精浆外泌体miR-26a-5p靶向PTEN调控特发性畸形精子症的分子机制及诊断价值

doi:10.3969/j.issn.1006-5725.2025.20.016

Abstract

Abstract:

Objective This study aims to elucidate the functional mechanism through which seminal plasma exosomal miR-26a-5p and its target genes contribute to idiopathic teratozoospermia， with emphasis on their regulatory pathways in sperm morphogenesis defects， thereby establishing a theoretical foundation for novel diagnostic markers and targeted therapies. Methods A case-control study design was adopted， enrolling 154 male subjects （84 in the teratozoospermia group ［TZS］ and 70 in the normal control group ［NC］）. Seminal plasma exosomes were isolated to screen differentially expressed miRNAs， followed by prediction and analysis of target genes and signaling pathways. Dual-luciferase reporter gene assays were employed to validate the direct binding of miR-26a-5p to PTEN. Quantitative real-time PCR and Western blot were used to measure miRNA and protein expression levels. Spearman correlation analysis evaluated relationships between miR-26a-5p， PTEN， and sperm morphological parameters， while ROC curve analysis assessed diagnostic efficacy. Results Seminal plasma exosomal miRNA sequencing identified 11 differentially expressed miRNAs， with miR-26a-5p significantly upregulated in the TZS group （P < 0.05） and negatively correlated with the percentage of normal sperm morphology in TZS （r = -0.762， P < 0.05）. PTEN was confirmed as a direct target of miR-26a-5p， with its expression significantly reduced in the TZS group （P < 0.05） and positively correlated with normal sperm morphology （r = 0.821， P < 0.05）. A negative correlation was observed between miR-26a-5p and PTEN （r = -0.878， P < 0.05）. Dual-luciferase assays demonstrated that miR-26a-5p specifically inhibited PTEN 3'UTR activity （45% reduction in luciferase activity， P < 0.05）. Functional enrichment analysis revealed that the miR-26a-5p-PTEN axis exacerbates oxidative stress， DNA damage， and abnormal spermatocyte division by regulating the PI3K/AKT/mTOR， p53， Wnt/β-catenin， and NF?κB pathways. ROC analysis showed diagnostic efficacy for teratozoospermia with AUC^ROC values of 0.785 （sensitivity 78.9%， specificity 71.3%） for miR-26a-5p and 0.754 （sensitivity 73.3%， specificity 75.1%） for PTEN. Conclusions miR-26a-5p suppresses PTEN through targeted inhibition， activating PI3K/AKT/mTOR signaling pathways while impairing DNA repair functions. This cascade leads to accumulated oxidative stress and sperm morphological abnormalities. The combined pattern of elevated seminal plasma exosomal miR-26a-5p expression and reduced PTEN levels demonstrates diagnostic potential for idiopathic teratozoospermia， with its molecular mechanism providing a theoretical foundation for biomarker development. Targeted silencing of miR-26a-5p， either alone or in combination with AKT/mTOR inhibitors and antioxidant therapy， may represent a novel strategy for improving sperm morphology.

Key words: seminal plasma exosomes, teratozoospermia, miR-26a-5p, PTEN, molecular mechanisms

CLC Number:

R697

Chunhui LIU,Ruipeng WU,Zhiqiang WANG,Wensheng SHAN,Shaojun. LI. Molecular mechanisms and diagnostic value of seminal plasma exosomal miR-26a-5p targeting PTEN in idiopathic teratozoospermia[J]. The Journal of Practical Medicine, 2025, 41(20): 3256-3266.

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miRNAs	MI编号	功能	表型/疾病	Up/Down
hsa-miR-23b-3p	MIMAT0000418	抑制癌细胞增殖，促进转移性乳腺癌休眠	转移性乳腺癌	Down
hsa-miR-212-5p	MIMAT0000269	抗血管平滑肌细胞增殖，减轻肺血管重构	抗血管平滑肌细胞增殖，抗肺血管重构	Down
hsa-miR-381-3p	MIMAT0000736	参与神经功能调控，影响MAPK信号通路	铝暴露导致的认知障碍	Up
hsa-miR-370-3p	MIMAT0000722	调控靶基因FGF7，影响神经元功能	铝暴露导致的认知障碍	Up
hsa-miR-26a-5p	MIMAT0000082	调控氧化应激、细胞增殖与凋亡，调控DNA修复，拮抗磷酸化	精子异常，肝癌，肺纤维化	Up
hsa-miR-29a-5p	MIMAT0004516	靶向胶原蛋白基因，抑制纤维化；调节DNA甲基化相关酶	肝纤维化、慢性淋巴细胞白血病	Down
hsa-miR-92a-3p	MIMAT0000092	抑制抑癌基因表达；参与血管内皮功能调控	结直肠癌、动脉粥样硬化	Up
hsa-miR-155-5p	MIMAT0000646	促炎性调控，增强免疫应答及B细胞分化	淋巴细胞白血病、自身免疫性疾病	Up
hsa-miR-17-5p	MIMAT0000070	调控细胞周期；促进血管生成	胃癌、肺癌（与EGFR突变相关）	Down
hsa-miR-139-5p	MIMAT0000250	调控TET2和TET3表达，参与基因沉默	空腔脏器肿瘤（调控表观遗传修饰）	Up
hsa-miR-15a-5p	MIMAT0000068	抑制细胞增殖，促进凋亡；调控免疫检查点PD-1/PD-L1通路	慢性淋巴细胞白血病、心血管疾病	Up

