|Year : 2019 | Volume
| Issue : 3 | Page : 63-72
Thymopentin promotes ovarian angiogenesis in mice by activating N6-methyladenosine (m6A) RNA modification of key factors in the Notch/Tie1 pathway
Junjun Tao1, Jiajia Lin2, Xiaoli Nie2, William Huang3, Jianming Guo4, Te Liu2
1 Department of Nursing, Huashan Hospital North, Shanghai, China
2 Central Laboratory, Shanghai Geriatric Institute of Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
3 Hainan Zhonghe Pharmaceutical Co., Ltd, Hainan, China
4 Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University, Beijing, China
|Date of Submission||20-May-2019|
|Date of Acceptance||26-Jul-2019|
|Date of Web Publication||28-Nov-2019|
Prof. Te Liu
Shanghai Geriatric Institute of Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 365 South Xiangyang Road, Shanghai 200031
Source of Support: None, Conflict of Interest: None
BACKGROUND: Premature ovarian failure (POF) is a typical condition of pathological ovarian ageing. Injuries and atrophies of blood vessels in the ovaries can lead to ovarian insufficiency, but the underlying mechanism remains unclear.
AIMS: This study investigated the epigenetic mechanism by which thymopentin (TP-5) activated ovarian angiogenesis to achieve its effects on POF.
MATERIALS AND METHODS: The qPCR, western blot and immunofluorescence staining were used to detecte the expression levels of gene. The hematoxylin and eosin stainingwas used to histopathological examination. The RNA-Seq sequencing was used to transcriptome detection.
RESULTS: First, pathological examination revealed that TP-5 significantly increased ovary weight in the cyclophosphamide-induced POF mouse model. The proportion of atretic follicles was reduced, and the level of E2 in the peripheral blood was elevated. Meanwhile, immunofluorescence staining showed that TP-5 increased protein expression of CD31 and Tie1 in the ovarian tissues of mice with POF, suggesting that TP-5 could induce ovarian angiogenesis. RNA-Seq sequencing results suggested that TP-5 could induce the high level of Notch/Tie2 pathway expression that is associated with angiogenesis in ovarian tissues. qPCR and western blotting showed significantly increased expression of METTL3, a methyltransferase that catalyses the formation of N6-methyladenosine (m6A) in RNA. Meanwhile, the results of the dot blot suggested that the overall RNA methylation level was significantly higher in the ovarian tissues of mice in the TP-5 group than in the control group. Finally, RIP-PCR analysis showed that specific sites in the 3'-untranslated region (3'UTR) of the mRNA of key factors in the Notch/Tie2 pathway were hypermethylated in the ovaries of mice in the TP-5 treatment group.
CONCLUSION: Therefore, this study demonstrated that TP-5 exerted its therapeutic effect on POF by stimulating angiogenesis. The mechanism of action is the promotion of the expression of RNA m6A methyltransferases by TP5, which increases the overall RNA m6A methylation level in mouse ovarian tissue. Specifically, the methylation ofspecific m6A sites in the 3'UTR of the RNA of key factors in the Notch/Tie2 pathway increased, which enhanced their stability and expression levels, leading to the induction of angiogenesis.
Keywords: Angiogenesis, Notch/Tie2 pathway, premature ovarian failure, RNA N6-methyladenosine, thymopentin
|How to cite this article:|
Tao J, Lin J, Nie X, Huang W, Guo J, Liu T. Thymopentin promotes ovarian angiogenesis in mice by activating N6-methyladenosine (m6A) RNA modification of key factors in the Notch/Tie1 pathway. Vasc Invest Ther 2019;2:63-72
|How to cite this URL:|
Tao J, Lin J, Nie X, Huang W, Guo J, Liu T. Thymopentin promotes ovarian angiogenesis in mice by activating N6-methyladenosine (m6A) RNA modification of key factors in the Notch/Tie1 pathway. Vasc Invest Ther [serial online] 2019 [cited 2020 Jan 29];2:63-72. Available from: http://www.vitonline.org/text.asp?2019/2/3/63/271907
Junjun Tao, Jiajia Lin and Xiaoli Nie contributed equally to this work and shared the first authorship.
