rno-miR-90 promotes chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SPARC-related modular calcium binding 2.

Bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into chondrocytes. In the differentiation of BMSCs into chondrocytes, micro-RNAs (miRNAs) play an important role. rno-miR-90 is a new miRNA discovered by our research team, and its role in chondrogenic differentiation of BMSCs is unknown. This study aimed to investigate whether rno-miR-90 could promote chondrogenic differentiation of BMSCs by regulating secreted protein acidic and rich in cysteine-related modular calcium binding 2 (Smoc2). First, BMSCs chondroblast differentiation was successfully induced in vitro by classical induction method of transforming growth factor (TGF)-ß3. On this basis, we transfected rno-miR-90 mimic and inhibitor, and confirmed that rno-miR-90 mimic could promote the differentiation of BMSCs into chondrocytes by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. In addition, we demonstrated that Smoc2 was a target gene of rno-miR-90 by dual-luciferase reporter assay, and confirmed that rno-miR-90 mimic could inhibit the expression of Smoc2 by RT-qPCR and western blotting. In order to further prove the targeting relationship between rno-miR-90 and Smoc2, we constructed three interfering fragments of Smoc2, and proved that silencing Smoc2 could promote the differentiation of BMSCs into chondrocytes at the transcriptional and protein levels. Finally, we constructed a carrier scaffold for ectopic chondrogenic differentiation in vivo, and confirmed that rno-miR-90 mimic and siSmoc2 could promote chondrogenic differentiation of BMSCs by Alcian blue staining and immunohistochemistry. In summary, our results suggested that rno-miR-90 could promote chondrogenic differentiation of BMSCs by down-regulating the expression of Smoc2. rno-miR-90 mimic and Smoc2 may be therapeutic targets of osteoarthritis.

The changes of the cardiac structure and function in cTnTR141W transgenic mice.

OBJECTIVE: To establish the transgenic mouse of cTnT(R141W) gene to make an animal model of dilated cardiomyopathy. METHODS: A transgenic plasmid was constructed by inserting the cTnT(R141W) gene driven by the alpha-MHC promoter. The expression level of the gene was determined with Northern blotting. Pathologic changes were observed by light microscopy and transmission electronic microscopy and analyzed with echocardiography. The localization of the mutant human cTnT protein was detected by immunohistochemistry. The hypertrophy markers were analyzed by RT-PCR. RESULTS: Transgenic mice carrying the cTnT(R141W) mutation were established. The cTnT(R141W) was expressed by 1.5- to 2.0-fold that of the endogenous cTnT gene and was showed to assemble in the sarcomere. The transgenic heart exhibited a thinner ventricular wall and an enlarged ventricular chamber. Interstitial fibrosis and the elongated and lysed myofrils were also observed in the transgenic heart tissue. The function on EF%, FS% and movement of the ventricular wall was significantly decreased. The immature death occurred after 4 months of age and the immature death rate was 11.1% before 8 months of age in the cTnT(R141W) mice. The increased NPPB, ACTA1 and decreased ATP2A2 were detected in the transgenic heart. CONCLUSIONS: The expression of mutant cTnT(R141W) in the mouse heart caused ventricular chamber enlargement, systolic dysfunction, myocardial hypertrophy, and interstitial fibrosis, suggesting that the cTnT(R141W) gene is a causal factor for DCM and that the cTnT(R141W) transgenic mouse is a useful animal model for the study of human DCM.