MeRIP-seq results (Fig. 2D). We also performed RT-qPCR analysis on VC and control groups to determine the transcript levels of these genes (Fig. 2E). Results showed that PLOD2, CASP3, and CASP9 were upregulated, and ATG3 and SLC22A13 were downregulated in the VC group compared with the control group. As a result, PLOD2 was the gene with the highest level of methylation and expression in VC group. Based on these results, we investigated the general locations of differentially methylated m6A sites within PLOD2 in VC group and control group. Our data indicated that the m6A peak was enriched around PLOD2’s 3′UTR in two groups, and it was higher in the VC group than in the control group (Fig. 2F). In summary, we found PLOD2 was hypermethylated and upregulated in the testes of VC rats.
PLOD2 expression and m6A methylation level under oxidative stress in GC-2 cell
For further investigation of how m6A methylation modification regulates PLOD2 expression in varicocele, the spermatocyte cell line GC-2 was used. To mimic varicocele’s oxidative stress, H2O2 (0.5 mM, 24 h) was added to the GC-2 cell culture medium. According to previous studies, H2O2 reduced the viability of GC-2 cells in culture in a dose-dependent manner . In comparison with control cells, GC-2 cells expressed higher levels of PLOD2 mRNA after H2O2 treatment (Fig. 3A). In addition, m6A enrichment of PLOD2 was also significantly greater in GC-2 cells treated with H2O2 than in control cells, as determined by MeRIP-qPCR (Fig. 3B).
Next, we investigated the mechanisms via which m6A modification affects PLOD2 expression in GC-2 cell. Results of western blots and RT-qPCR analysis showed that after H2O2 treatment, ALKBH5-overexpressing GC-2 cells expressed less PLOD2 protein and mRNA (Fig. 3C and D). In addition, when GC-2 cells were treated with H2O2, overexpression of ALKBH5 significantly inhibited m6A antibody-enriched PLOD2 mRNA (Fig. 3E and Additional file 1: Fig. S1A). Additionally, Act-D was used to block transcription in normal control and ALKBH5-overexpressing GC-2 cells. It has been shown that PLOD2 mRNA half-lives are shortened by overexpression of ALKBH5 (Fig. 3F). As a result, m6A modification might delay PLOD2 mRNA degradation in GC-2 cells.
IGF2BP2 affected the mRNA stability of PLOD2 by participating in the PLOD2 m6A modification
In PLOD2 mRNA, several differentially methylated m6A peaks (DMMPs) were detected by MeRIP-seq (Fig. 1F). With an anti-m6A antibody, fragmented RNA from GC-2 cells was immunoprecipitated to characterize m6A methylation. According to MeRIP-qPCR results, the 3′UTR exhibited the highest level of m6A methylation, followed by the CDS and the 5'UTR region (Fig. 4A). Thus, in GC-2 cells overexpressing ALKBH5, m6A enrichment of PLOD2 3'UTR was decreased, which indicated m6A modifications are more dynamic in the 3'UTR than in the CDS (Fig. 4B). Based on luciferase assays in GC-2 cells using reporters containing PLOD2-3′UTR-WT or -MUT, we investigated the potential role of m6A methylation in the 3′UTR region of PLOD2 (Fig. 4B). According to the dual-luciferase assay, PLOD2–3’UTR-WT translated more efficiently in GC-2 cells overexpressing ALKBH5 compared with controls (Fig. 4C).
Further investigations were conducted into mechanisms regulating mRNA stability by m6A methylation modification. Researchers found that m6A modification regulates mRNA stability by binding to YTHDF2, YTHDF3, and IGF2BP1 ~ 3. Therefore, we explored which m6A reader protein enhances PLOOD2 mRNA stability in a manner dependent on m6A modification. To determine whether IGF2BP2 binds significantly to PLOD2 mRNA in GC-2 cells, RIP-qPCR assays were performed. Results showed IGF2BP2 bound significantly to PLOD2 mRNA (Fig. 4D and Additional file 1: Fig. S1B). Thus, we knocked down IGF2BP2 in GC-2 cells and found that IGF2BP2 downregulation inhibited the mRNA and protein levels of PLOD2 (Fig. 4E–G). It has also been shown that downregulation of IGF2BP2 can decrease the stability of PLOD2’s mRNA (Fig. 4H). Based on these results, IGF2BP2 may be involved in the m6A methylation modification and in regulating the stability of PLOD2 mRNA.
Targeting m6A demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 to regulate GC-2 cell proliferation and apoptosis under oxidative stress
In the next step, we demethylated the m6A of PLOD2 by fusing the catalytically dead type VI-B Cas13 enzyme with the m6A demethylase ALKBH5 (dCas13b-A5) enzyme . To target the PLOD2 mRNA, specific guide RNAs were designed around the m6A site (Fig. 5A). The dCas13b-A5 induced demethylation of PLOD2 in GC-2 cells was first confirmed by MeRIP-qPCR (Fig. 5B). As a result of dCas13b-A5 targeting PLOD2, we observed a significant decrease in PLOD2 mRNA and protein levels in GC-2 cells (Fig. 5C). There might be a reason for this, since dCas13b-A5 with a gRNA for PLOD2 significantly decreases PLOD2 mRNA binding to IGF2BP2 protein. To investigate whether dCas13b-A5 targeting PLOD2 can modulate GC-2 cell homeostasis, we monitored GC-2 cell proliferation and apoptosis after transfection with control or gRNA for PLOD2 combined with dCas13b-A5. Compared with non-targeted control gRNA combined with dCas13b-A5, gRNA targeting PLOD2 significantly promoted cell proliferation and decreased apoptosis in GC-2 cells (Fig. 5D–F). In addition, western blot analysis showed that PLOD2-dymethylated GC-2 cells had decreased Bax protein level and increased Bcl-2 level. In GC-2 cells, p-Akt expression was also decreased after the PLOD2 mRNA demethylation (Fig. 5G, H). These results suggest that a decreased m6A methylation level of PLOD2 can promote cell proliferation, reduce cell apoptosis, and further inactivate the PI3K/AKT/mTOR pathway.