| Introduction|| |
Premature ovarian failure (POF) is a disease characterized by amenorrhea, infertility, low estrogen levels, high gonadotropin levels, and deficiency in mature follicles in women before the age of 40 years. It is one of the common etiologies of female infertility.,,,, The development of POF is closely associated with the status and quality of ovarian granulosa cells (OGCs).,,,, Aging and apoptosis of OGCs are one of the important causes contributing to diminished ovarian reserves.,,,, However, in our preliminary experiments, we found that the number of blood vessels in ovarian tissues from a POF mouse model was significantly reduced. There was also evidence of atrophy, suggesting that POF was largely related to the status of blood vessels. Angiogenesis refers to the development of new blood vessels from existing capillaries or postcapillary venules. The process primarily includes degradation of the vascular basement membrane during the sprouting stage; activation, proliferation, and migration of vascular endothelial cells; and regeneration of new blood vessels and vascular networks. Angiogenesis is a complex process that involves multiple molecules from multiple cells coordinated by pro- and anti-angiogenic factors.,,, Under normal conditions, the two types of factors are in equilibrium. Disturbances to this balance activate the vascular system, leading to excessive angiogenesis or inhibition of the vascular system, which results in vascular degeneration.,,, The mechanism of angiogenesis is complicated, and many factors are implicated in its promotion. For example, during the epithelial–mesenchymal transition of tumors, tumor necrosis factor-α and interleukin-8 secreted by tumor-associated macrophages can promote the proliferation of vascular endothelial cells; transforming growth factor-β, platelet-derived endothelial cell growth factor, heparanase, angiopoietin, bone morphogenetic protein, cyclooxygenase, hypoxia-inducible factor-1, laminin, placental growth factor, survivin, and erythropoietin, are all implicated in the process of angiogenesis.,,, However, to date, no reports have clearly demonstrated the relationship between angiogenesis and POF.
RNA N-6 methyladenosine (m6A) refers to the methylation of adenosine at the nitrogen-6 position in RNA. RNA m6A methylation is widely present in most eukaryotic species (from yeast, plants, and fruit flies, to mammals) and viral mRNA, and it plays a key modulatory role in the posttranscriptional regulation and metabolism of mRNA.,,,, The m6A methyltransferases, METTL14 and METTL3, are two components of the m6A methyltransferase complex. They form a stable complex at a 1:1 ratio to complete the modification of m6A on RNA, as part of the writer complex.,,, In contrast, the FTO alpha-ketoglutarate dependent dioxygenase (FTO) proteins remove the RNA methylation on m6A and play the role of erasers.,,, Therefore, the modification of m6A RNA is a dynamic and reversible enzymatic reaction.,,, Studies have suggested that m6A modification of RNA can improve the stability of mRNA, increase its rate of transcription and translation, promote tumorigenesis and tumor invasion, and improve the reprogramming efficiency of stem cells.,,,, However, the mechanism by which dynamic modification of m6A RNA regulates angiogenesis during the development of POF has not been elucidated.,,,,,,,,,
In addition, thymopentin (TP-5) is composed of five amino acids, arginine, lysine, aspartic acid, valine, and tyrosine,,,,, and has the chemical name N-[N-[N-[NL-arginyl-L-lysyl]-L-α-aspartyl]-L-valyl]-L-tyrosine.,,,, Its molecular formula is C30H49N9O9, and its molecular weight is 679.77. TP-5 is an active portion of thymopoietin II, which is secreted by the thymus.,,,, Thymopoietin II is a monomeric polypeptide isolated from a thymus hormone. It is composed of 49 amino acids, and one of its peptide chains consisting of five of the amino acids produces the same physiological functions as thymopoietin II and is therefore named TP-5.,,,, Studies have shown that TP-5 possesses significant immunomodulatory effects. It can promote T-cell differentiation by increasing the levels of Cyclic Adenosine monophosphate (CAMP). It can also bind to T-cell-specific receptors, which increases the levels of intracellular cyclic guanosine monophosphate, thereby inducing a series of intracellular reactions through which TP-5 exerts its regulatory effects on immune function.,,,, In addition, TP-5 can induce T-cell differentiation and promote the development, maturation, and activation of subsets of T-cells. It can also regulate the ratio of CD4/CD8 T-cells toward their normal levels.,,,, TP-5 has displayed notable effects on the reestablishment of immune function in cancer patients who received chemoradiotherapy, as well as the enhancement of the immune system in the elderly or people with weak or reduced immunity.,,,, At the same time, TP-5 also has considerable therapeutic effects on autoimmune diseases, such as rheumatoid arthritis, lupus, Type II diabetes, and female climacteric syndrome.,,,, However, to date, there have been no reports on the use of TP-5 in the treatment of POF.,,,,
Based on this evidence, we established a cyclophosphamide-induced POF mouse model, which we treated with TP-5. We aimed to investigate the therapeutic effects of TP-5 on POF through studying angiogenesis. Meanwhile, we attempted to delineate the mechanism of action of TP-5 through evaluating m6A modifications on the RNA of key factors in the Notch/Tie1 pathway, which is a stimulatory pathway associated with angiogenesis.
