L-type Ca 2+ channels (LTCCs) are the main mediators of Ca 2+ influx. LTCCs are multi-subunit complexes, of which CaV1.2 subunit forms the channel pore for Ca 2+ movement. Ca 2+ influx through LTCC CaV1.2 is a proximal signal for pathological cardiomyocyte hypertrophy. However, very little is known regarding the molecular mechanism of LTCC transcription regulation. Myocardin, a co-activator of serum response factor (SRF) that potently transactivates CArG box-containing cardiac and smooth muscle target genes, can stimulate neonatal rat cardiomyocyte hypertrophy. In the present study, the gene fragment of rat LTCC CaV1.2 promoter containing five CArG boxes or no CArG box was amplified by PCR and cloned into empty vector pGL3-Basic to construct luciferase reporter plasmids, then luciferase activity assay was performed in HEK293T cells. The results showed that the luciferase reporter plasmids were constructed successfully, Myocardin potently activated CArG boxcontaining LTCC CaV1.2 reporter, by contrast, Myocardin had a weaker transcriptional activation on LTCC CaV1.2 reporter containing no CArG box. The results suggest that Myocardin may play a critical role in regulating the expression of LTCC CaV1.2 in cardiomyocyte hypertrophy.
Background Promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the main therapeutic goal for postmenopausal osteoporosis (PMOP). Recently, several long non-coding RNAs (lncRNAs) have been reported to be involved in PMOP; however, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIM3) remains to be investigated. Methods The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIM3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay. Results The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and OCN were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIM3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIM3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIM3 exhibited the reverse results. Subsequently, lncTIM3 was confirmed to be located in the cytoplasm of BMSC, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIM3 and Smad4 were elucidated in vitro. Conclusion lncTIM3 attenuated PMOP via miR-214/Smad4. Possibly, these findings provided lncTIM3 as a therapeutic molecule for PMOP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.