Panoply™ Human CDK9 Over-expressing Stable Cell Line

Cdk9 is the catalytic core of positive transcription elongation factor b (P-TEFb), regulating transcription elongation by phosphorylating RNA polymerase II. Besides its role in myogenic gene expression, the regulatory mechanism of Cdk9 on muscle-specific microRNAs during early cardiac myogenesis remains unclear. Here, researchers show that Cdk9 not only regulates myogenic transcription factors but also controls the expression of muscle-specific microRNAs. During cardiac differentiation of mouse embryonic stem cells, high expression of Cdk9 precedes the upregulation of miR-1. To investigate the potential regulatory role of Cdk9 on cardiac microRNAs and myogenesis genes, researchers overexpressed Cdk9 in C2C12 myoblasts, resulting in a significant upregulation of miR-1 and miR-206, while miR-133 was downregulated. Furthermore, the expression levels of key myogenic regulatory factors MyoD and Srf were also increased in Cdk9-overexpressing cells. It was further observed that Cdk9-mediated apoptosis in C2C12 cells was associated with increased miR-1 expression levels. Therefore, Cdk9 plays a complex role in myoblast differentiation and apoptosis by regulating the expression of myogenic proteins and muscle-specific microRNAs.

Previous studies have reported that miR-1 can inhibit cell growth and induce apoptosis. Since Cdk9 overexpression leads to the upregulation of miR-1, researchers examined the viability of Cdk9-transfected cells. As shown in Figure 1A, the transfected cells exhibited morphological changes, including a decrease in cell number and an increase in cell death rate. Compared to the control group, the proliferation rate of Cdk9-overexpressing cells was inhibited (Figure 1B). The growth rate of Cdk9-overexpressing cells was 30-40% lower than that of the control group (Figure 1B). Furthermore, MTT assays showed that cell proliferation was significantly inhibited in Cdk9-overexpressing cells compared to the control group (Figure 1C). Researchers used FITC-Annexin V and PI staining combined with flow cytometry to determine the percentage of apoptotic cells. As shown in Figure 1D, the number of apoptotic cells was significantly increased in Cdk9-overexpressing cells compared to the control group (approximately 50%). To further analyze Cdk9-mediated apoptosis, they partially inhibited the function of miR-1 by transfecting Cdk9-overexpressing cells with miR-1 antisense oligonucleotides. Interestingly, flow cytometry analysis showed a significant decrease in the number of apoptotic cells in cells transfected with miR-1 antisense oligonucleotides (Figure 1E). These results indicate that Cdk9 regulates the apoptosis of myoblast progenitor cells by regulating the expression of microRNA-1.

Figure 1. Cdk9 induces apoptosis by modulating miR-1 expression in C2C12 cells. (Tarhriz V, et al., 2018)