Exploration of small RNA biomarkers for testicular injury in the serum exosomes of rats

Testicular injury is often observed in drug development. Serum hormones are usually used as non-invasive biomarkers for testicular injury; however, their sensitivity is low. Therefore, it is difficult to monitor testicular injury in pre-clinical and clinical drug developments. In recent years, molecules in body fluid exosomes are attracting attention as disease biomarkers such as cancer. In this study, small RNAs in serum exosomes were analyzed for identifying non-invasive biomarkers of testicular injury in rats, which are mainly used in a pre-clinical drug development. Testicular injury models in rats were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether in which spermatocytes degeneration and sertoli cells vacuolation were observed, or 400 mg/kg carbendazim in which sertoli cells vacuolation and seminiferous tubules dilation were observed. Serum exosome small RNA-seq was performed in these models. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For qPCR validation of the candidates, testicular injury models were further prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone in which spermatocytes degeneration and sertoli cells vacuolation were observed. In qPCR analysis, the selected two miRNAs in exosomes were upregulated in the all models except for 1,3-dinitrobenzene model in which severe hemolysis was observed. On the other hand, the miRNAs in serum did not significantly change in the any models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosome as non-invasive biomarkers for testicular injury in rats.药物研发过程中常可观察到睾丸损伤。血清激素通常被用作睾丸损伤的无创生物标志物，但其灵敏度较低，因此在临床前与临床药物研发中难以对睾丸损伤进行有效监测。近年来，体液外泌体（exosome）中的分子作为癌症等疾病的生物标志物受到广泛关注。本研究针对临床前药物研发中常用的大鼠模型，分析其血清外泌体中的小分子RNA（small RNA），以期筛选出睾丸损伤的无创生物标志物。本研究通过两种方式构建大鼠睾丸损伤模型：单次口服给予2000mg/kg的乙二醇单甲醚（ethylene glycol monomethyl ether），该模型可见生精细胞（spermatocytes）变性与支持细胞（Sertoli cells）空泡化；或单次口服给予400mg/kg的多菌灵（carbendazim），该模型可见支持细胞空泡化与生精小管（seminiferous tubules）扩张。对上述模型开展血清外泌体小分子RNA测序（small RNA-seq）分析，共筛选出3种在两类模型中均出现表达波动的小分子RNA，并选定miR-423-5p与miR-128-3p作为候选标志物。为验证候选标志物，本研究进一步构建了另外两种大鼠睾丸损伤模型：单次口服给予60mg/kg的1,3-二硝基苯（1,3-dinitrobenzene），或500mg/kg的呋喃唑酮（nitrofurazone），两类模型均可见生精细胞变性与支持细胞空泡化。实时定量PCR（qPCR）验证结果显示，除观察到严重溶血的1,3-二硝基苯模型外，其余模型中外泌体中的选定两种miRNA均呈现表达上调；而血清中的miRNA在所有模型中均未出现显著表达变化。综上，本研究筛选出血清外泌体中的miR-423-5p与miR-128-3p，可作为大鼠睾丸损伤的无创生物标志物。