miRNA靶基因	预测的碱基对序列	位点性质
PTEN 3'UTR	5'-CAUCAUAAGAAACAUACUUGAA-3'	8mer
miR-26a-5p	3'-UCGGAUAGGACCUAAUGAACUU-5'	8mer

组别	例数	年龄/岁	精子浓度/ （×10⁶·mL^-1）	PR/%	T/(ng/mL)	FSH/ (mIU/mL)	LH/ (mIU/mL)	miR-26a-5p	PTEN
NC组	70	28.32 ± 5.85	69.25 ± 12.21	37.18 ± 5.28	4.26 ± 1.36	5.34 ± 1.65	4.19 ± 1.56	1.000 ± 0.000	1.000 ± 0.000
TZS组	84	27.26 ± 6.31	76.75 ± 10.35	39.34 ± 6.34	4.13 ± 1.05	6.18 ± 1.88	4.53 ± 1.16	1.625 ± 0.213 ^*	0.452 ± 0.121 ^*
F值		1.125	3.842	1.477	1.395	2.654	2.639	16.361	24.762
P值		0.784	0.521	0.845	0.698	0.593	0.498	< 0.001	< 0.001

变量	miR-26a-5p	PTEN	正常形态精子占比（NC组）	正常形态精子占比（TZS组）
miR-26a-5p	—	r = -0.878 P = 0.000	r = -0.585 P = 0.036	r = -0.762 P = 0.016
PTEN	r = -0.878 P = 0.000	—	r = 0.638 P = 0.023	r = 0.821 P = 0.002
正常形态精子占比（NC组）	r = -0.585 P = 0.036	r = 0.638 P = 0.023	—	r = -0.214 P = 0.437
正常形态精子占比（TZS组）	r = -0.762 P = 0.016	r = 0.821 P = 0.002	r = -0.214 P = 0.437	—

项目		WT + NC mimic	WT + miR-26a-5p mimic	MUT + NC mimic	MUT + miR-26a-5p mimic
Firefly luciferase	Group 1	321 514	241 265	168 549	125 489
	Group 2	315 284	236 584	171 594	121 687
	Group 3	312 541	231 984	163 658	119 864
	AVG	316 446.333	236 611.0	167 933.666	122 346.666
Renilla luciferase	Group 1	313 548	136 847	158 954	129 824
	Group 2	318 744	131 654	168 964	126 846
	Group 3	316 954	125 875	169 896	122 364
	AVG	316 415.333	131 458.666	165 938.0	126 344.666
Ratio RLUC/FLUC	Group 1	0.9 752 234 739	0.5 672 061 841	0.9 430 729 343	1.0 345 448 605
	Group 2	1.0 109 742 327	0.5 564 788 828	0.9 846 731 238	1.0 423 956 544
	Group 3	1.0 141 197 474	0.542 602 076	1.0 381 160 713	1.0 208 484 545
	AVG	1.0 016 053 275	0.5 555 898 331	0.9 881 163 435	1.0 326 776 375
Normalization	AVG	1.000 ± 0.107	0.552 ± 0.076 ^*	0.985 ± 0.064	1.025 ± 0.086

Molecular mechanisms and diagnostic value of seminal plasma exosomal miR-26a-5p targeting PTEN in idiopathic teratozoospermia

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组别	指标	AUC^ROC	95%CI	P值	灵敏度/%	特异度/%
NC组	miR-26a-5p	0.589	0.538 ~ 0.636	0.569	36.7	29.3
NC组	PTEN	0.564	0.512 ~ 0.596	0.694	32.9	26.4
TZS组	miR-26a-5p	0.785	0.719 ~ 0.894	< 0.000 1	78.9	71.3
TZS组	PTEN	0.754	0.696 ~ 0.826	< 0.000 1	73.3	75.1

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