| Materials and Methods|| |
Premature ovarian failure model and thymopentin-5 treatment
Thirty C57BL/6 mice (10-week-old female) were purchased from the Experiment Animal Center of Shanghai University of Traditional Chinese Medicine. According to our previously established protocol, the mice were divided into three groups: a wild-type group untreated any reagents; negative control group treated with saline and 70 mg/kg CTX daily by intraperitoneal injection; and an experimental group treated with 5 mg/kg TP-5 (Hainan Zhonghe Pharmaceutical Co., Ltd., Hainan, China) and 30 mg/kg CTX (Sigma-Aldrich) daily by intraperitoneal injection. Following treatment, experiments using the animal model were conducted within 30 days. All the animal experiments were conducted in accordance with the guidelines of the NIH for the care and use of laboratory animals. The study protocol was also approved by the Committee on the Use of Live Animals in Teaching and Research, Experiment Animal Center of Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Extraction of total RNA and quantitative polymerase chain reaction
Total RNA from each group of cells was extracted using Trizol reagent according to the manufacturer's instructions. Total RNA was treated with DNase I (Sigma-Aldrich), quantified, and reverse transcribed into cDNA using the ReverTra Ace-αFirst Strand cDNA Synthesis Kit (TOYOBO). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with a RealPlex4 real-time PCR detection system from Eppendorf Co., Ltd., (Germany). SYBR Green Real-Time PCR Master Mix (TOYOBO) was used as the fluorescent dye in the nucleic acid amplification. qRT-PCR was completed with 40 amplification cycles as follows: denaturation at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 42 s. The relative gene expression levels were calculated using the 2− ΔΔ Ct method (ΔCt = Ct_genes–Ct_18sRNA; ΔΔCt = ΔCt_all_groups–ΔCt_blankcontrol_group). The mRNA expression levels were normalized to the expression level of 18 s rRNA. The primers for amplification of each gene are shown in [Supplementary Table 1][Additional file 1].
Briefly, total proteins from the cells in each group were subjected to 12% denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred onto a polyvinylidene fluoride membrane (Millipore). The membrane was blocked, washed, and incubated with primary antibodies at 37°C for 45 min [Table 1]. After the membrane was fully washed, it was incubated with secondary antibodies at 37°C for 45 min [Table 1]. The membrane was washed with Tris-buffered saline/Tween 20 4 times at room temperature for 14 min each time. Next, the samples were exposed and imaged (Sigma-Aldrich Chemical) using the enhanced chemiluminescence (ECL) method (Pierce Biotechnology).
Hematoxylin and eosin staining
According to a previously reported method, tissues were fixed in 4% paraformaldehyde at room temperature for 12 h. Frozen tissue sections were prepared at thicknesses of approximately 5 μm. Sections were fixed in 95% anhydrous ethanol for 2 min, stained in hematoxylin for 5 min, and differentiated in differentiation solution for 2 min. Sections were immersed in weak ammonia solution for 3 min, washed with deionized water for 5 min, stained with eosin for 5 min, and washed with deionized water for 5 min. Tissue sections were immersed in 70%, 80%, and 90% alcohol solution once for 1 min, washed with anhydrous ethanol twice for 1 min each wash, cleared in xylene twice for 1 min each wash, and mounted using neutral balsam. These reagents and materials were all purchased from Beyotime Biotechnology Co., Ltd., Zhejiang, China.
Establishment of cDNA sequencing libraries and high-throughput RNA-Seq
The following analysis was conducted by KangChen Bio-tech (Shanghai, China). According to their experimental procedures, a random fragment sequencing library was constructed using a SOLiD Whole Transcriptome Analysis Kit (Life technologies). Nucleic acid-cleaving reagents were added, and the mRNA was randomly disrupted into short segments in a shaking incubator. First-strand cDNA was reverse transcribed using the fragmented mRNA as the template. Second-strand cDNA was synthesized using a second-strand DNA synthesis reaction system consisting of DNA polymerase I, dNTPs and RNase H (Sigma). The synthesized DNA was purified using a DNA purification kit and recovered. Thebase “A” was added to the 3' end of the cDNA, followed by ligation to the adapter, to complete the blunt end repair reaction. Subsequently, DNA fragment size selection was performed. Finally, the cDNA was used for PCR amplification to obtain a sequencing library. The constructed library was qualified using an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System and was subjected to high-throughput sequencing using an Illumina HiSeq ™ 2000 Sequencer after passing quality control.
