ABSTRACT
Objective@#To screen differentially expressed miRNAs in the testis of male rats exposed to cigarette smoke (CS) and identify the early molecular markers of CS-induced apoptosis of testicular cells.@*METHODS@#We randomly divided 200 SPF male SD rats into blank control and low-dose (10 non-filter cigarettes/d), medium-dose (20 non-filter cigarettes/d) and high-dose (30 non-filter cigarettes/d) CS exposure groups. After 2, 4, 6, 8 and 12 weeks of CS exposure, we observed the histopathological changes of the testis by HE staining, detected the apoptosis of the testicular cells by TUNEL, and determined the expressions of caspase-3 and caspase-9 in the testis tissue by immunohistochemistry, RT-PCR and Western blot. Based on the laboratory results, we selected 4 testicular samples from the 12-week high-dose group and another 4 from the control for miRNA microarray-based screening, bioinformatics analysis, and verification of differentially expressed miRNAs in all the animals by RT-PCR.@*RESULTS@#Compared with the controls, the CS-exposed rats showed dose- and time-dependent increase in the atrophy of the testis and significantly increased number of apoptotic testis cells from the 6th week of exposure (P < 0.05), with dramatically up-regulated expressions of caspase-3 (P < 0.01) and caspase-9 protein and mRNA (P < 0.05) in the testis tissue. Microarray-based screening and RT-PCR revealed 5 differentially expressed miRNAs in the testis of the CS-exposed rats, of which miR-138-5p, miR-181d-5p, miR-19a-3p and miR-3588 were down-regulated, and miR-155-5p up-regulated, and the target genes of the differentially expressed miRNAs positively regulated the apoptosis of the testicular cells.@*CONCLUSIONS@#The differentially expressed miRNAs miR-155-5p, miR-138-5p, miR-181d-5p, miR-19a-3p and miR-3588 regulate CS-induced apoptosis of testicular cells, and may become biomarkers for early diagnosis and prognosis of CS-induced spermatogenesis obstruction.《.