Method for activating ASXL2-PRC2 axis and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a method for activating an ASXL2-PRC2 axis, which is characterized in that adenovirus is injected into seminiferous tubules of a living body through an outgoing tube, and expression of endogenous EZH2 and spermatogenic cell specificity ASXL2 of the living body is activated. The method disclosed by the invention can be used for alleviating damaged sperm motility, increasing sperm penetration and tubulin polymerization, inhibiting sperm abnormality, down-regulating the transcriptional level of a TZ marker, increasing the enrichment of SUZ12, EED and H3K27me3 in CEP162 and CEP290 promoters under an anoxic condition, and retarding hypoxia-induced abnormal spermatogenesis in a germ cell line of an organism and spermatogenic cell activation of an ASXL2-PRC2 axis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for activating an ASXL2-PRC2 axis and an application thereof. Background Art

[0002] It is well known that hypoxia affects spermatogenesis, however, its underlying mechanism remains largely unclear. To explore its potential mechanism, we placed 8-week-old SD male rats in a hypobaric oxygen chamber with an oxygen concentration of 11.7% (simulating an altitude of 5800 meters), while age- and sex-matched control rats were placed in a normal oxygen concentration (21%) for 10 weeks to study the effects of severe chronic hypoxia on spermatogenesis in rats. The results showed that long-term hypoxic exposure impaired the development of elongated spermatids. It was also observed that under normal conditions, the expression of Asxl2 gradually decreased from spermatogonia to spermatocytes, suddenly increased at the round spermatid stage, and then gradually decreased again. However, in hypoxic-treated cells, the expression of Asxl2 decreased throughout spermatogenesis, and there was a significant difference in expression between normal and hypoxic treatments, especially at the round spermatid stage. Subsequent analysis showed that chronic hypoxic exposure may increase the expression of CEP162 by downregulating the Asxl2 / PRC2 axis, thereby leading to abnormal spermatogenesis.

[0003] At the same time, we further studied the relationship between ASXL2-PRC2 and male infertility diseases by screening the Male Infertility Knowledge Base (MKI) and found that ASXL2 and PRC2 are jointly involved in regulating the occurrence and development of teratozoospermia and cryptorchidism. In order to demonstrate the clinical relevance of ASXL2 and PRC2 in spermatogenesis, we analyzed the correlation between ASXL2 and PcG gene expression using the sequence archive read (SRA) dataset, which consists of expression data from 3 non-obstructive azoospermia (NOA) samples, 3 non-obstructive azoospermia samples, 13 normospermic individuals, and 8 teratozoospermic individuals. Figure 1 As shown, the analysis revealed that the expression of ASXL2 or PRC2 was downregulated in patients with teratozoospermia and NOA.

[0004] In conclusion, the ASXL2-PRC2 axis is closely related to abnormal spermatogenesis. Summary of the invention

[0005] The present invention provides a method for activating the ASXL2-PRC2 axis, aiming to activate the ASXL2-PRC2 axis by an external method, reduce the expression of CEP162, and thus prevent sperm abnormality.

[0006] To achieve the above object, the present invention provides a method for activating the ASXL2-PRC2 axis, and the specific steps are as follows:

[0007] Adenovirus is injected into the seminiferous tubules of the organism through the efferent ductus, and the adenovirus is used to activate the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2 in the organism.

[0008] Furthermore, the adenovirus is pAAV2-EFS-mAsxl2-3xFLAG-pA virus or pAAV2-EFS-3xFLAG-pa virus.

[0009] The present invention also provides an application of a method for activating the ASXL2-PRC2 axis, wherein the method for activating the ASXL2-PRC2 axis is used to downregulate the transcription level of TZ markers and to increase the enrichment of SUZ12, EED and H3K27me3 in CEP162 and CEP290 promoters under hypoxic conditions;

[0010] Wherein, the TZ markers include CEP162 and CEP290.