Enzyme-linked immunosorbent assay
The mouse estradiol (E2) enzyme-linked immunosorbent assay kit (Westang Bio, Shanghai, China) was used according to the manufacturer's instructions to determine the level of E2 in mouse plasma. Briefly, 100 μL of mouse E2 standardized to 8000, 4000, 2000, 1000, 500, 250 and 125 pg/mL or diluted mouse plasma were added to anti-E2 antibody-precoated microwells and incubated for 60 min. After three washing steps, (HRP) Horseradish Peroxidase-conjugated detection antibodies were added, followed by the substrate solution. The absorbance was measured at 450 nm.
Five mice were randomly selected from each group. From each mouse, a specimen containing plaques at the aortic root was resected, dehydrated, cleared, embedded in paraffin, sectioned, dewaxed, and rehydrated. After treatment with citrate buffer in a boiling water bath for 10 min for antigen retrieval, the specimen was cooled to room temperature, subjected to the same treatment again, and cooled to room temperature. Blocking was performed at room temperature for 2 h using 5% bovine serum albumin (BSA) blocking solution. The primary antibodies [Table 1] were diluted 1:500, and blocking was performed for 1 h. After overnight shaking at 4°C, the specimen was washed with phosphate buffered saline (PBS) for 15 min, the secondary antibodies [Table 1] were diluted in a blocking solution at 1:500 ratio. Sections were incubated with secondary antibody at room temperature for 1 h. After washing with PBS for 45 min, the specimen was mounted using anti-fade medium containing 4',6-diamidino-2-phenylindole and observed and photographed under a fluorescence microscope with excitation at 488 nm using a laser.
Different doses of genomic DNA from each group were spotted on Hybond-N + membrane, then spotted DNA was cross-linked to the membrane by UV Cross-linker, then membrane was blocked in 5% BSA and subsequently incubated with anti-m6A antibody Cell Signaling Technology (CST) and HRP-conjugated anti-mouse secondary antibody (CST), and finally developed with ECL reagents and exposed to the imaging film.
RNA immunoprecipitation-polymerase chain reaction
RIP experiments were performed by using the Magna RNA-Binding Protein Immunoprecipitation (RIP) Kit (Millipore, Bedford, MA, USA). All the steps of RIP were performed as previously described., Briefly, cells from all groups were lysed (500 μL/plate) in a modified cell lysis buffer used for western blotting and IP (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, sodium pyrophosphate, β-glycerophosphate, Na3 VO4 and leupeptin) (Beyotime institute of Biotechnology). After lysis, each sample was centrifuged to clear the insoluble debris and was preincubated with 20 μg protein A agarose beads (Beyotime institute of Biotechnology) by rocking for 30 min at 4°C, followed by centrifugation and transfer to a fresh 1.5 mL tube. The mouse anti-human AgO2 monoclonal antibody (1:100; Santa Cruz biotechnology, California, USA) was added and incubated for 90 min before the readdition of 20 μg of protein A agarose beads to capture the immune complexes. The agarose beads were then washed three times with ice-cold homogenization buffer. The specific primers were designed as [Table 2].
Each experiment was performed as least three times, and data were shown as the mean ± SE where applicable, and differences were evaluated using Student's t-tests. P < 0.05 was considered significant.
| Results|| |
Thymopentin significantly ameliorated premature ovarian failure symptoms
Histopathological examination performed on the ovarian tissues of mice in each group showed that follicles of different phases were rarely observed in the ovarian tissues of mice in the CTX model group (POF), while multiple mature oocytes and multiple follicular tissues of different phases were observed in the ovaries of mice in the control group Widetype (WT) [Figure 1]a and [Figure 1]c. The proportion of atretic follicles in ovarian tissue was significantly reduced in mice treated with TP-5. The follicle counts indicated that the ratio of atretic follicles was significantly lower in the ovaries of mice treated with TP-5 than in the saline-treated group [Figure 1]a and [Figure 1]c. The ovary weights in the mice with POF were significantly lower those from mice in the WT group, while the ovary weights of mice in the TP-5 group were significantly higher than those of mice in the POF group [Figure 1]b. The level of E2 in the peripheral blood of mice with POF was significantly lower than that in mice from the WT group, while the level of E2 in the peripheral blood of mice in the TP-5 group was significantly higher than that of mice in the saline-treated group [Figure 1]d. Furthermore, the level of follicle-stimulating hormone in the peripheral blood of mice in the TP-5 group was significantly lower than that in mice in the saline-treated group [Figure 1]e. These results indicated that TP-5 could significantly ameliorate the symptoms of POF.