Varicocele is the abnormal expansion and tortuosity of the tendriform venous plexus in the spermatic vein, with the left side being the most common . It was first described by Saypol et al. nearly a century ago that partial ligation of the left renal vein simulating varicocele could be performed. Hence, the left renal vein was partially ligated in this study to create the VC model. VC has been proved by a number of studies to cause serious damage to testicular spermatogenic function, which can then cause male infertility. The main pathophysiological cause of VC is persistent hypoxia at the site of testicular spermatogenesis, as oxygen is essential for spermatogenic tubules to produce normal spermatogenesis . As a result of VC, the one-way blood flow system between the spermatic vein and the testicular vein is damaged, resulting in venous blood stasis. As a result, it causes the hydrostatic pressure inside the testicle to increase and overtake the microcirculation pressure inside the testicular artery, causing a relatively anoxic situation . Hypoxia in testicular tissue can inhibit HIF-1α protein degradation, leading to increased expression levels and downstream gene activation [33, 34]. It has also been demonstrated that hypoxia controls sarcoma metastasis by enhancing the expression of the intracellular enzyme PLOD2 .
In eukaryotes, the dynamic and reversible m6A RNA modification is mediated by methyltransferases and demethylases. In addition to heat shock , ultraviolet light , hypoxic stress , and oxidative stress , m6A modification participates in many cellular activities and reactions. Although numerous studies have confirmed the hypoxic stabilization of specific mRNAs is dependent on m6A modification, less is known about their role in VC. By activating hydroxylation of collagen fiber molecules, PLOD2 plays an important role in fibrotic processes and tissue remodeling . Overexpression of PLOD2 can lead to collagen crosslinking, an increase in extracellular matrix hardness, and cancer cell proliferation and metastasis . However, whether m6A methylation modification could influence PLOD2 expression to alter epigenetic remodeling, or contribute to the features of VC, is unclear and worthy of investigation.
Based on the results from this study, PLOD2 is likely to be a modulatory biomarker in varicocele. Based on MeRIP-seq technology, we identified many differentially methylated genes among VC and control rats and found that PLOD2 was frequently upregulated in VC groups. Furthermore, our data indicate that activation of PLOD2 might be due to an increase in m6A methylation modification in the 3′UTR regions of mRNA in the VC group. When ALKBH5 is overexpressed in GC-2 cells, it can decrease m6A methylation level, thus decreasing PLOD2 mRNA stability and expression levels.
In addition to RNA processing, nuclear export and translation modulation, the m6A modification can regulate many stages of RNA’s life cycle [42, 43]. According to our findings, the luciferase reporter system showed that m6A in the 3′UTR of mRNA positively regulated PLOD2 m6A methylation modification. Furthermore, m6A-regulated mRNA stability of PLOD2 was dependent on IGF2BP2 in GC-2 cells. It has been demonstrated that IGF2BPs can bind the GG(m6A)C sequence of mRNA to promote its stability and storage . Overall, our data provide a new insight into the function of IGF2BP2 regulating PLOD2 mRNA stability under hypoxic stress. A newly developed method called CRIPSR/dCas13b-ALKBH5 system regulates m6A modification level of target genes by targeting demethylation of specific mRNA in the transcriptome . With CRIPSR/dCas13b-ALKBH5, we specifically demethylated the m6A modification of PLOD2 mRNA. m6A methylation level was decreased about two-fold and PLOD2 expression was also significantly decreased by this system. In GC-2 cells, dCas13b-A5 promoted cell proliferation while inhibiting apoptosis and inactivating PI3K/AKT/mTOR signaling.
In summary, we found that m6A methylation modification played va ital role in oxidative stress-induced apoptosis in varicocele, and may be used as a novel target for oxidative stress-related male infertility. Furthermore, the regulatory network involving the new complex ALKBH5/PLOD2 may provide insight into the pathogenesis and development of male infertility.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
M6A methylated RNA immunoprecipitation-sequencing
3′ Untranslated region
Hypoxic inducible factor
5′ Untranslated region
Translation start site
Differentially methylated m6A peaks
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This study followed the Helsinki declaration and was approved by the Institutional Ethical Review Board of Peking University Shenzhen Hospital and animal center of Shenzhen PKU-HKUST Medical Center (no. 4129-16, date: 22 May 2020).
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Factors involved in m6A-regulated expression of PLOD2. A MeRIP-qPCR analysis of PLOD2 m6A levels in control and overexpression of ALKBH5 or FTO cells. B RIP-qPCR analysis of PLOD2 enrichment levels using IGF2BP1, IGF2BP2, and IGF2BP3 cells
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Li, H., Zhao, J., Deng, H. et al. N6-methyladenosine modification of PLOD2 causes spermatocyte damage in rats with varicocele. Cell Mol Biol Lett 28, 72 (2023). https://doi.org/10.1186/s11658-023-00475-4