MiR-590-5p inhibits pathological hypertrophy mediated heart failure by targeting RTN4.

Heart failure (HF) is a rising epidemic and public health burden in modern society. It is of great need to find new biomarkers to ensure a timely diagnosis and to improve treatment and prognosis of the disease. The mouse model of HF was established by thoracic aortic constriction. Color Doppler ultrasound was performed to detect left ventricular end-diastolic diameter. Hematoxylin and eosin staining was conducted to observe the pathological changes of mouse myocardium. The RT-qPCR analysis was performed to detect miR-590-5p and RTN4 expression levels. Western blot was conducted to detect protein levels of the indicated genes. We found that the expression of miR-590-5p was downregulated in cardiac tissues of HF mice. Injection of AAV-miR-590-5p attenuated myocardium hypertrophy and myocyte apoptosis. Additionally, miR-590-5p overexpression promoted viability, inhibited apoptosis, and decreased ANF, BNP and beta-MHC protein levels in H9c2 cell. Mechanistically, miR-590-5p binds to RTN4 3'-untranslated region, as predicted by starBase online database and evidenced by luciferase reporter assay. Furthermore, miR-590-5p negatively regulates RTN4 mRNA expression and suppresses its translation. The final rescue experiments revealed that miR-590-5p modulated cardiomyocyte phenotypes by binding to RTN4. In conclusion, miR-590-5p modulates myocardium hypertrophy and myocyte apoptosis in HF by downregulating RTN4.

ß-Glucanase specific expression in the parotid gland of transgenic mice.

The feasibility of using the pig parotid secretory protein promoter to drive the ß-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing ß-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the ß-glucanase gene and the ß-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The ß-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % ß-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign ß-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of ß-glucans in feed.

Production of transgenic pigs mediated by pseudotyped lentivirus and sperm.

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.