|Figure 1: Thymopentin alleviated the symptoms of premature ovarian failure. (a) H and E staining of mouse ovarian tissues; GC: ovarian granulosa cells; OC: oocyte; AF: atretic follicles; (b) ovary weights; (c) atretic follicle percentage; (d) enzyme-linked immunosorbent assay for E2 in peripheral blood; (e) enzyme-linked immunosorbent assay for follicle-stimulating hormone in peripheral blood|
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Thymopentin significantly promoted ovarian angiogenesis in mice
Immunofluorescence staining showed that the ratio of newly formed vascular endothelial cells (CD31+) in the ovarian tissues of mice in the TP-5 treatment group was significantly higher than that of mice in the saline-treated group [Figure 2]. Meanwhile, the fraction of cells positive for receptor tyrosine kinase Tie1 (CD31+/Tie1+) was significantly higher in the newly formed vascular endothelial cells of the TP-5 group than those of the saline-treated group [Figure 2]. These results indicate that TP-5 promotes ovarian angiogenesis in mice with POF.
|Figure 2: Immunofluorescence staining of angiogenesis markers in mouse ovarian tissues|
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Thymopentin promoted Notch/Tie2 molecular pathway expression in ovarian tissues
RNA-Seq was used to analyze the overall differences in the RNA transcriptomes of the ovarian tissues from mice in the TP-5 treatment group and those in the saline-treated group. We focused on 106 pathways and genes associated with angiogenesis and analysed the differences in their expression [Figure 3]a and [Supplementary Table 1]. In particular, the mRNAs of 10 genes were significantly upregulated in the TP-5 treatment group compared to the saline-treated group (CTX + TP-5/CTX + Saline >2; P < 0.01). The results were validated with qPCR confirming that the 10 genes were in fact expressed at significantly higher levels in the TP-5 treatment group than in the saline-treated group [Figure 3]b. Subsequently, we performed bioinformatic analysis (STRING: functional protein association networks; https://string-db.org/) and identified the interaction network of the angiogenesis-associated proteins. The software prediction results indicated that the proteins encoded by the three genes involved in the Notch pathway (Notch3, Jag2, Dll4) and the three vascular endothelial cell–associated growth factors (Tie1, Vwf, F8) were intrinsically linked and could form a complete pathway [Figure 3]c.
|Figure 3: Thymopentin-5 activated the expression of angiogenesis-associated factors. (a) RNA-Seq results; (b) quantitative polymerase chain reaction results; P < 0.01, t-test, n = 6; (c) Predicted interaction network for proteins in the Notch/Tie1 pathway|
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Thymopentin promoted the expression of RNA m6A methyltransferases and the RNA m6A methylation of key factors in the Notch/Tie2 molecular pathway
We initially examined changes in the expression levels of RNA m6A methyltransferases in each sample. Both the qPCR and Western blot results showed that the expression levels of Mettle3 and Mettle14 were significantly higher in the ovaries of mice in the TP-5 treatment group than in those in the saline group [Figure 4]a and [Figure 4]b. The immunofluorescence staining results were also consistent with these results [Figure 4]c. Dot blotting then showed that the overall level of RNA m6A modification in the ovaries of mice in the TP-5 treatment group was significantly higher than that in the ovaries of mice in the saline group and was similar to that in ovaries from the normal control group [Figure 4]d. RIP-PCR on the ovaries of mice treated with TP-5 also showed that with compounds that could be cross-linked with anti-m6A antibody (α M6A ab), specific products could be amplified from the 3'-untranslated region (3'UTR) of the mRNAs of six key factors in the Notch/Tie2 molecular pathway using PCR [Figure 4]e. In contrast, with compounds that could be cross-linked with α M6A ab, the aforementioned 3'UTR specific products were hardly amplified in the ovaries of mice in the saline group using PCR [Figure 4]e. These findings demonstrated that TP-5 can increase the overall RNA m6A methylation level in mouse ovarian tissues through promoting the expression of RNA m6A methyltransferases. Furthermore, TP-5 can increase the methylation levels of specific m6A sites in the 3'UTR of the RNA of key factors in the Notch/Tie2 pathway, thereby enhancing their stability and expression.