[0011] The beneficial effects of the present invention are:

[0012] The present invention provides a method for activating the ASXL2-PRC2 axis, wherein an adenovirus is injected into the seminiferous tubules of an organism through the efferent ducts to activate the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2 in the organism. The method of the present invention can be used to alleviate impaired sperm motility, increase sperm penetration and tubulin polymerization, inhibit sperm abnormalities, downregulate the transcription level of TZ markers, increase the enrichment of SUZ12, EED and H3K27me3 in CEP162 and CEP290 promoters under hypoxia, and slow down hypoxia-induced abnormal spermatogenesis in the germ cell line of an organism and spermatogenic cell activation of the ASXL2-PRC2 axis.

[0013] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For clinical outcomes;

[0015] Figure 2 for exogenous overexpression of ASXL2 / EZH2 in mice;

[0016] Figure 3 It is used to show that exogenous activation of Asxl2-PRC2 axis can significantly reduce sperm tail deformity and alleviate hypoxia-induced abnormal spermatogenesis;

[0017] Figure 4 Figure 3 Gene expression data analysis of the median level of teratozoospermia / NOA in the GEO / SRA dataset. DETAILED DESCRIPTION

[0018] In order to make the technical solutions, advantages and purposes of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of this application.

[0019] This embodiment provides a method for activating an ASXL2-PRC2 axis, comprising the following steps:

[0020] Adenovirus pAAV2-EFS-mAsxl2-3xFLAG-pA virus or pAAV2-EFS-3xFLAG-pa virus was injected into the seminiferous tubules of mice through the efferent ductus to activate the expression of mouse endogenous EZH2 and spermatogenic cell-specific ASXL2, respectively, and their effects on spermatogenesis were detected.

[0021] Comparative experiment

[0022] The mice were exposed to hypoxia for 10 weeks, during which time injections were given every three weeks.

[0023] Experimental group: 8-week-old SD male rats were placed in a hypoxic environment (a hypobaric oxygen chamber with an oxygen concentration of 11.7% to simulate an altitude of 5,800 meters) for 10 weeks. During this period, adenovirus was injected once every three weeks into the seminiferous tubules of the mice through the efferent ductus.

[0024] Control group: 8-week-old SD male rats (control rats) were selected and placed under normal oxygen concentration (21%) for 10 weeks.

[0025] Experimental analysis

[0026] pAAV2-EFS-mAsxl2-3xflag-pA and pAAV2-CAG-EZH2-EGFP-3xflag-WPRE-pA were used to stain mouse testicular sections 3 weeks after virus injection. Figure 2 It can be seen that it successfully activated the ASXL2-PRC2 axis.

[0027] Figure 2Exogenous overexpression of ASXL2 / EZH2 in mice, where A is the immunostaining of ASXL2 10 weeks after microinjection of AAV2; B is the immunostaining of EZH2 10 weeks after microinjection of AAV2; C is the WB analysis of ASXL2 expression in wild-type untransplanted and microinjected testes within 10 weeks using AAV2; D is the WB analysis of EZH2 expression in wild-type untransplanted and microinjected testes within 10 weeks using AAV2.

[0028] Mice were exposed to 11.7% oxygen for 10 weeks and injected with ASXL2-AAV or EZH2-AAV.

[0029] like Figure 3 As shown in A-3H, activation of the ASXL2-PRC2 axis significantly alleviated impaired sperm motility.

[0030] We also found that activation of the ASXL2-PRC2 axis significantly increased sperm penetration ( Figure 3 I) and tubulin polymerization, and significantly inhibited sperm abnormalities ( Figure 3 J).

[0031] Next, we detected the expression of free tubulin and polymerized microtubules in spermatogenic cells. Western blot analysis showed that after ASXL2 / PRC2 activation, free tubulin decreased and polymerized microtubules increased significantly in spermatogenic cells ( Figure 3 K).

