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Background Recent studies have characterized that microRNA (miRNA) is a suitable candidate for the study of bleomycin/LPS-induced pulmonary fibrosis, but the knowledge on miRNA in bacteria-induced pulmonary fibrosis (BIPF) is limited. Forest musk deer (Moschus berezovskii, FMD) is an important endangered species that has been seriously affected by BIPF. We sought to determine whether miRNA exist that modulates the pathogenesis of BIPF in FMD. Methods High-throughput sequencing and RT-qPCR were used to determine the differentially expressed miRNAs (DEmiRNAs) in the blood of BIPF FMD. The DEmiRNAs were further detected in the blood and lung of BIPF model rat by RT-qPCR, and the targeting relationship between candidate miRNA and its potential target gene was verified by dual-luciferase reporter activity assay. Furthermore, the function of the candidate miRNA was verified in the FMD lung fibroblast cells (FMD-C1). Results Here we found that five dead FMD were suffered from BIPF, and six circulating miRNAs (miR-30g, let-7f-5p, miR-27-3p, miR-25-3p, miR-9-5p and miR-652) were differentially expressed in the blood of the BIPF FMD. Of these, let-7f-5p showed reproducibly lower level in the blood and lung of the BIPF model rat, and the expression levels of PI3K/AKT/COX2 signaling pathway genes (PIK3CA, PDK1, Akt1, IKBKA, NF-κB1 and COX2) were increased in the lung of BIPF model rats, suggesting that there is a potential correlation between BIPF and the PI3K/AKT/COX2 signaling pathway. Notably, using bioinformatic prediction and experimental verification, we demonstrated that let-7f-5p is conserved across mammals, and the seed sequence of let-7f-5p displays perfect complementarity with the 3’ UTR of PIK3CA gene and the expression of the PIK3CA gene was regulated by let-7f-5p. In order to determine the regulatory relationship between let-7f-5p and the PI3K/AKT/COX2 signaling pathway in FMD, we successfully cultured FMD-C1, and found that let-7f-5p could act as a negative regulator for the PI3K/Akt/COX2 signaling pathway in FMD-C1. Collectively, this study not only provided a study strategy for non-invasive research in pulmonary disease in rare animals, but also laid a foundation for further research in BIPF.

The screening of reference genes for real-time quantitative PCR (qPCR) in forest musk deer (FMD) tissue is of great significance to the basic research on FMD.However, there are few reports on the stability analysis of FMD reference genes so far. In this study, We used qPCR to detect the expression levels of 11 reference gene candidates (18S rRNA, beta-actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box-binding protein [TBP], hypoxanthine phosphoribosyltransferase 1 [HPRT1], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide [YWHAZ], hydroxymethylbilane synthase [HMBS], eukaryotic translation elongation factor 1 alpha 1 [EEF1A1], succinate dehydrogenase complex flavoprotein subunit A [SDHA], peptidylprolyl isomerase B [PPIB], and ubiquitin C [UBC]) in heart, liver, spleen, lung and kidney of FMD. After removing 18S rRNA on account of its high expression level, geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to evaluate the expression stability of the remaining genes in the five organs, and further comprehensive ranking was calculated by RefFinder. According to the results, the selected reference genes with the most stable expression in the heart of FMD are SDHA and YWHAZ, while in the liver are ACTB and SDHA; in the spleen and lung are YWHAZ and HPRT1; in the kidney are YWHAZ and PPIB. The use of common reference genes in all five organs is not recommended. The analyses showed that tissue is an important variability factor in genes expression stability. Meanwhile, the result can be used as a reference for the selection of reference genes for qPCR in further study.

Background: Forest musk deer (Moschus berezovskii) is a national first-level protected and endangered wild animals in China. The intestinal coccidiosis of captive forest musk deer is one of the most important diseases. However, few studies have been conducted to quantify Eimeria sp. infection and to identify its molecules. Thus, the objective of this study was to investigate the Eimeria sp. infection in the intestinal tract of forest musk deer in Sichuan and Shaanxi, China, and to identify the 18S rRNA gene fragment of Eimeria sp. , which provides scientific basic experimental data for the molecular epidemiological investigation and population genetic analysis of Eimeria sp. of captive forest musk deer in 7 regions.Methods: 328 faecal samples of forest musk deer were collected from 7 farms. The DNA of Eimeria sp. in the positive samples was extracted and used as template for nested PCR amplification. The 18S rRNA gene fragment was connected with the plasmid vector, and the products were introduced into Escherichia coli (DH5α). The culture bacterial solution was used as a PCR reaction template for identification.Results: In total, we collected 328 faecal samples from forest musk deer in Lixian (n=54), Maoxian (n=52), Ma’erkang (n=49), Dujiangyan (n=55), Hanyuan (n=41), Luding (n=36) and Weinan (n=41). 198 (60.37%) faecal samples were positive for Eimeria sp. We analyzed the 18S rRNA gene sequence of Eimeria sp, and determined 34 types with similarity of 90.5% ~ 100%. A phylogenetic tree constructed based on the 18S rRNA gene sequence of Eimeria sp., it was confirmed that Eimeria sp. parasitized in the intestinal tract of forest musk deer was closely related to Eimeria alabamensis from Bos taurus, Eimeria faurei from Ovis aries and Eimeria ahsata from Ovis aries.Conclusions: To our knowledge, this was the first molecular identification of Eimeria sp. in the intestinal tract of forest musk deer. The study suggested that Eimeria sp. parasitizes in the intestinal tract of forest musk deer, which is their carrier.