|Figure 4: Thymopentin5 promoted RNA methylation (N6-methyladenosine) of key factors in the Notch/Tie1 pathway. (a) quantitative polymerase chain reaction results; **P < 0.01 versus WT; ##P < 0.01 versus cerebrotendinous xanthomatosis + Saline; t-test; n = 6; (b) Western blot results; *P < 0.05 versus WT; ##P < 0.01 versus cerebrotendinous xanthomatosis + Saline; t-test; n = 6; (c) Immunofluorescence staining; (d) Sot blot; (e) RNA immunoprecipitation-polymerase chain reaction results|
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| Discussion|| |
Studies have determined that the factors that play important roles in the pathogenesis of POF include inflammation, genetic mutations, mood swings, endocrine disorders, and pathological aging.,,,,, However, ovarian insufficiency caused by injury to and atrophy of blood vessels in the ovaries has not been reported. Nonetheless, considering the importance of blood vessels for tissues and organs, we decided to investigate its intrinsic link with POF pathogenesis. In this study, we focused on two aspects: the first pathways related to angiogenesis and the second epigenetic modifications of factors in angiogenesis-related pathways. We found that the expression of the Notch/Tie1 signaling pathway was significantly reduced in the ovaries of mice with POF, while treatment with TP-5 significantly increased its expression, suggesting that TP-5 likely promotes ovarian angiogenesis. The Notch signaling pathway is broadly present in vertebrates and invertebrates and is highly conserved. This pathway regulates the differentiation and development of cells, tissues, and organs through modulating interactions between adjacent cells.,, The Notch signaling pathway is comprised of the Notch receptor, Notch ligand (Jagged canonical Notch ligand protein), CBF-1, Suppressor of hairless, Lag (CSL = CBF-1, Suppressor of hairless, Lag) DNA-binding protein, other effectors, and molecular regulators of Notch.,, Mammals contain four Notch receptors (Notch 1–4) and five Notch ligands (DLL1, 3, 4, Jagged1, Jagged2). The notch signal is generated by the interaction of the Notch ligand and receptor present on adjacent cells. The Notch protein is cleaved three times, after which the notch intracellular domain (NICD) is released into the cytoplasm and enters the nucleus. Within the nucleus, NCID binds to the transcription factor CSL, forming the NICD/CSL transcription activation complex, which activates the target genes in the Basichelix-loop-helix transcription repressor family, such as HES, HEY, and HERP.,, The TIE1 protein belongs to the Tie family of receptor tyrosine kinases. TIE1 is structurally similar to its homolog TIE2, but TIE1 is an orphan receptor while TIE2 is not.,,,,,, A key function of TIE1 is regulating TIE2 signalling through heterodimerization with TIE2 on the cell surface.,,,,,, The effect of the TIE1-TIE2 interaction is dependent on the environment; heterodimerization can promote or inhibit downstream TIE2 signaling based on local TIE2 levels.,,,,,, Many studies have shown that the Notch pathway and TIE1 are closely related to angiogenesis, vascular maturation, tissue remodeling, and inflammation. However, there have been no reports showing intrinsic links between these factors and POF pathogenesis. The present study found that the expression of the Notch/Tie2 signaling pathway was significantly reduced during the development of POF. However, treatment with TP-5 significantly increased expression of the Notch/Tie2 signaling pathway in ovarian tissues from the POF mouse model. These findings suggested that TP-5 likely promoted angiogenesis in ovarian tissues through increasing the expression of the Notch/Tie2 signaling pathway, thereby exerting its therapeutic effects.,,,,,,,,,,,,,,
This study showed how TP-5 promoted the stable expression of the Notch/Tie2 signaling pathway by regulating RNA methylation. RNA m6A refers to the methylation of adenosine at the nitrogen-6 position in RNA. RNA m6A methylation is widely present in most eukaryotic species (from yeast, plants, and fruit flies, to mammals) and viral mRNA, and it plays a key modulatory role in the posttranscriptional regulation and metabolism of mRNA.,,,, Meyer et al. and Dominissini et al. both utilized m6A-specific immunoprecipitation high-throughput sequencing to analyze human and mouse genes. They studied the distribution of m6A RNA modifications in a transcriptome-wide manner. They found that m6A was mainly distributed in the 3'-UTR of mRNA, as well as near the stop codon in the coding regions (CDS) of mRNA. They