MiR-429及其目标基因HSPA4L在弱精症低活力精子中的表达

Expression of MiR-429 and Its Target Gene HSPA4L in Sperms from Asthenospermia Patients

目的探讨miR-429及其目标基因热休克蛋白A4L(HSPA4L)在弱精症患者低活力精子中的表达。方法收集20份成年健康生育能力的精液标本和20份弱精症患者精液标本,按照WHO第五版规定的精液分级标准,对液化后的精液进行常规参数分析;利用qRT-PCR检测精子中miR-429的表达水平;通过生物信息学手法预测miR-429的靶点;构建HSPA4L3′UTR双萤光素酶报告载体,采用双萤光素酶报告实验验证miR-429的靶基因;利用qRT-PCR和Western blot分别检测HSPA4L mRNA和蛋白的表达水平。结果弱精症精子活力低于健康对照组,同时miR-429在弱精症精子中表达上调。通过生物信息学手法、双萤光素酶报告基因基检测以及转染实验发现miR-429通过作用于HSPA4L3′UTR来抑制HSPA4L mRNA和蛋白的表达。进一步的qRT-PCR分析发现弱精症组的HSPA4L mRNA和蛋白的表达水平下调,并且HSPA4L mRNA表达水平与miR-429的表达水平呈负相关(r=-0.725,P<0.05)。结论 MiR-429在低活力精子中的表达上调,可能通过调控HSPA4L基因来影响精子的活力。

Objective To investigate the expression of miR-429 and its target gene heat shock protein A4L （HSPA4L） in sperms from asthenospermia patients. Methods Twenty semen samples from healthy and fertile adults and 20 semen samples from asthenospermia patients were collected, and normal sperm parameters were defined according to World Health Organization criteria. The expression levels of miR-429 and HSPA4L mRNA were determined by qRT-PCR, and the bioinformatics tool （Targetscan） was used to predict the target of miR-429. Luciferase reporter assay and transfection study were performed to confirm target gene of rniR-429. The expression levels of HSPA4L mRNA and protein were further determined by qRT-CPR and Western blot, respectively. Results The motility and viability of sperms from asthenospermia patients were lower than that in control group, and miR- 429 was up-regulated in sperms from asthenospermia patients. Bioinformatics analysis revealed that HSPA4L was a target of miR-429. Luciferase reporter assay and transfection study further confirmed that rniR-429 suppresses the expressions of HSPA4L mRNA and protein via directly targeting HSPA4L 3＇UTR. Results from clinical samples also demonstrated that HSPA4L mRNA and protein were down-regulated in sperms from asthenospermia patients and the expression level of miR-429 was inversely correlated with the expression level of HSPA4L mRNA （r=-0. 725,P〈0.05）. Conclusion MiR-429 is up-regulated in sperms from asthenospermia patients, and it may modulate the motility and viability of sperms via suppressing the expression of HSPA4L.