[0032] Currently, it is widely accepted that components of cilia, including the TZ (such as CEP162 and CEP290), play an important role in spermatogenesis and that Polycomb is involved in regulating the transition zone. Therefore, we investigated whether activation of the ASXL2-PRC2 axis would alter the levels of TZ components. Mice were injected with the same virus to activate the ASXL2-PRC2 axis. Indeed, qPCR analysis showed that activation of the ASXL2-PRC2 axis significantly downregulated the transcript levels of TZ markers, including CEP162 and CEP290 ( Figure 3 L and 3M, Figure 4 ). In addition, ChIP-qPCR analysis showed that activation of the ASXL2-PRC2 axis increased the enrichment of SUZ12, EED, and H3K27me3 in the CEP162 and CEP290 promoters under hypoxia ( Figure 3 N-3P). Taken together, these results indicate that activation of the ASXL2-PRC2 axis significantly rescues H3 histone 27 lysine methyltransferase activity and TZ transcription and significantly reverses the abnormal changes in TZ composition, slowing hypoxia-induced abnormal spermatogenesis and spermatogenic activation of the ASXL2-PRC2 axis in the mouse germ line.

[0033] Figure 3 Exogenous activation of the Asxl2-PRC2 axis significantly reduced sperm tail deformities and alleviated hypoxia-induced abnormal spermatogenesis. Figure 3 (A) Spermatozoa survived 10 weeks in 11.7% oxygen (n=12). Sperm motility parameters, including (B) progressive motility (n=12), (C) linear velocity (n=12). Figure 3 D to 3H represent the sperm curve velocity (D), average path velocity (E), forwardness (F), whipping frequency (G) and head swing amplitude (H) motion parameters detected by sperm automatic analyzer after ASXL2 and EZH2 were overexpressed in mouse sperm cells under hypoxia. (I) The entry of spermatogonia into oocytes was observed by acetic acid fuchsin staining, and the percentage of sperm entering the oocyte nucleus was calculated (%) (n=12). (J) Quantitative analysis of abnormalities between WT and ASXL2-AAV and EZH2-AAV (n=12). (K) Immunoblotting analysis of free tubulin and polymerized microtubules in spermatogenic cells (n=10). (L) qPCR analysis of ciliary assembly-related genes in primary spermatogenic cells between ASXL2-AAV group and control group (n=6). (M) qPCR was also used to compare the mRNA levels of CEP162 and CEP290 between ASXL2 overexpression treatment group and EZH2 overexpression treatment group and control group in GC2 cells. The mRNA expression values ​​of the treatment groups were normalized to that of β-actin and expressed as the ratio of the normalized mRNA expression value to the mRNA expression in control cells (n=10). Figure 3 After overexpression of ASXL2 and EZH2 in G2 cells, Cut&tag-qPCR was used to detect the binding of SUZ12 (N), EED (O), and H3K27me3 (P) on the CEP162 and CEP290 promoters. In G2 cells treated with control, AD-ASXL2, or AD-EZH2, (N) SUZ12 (N = 10) and (O) EED (N = 10) on the CEP162 (left) or CEP290 (right) promoters were detected. Data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 were determined by two-tailed, unpaired Student's multiple t-test (L) and one-way ANOVA with Tukey's post hoc test (AK, MP).

[0034] Figure 4 Analysis of gene expression data for the median of teratozoospermia / NOA in the GEO / SRA dataset, and the statistics of patients identified as having high or low expression of (A) teratozoospermia or (B) NOA-related genes.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the protection scope of the present invention.

Claims

1. A method for activating an ASXL2-PRC2 axis, characterized in that: The specific steps are as follows: Adenovirus is injected into the seminiferous tubules of the organism through the efferent ductus, and the adenovirus is used to activate the expression of endogenous EZH2 and spermatogenic cell-specific ASXL2 in the organism.

2. A method for activating an ASXL2-PRC2 axis according to claim 1, characterized in that: The adenovirus is pAAV2-EFS-mAsxl2-3xFLAG-pA virus or pAAV2-EFS-3xFLAG-pa virus.

3. An application of a method for activating the ASXL2-PRC2 axis, using the method for activating the ASXL2-PRC2 axis according to any one of claims 1-2, for downregulating the transcription level of TZ markers and increasing the enrichment of SUZ12, EED and H3K27me3 in the CEP162 and CEP290 promoters under hypoxic conditions; in, The TZ markers include CEP162 and CEP290.

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