also demonstrated that the distribution of m6A in humans and mice was highly conserved. Furthermore, it has been reported that m6A modifications on RNA can increase mRNA stability.,,, Further investigations have shown that m6A is mainly enriched in the vicinity of stop codons, 3'UTRs, and long internal exons of mRNA, and the predominant conserved sequences are G (m6A) C (70%) and A (m6A) C (30%).,, Based on these clues, we examined m6A modifications in specific 3'UTR regions of the mRNA of key factors in the Notch/Tie2 signaling pathway. We found that the levels of m6A modifications in the specific 3'UTR regions of mRNA of key factors in the Notch/Tie2 signaling pathway were very low in the ovaries of the POF mouse model. Following TP-5 treatment, the levels of m6A modifications in the specific regions of the aforementioned genes increased significantly. We speculated that TP-5 achieved this effect through promoting the expression of the m6A methyltransferase METTL3 in ovarian tissues [Figure 5].,,,,,,,
|Figure 5: Thymopentin5 promotes ovarian angiogenesis in mice via inducing N6-methyladenosine RNA modification of key factors in the Notch/Tie1 pathway|
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Therefore, this study demonstrated that TP-5 exerted its therapeutic effect on POF through stimulating angiogenesis. The mechanism of action was promotion of the expression of RNA m6A methyltransferases by TP-5, which increased the overall level of RNA m6A methylation in mouse ovarian tissues. Specifically, methylation levels in specific m6A sites in the 3'UTR of RNA of key factors in the Notch/Tie2 pathway were increased, which enhanced their stability and expression levels, leading to the induction of angiogenesis.
| Conclusion|| |
The mechanism of TP-5 is the promotion of the expression of RNA m6A methyltransferases by TP5, which increases the overall RNA m6A methylation level in mouse ovarian tissue. Specifically, the methylation ofspecific m6A sites in the 3'UTR of the RNA of key factors in the Notch/Tie2 pathway increased, which enhanced their stability and expression levels, leading to the induction of angiogenesis.
This work was supported by grant from the National Natural Science Foundation of China (No. 81973899). And, grant from the projects sponsored by the development fund for Shanghai talents (2017054). And, grant from the projects sponsored by the fund for Xinglin talents of Shanghai University of TCM (201707081). We declared no potential conflicts of interest. Beijing Hospitals Authority Training Programme (PX2018035), and Beijing Hospitals Authority Youth Programme (QML20180804).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Liu T, Huang Y, Guo L, Cheng W, Zou G. CD44+/CD105+ human amniotic fluid mesenchymal stem cells survive and proliferate in the ovary long-term in a mouse model of chemotherapy-induced premature ovarian failure. Int J Med Sci 2012;9:592-602.
Xiong Y, Liu T, Wang S, Chi H, Chen C, Zheng J, et al.
Cyclophosphamide promotes the proliferation inhibition of mouse ovarian granulosa cells and premature ovarian failure by activating the lncRNA-Meg3-p53-p66Shc pathway. Gene 2017;596:1-8.
Ai A, Xiong Y, Wu B, Lin J, Huang Y, Cao Y, et al.
Induction of miR-15a expression by tripterygium glycosides caused premature ovarian failure by suppressing the Hippo-YAP/TAZ signaling effector lats1. Gene 2018;678:155-63.
Liu T, Wang S, Li Q, Huang Y, Chen C, Zheng J, et al.
Telocytes as potential targets in a cyclophosphamide-induced animal model of premature ovarian failure. Mol Med Rep 2016;14:2415-22.
Liu T, Huang Y, Zhang J, Qin W, Chi H, Chen J, et al.
Transplantation of human menstrual blood stem cells to treat premature ovarian failure in mouse model. Stem Cells Dev 2014;23:1548-57.
Sajib S, Zahra FT, Lionakis MS, German NA, Mikelis CM. Mechanisms of angiogenesis in microbe-regulated inflammatory and neoplastic conditions. Angiogenesis 2018;21:1-4.
Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298-307.
Mathew SA, Naik C, Cahill PA, Bhonde RR. Placental mesenchymal stromal cells as an alternative tool for therapeutic angiogenesis. Cell Mol Life Sci 2019;doi: 10.1007/s00018-019-03268-1.
Viallard C, Larrivée B. Tumor angiogenesis and vascular normalization: Alternative therapeutic targets. Angiogenesis 2017;20:409-26.
Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR, et al.
Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 2012;149:1635-46.
Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al.
Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 2012;485:201-6.
Yue Y, Liu J, He C. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev 2015;29:1343-55.
Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al.
N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 2011;7:885-7.
Chen T, Hao YJ, Zhang Y, Li MM, Wang M, Han W, et al.
M (6) A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell 2015;16:289-301.
Tang C, Klukovich R, Peng H, Wang Z, Yu T, Zhang Y, et al.
ALKBH5-dependent m6A demethylation controls splicing and stability of long 3'-UTR mRNAs in male germ cells. Proc Natl Acad Sci U S A 2018;115:E325-33.
Chen M, Wei L, Law CT, Tsang FH, Shen J, Cheng CL, et al.
RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2. Hepatology 2018;67:2254-70.
Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, et al.
A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 2014;10:93-5.
Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, et al.
N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 2014;505:117-20.
Barbieri I, Tzelepis K, Pandolfini L, Shi J, Millán-Zambrano G, Robson SC, et al.
Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control. Nature 2017;552:126-31.
Li J, Cheng Y, Zhang X, Zheng L, Han Z, Li P, et al.
The in vivo
immunomodulatory and synergistic anti-tumor activity of thymosin α1-thymopentin fusion peptide and its binding to TLR2. Cancer Lett 2013;337:237-47.
Liu Z, Zheng X, Wang J, Wang E. Molecular analysis of thymopentin binding to HLA-DR molecules. PLoS One 2007;2:e1348.
Fan YZ, Chang H, Yu Y, Liu J, Zhao L, Yang DJ, et al.
Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells. Biochim Biophys Acta 2006;1763:1059-66.
Xiaojing C, Yanfang L, Yanqing G, Fangfang C. Thymopentin improves cardiac function in older patients with chronic heart failure. Anatol J Cardiol 2017;17:24-30.
Wu C, Zhang M, Zhang Z, Wan KW, Ahmed W, Phoenix DA, et al
. Thymopentin nanoparticles engineered with high loading efficiency, improved pharmacokinetic properties, and enhanced immunostimulating effect using soybean phospholipid and PHBHHx polymer. Mol Pharm 2014;11:3371-7.
Liu T, Chi H, Chen J, Chen C, Huang Y, Xi H, et al.
Curcumin suppresses proliferation and in vitro
invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR. Gene 2017;631:29-38.
Liu T, Cheng W, Huang Y, Huang Q, Jiang L, Guo L, et al.
Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression. Exp Cell Res 2012;318:424-34.
Yan G, Schoenfeld D, Penney C, Hurxthal K, Taylor AE, Faustman D, et al.
Identification of premature ovarian failure patients with underlying autoimmunity. J Womens Health Gend Based Med 2000;9:275-87.
Wieland E, Rodriguez-Vita J, Liebler SS, Mogler C, Moll I, Herberich SE, et al.
Endothelial Notch1 activity facilitates metastasis. Cancer Cell 2017;31:355-67.
Ramasamy SK, Kusumbe AP, Wang L, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 2014;507:376-80.
D'Amico G, Korhonen EA, Anisimov A, Zarkada G, Holopainen T, Hägerling R, et al.
Tie1 deletion inhibits tumor growth and improves angiopoietin antagonist therapy. J Clin Invest 2014;124:824-34.
La Porta S, Roth L, Singhal M, Mogler C, Spegg C, Schieb B, et al.
Endothelial Tie1-mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis. J Clin Invest 2018;128:834-45.
Savant S, La Porta S, Budnik A, Busch K, Hu J, Tisch N, et al.
The orphan receptor Tie1 controls angiogenesis and vascular remodeling by differentially regulating Tie2 in tip and stalk cells. Cell Rep 2015;12:1761-73.
Leppänen VM, Saharinen P, Alitalo K. Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization. Proc Natl Acad Sci U S A 2017;114:4376-81.
Korhonen EA, Lampinen A, Giri H, Anisimov A, Kim M, Allen B, et al.
Tie1 controls angiopoietin function in vascular remodeling and inflammation. J Clin Invest 2016;126:3495-510.
Qu X, Zhou B, Scott Baldwin H. Tie1 is required for lymphatic valve and collecting vessel development. Dev Biol 2015;399:117-28.
Mueller SB, Kontos CD. Tie1: An orphan receptor provides context for angiopoietin-2/Tie2 signaling. J Clin Invest 2016;126:3188-91.
Bartosovic M, Molares HC, Gregorova P, Hrossova D, Kudla G, Vanacova S, et al.
N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing. Nucleic Acids Res 2017;45:11356-70.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]