MicroRNA miR-214-5p induces senescence of microvascular endothelial cells by targeting the JAG1/Notch signaling pathway.

During cellular senescence, irreversible cell cycle arrest is accompanied by morphological and genetic alterations. MicroRNAs (miRNAs) play a critical role in regulating senescence by modulating the abundance of crucial senescence regulatory proteins. Therefore, to identify novel senescence-associated miRNAs, we analyzed differentially expressed miRNAs in microvascular endothelial cells (MVEC). Among the 80 differentially expressed miRNAs in replicative senescent MVECs, 16 miRNAs of unknown gene ontology were used in the senescence-associated ß-galactosidase assay. Thus, we identified miR-214-5p as having high senescence-inducing activity, inhibiting the proliferation and angiogenesis activity of MVECs. To reveal the senescence-regulating mechanism of miR-214-5p, we searched for target genes through sequence- and literature-based analysis. Molecular manipulation of miR-214-5p demonstrated that miR-214-5p regulated the expression and function of Jagged 1 (JAG1) in senescent MVECs. Silencing JAG1 or downstream genes of JAG1-Notch signaling, accelerated the senescence of MVECs. Additionally, ectopic overexpression of JAG1 reversed the senescence-inducing activity of miR-214-5p. In conclusion, we identified miR-214-5p as a senescence-associated miRNA. Targeting miR-214-5p may be a potential strategy to delay vascular aging and overcome the detrimental effects of senescence and age-related diseases.

MicroRNA-Mediated Downregulation of HMGB2 Contributes to Cellular Senescence in Microvascular Endothelial Cells.

High mobility group box 2 (HMGB2) is a non-histone chromosomal protein involved in various biological processes, including cellular senescence. However, its role in cellular senescence has not been evaluated extensively. To determine the regulatory role and mechanism of HMGB2 in cellular senescence, we performed gene expression analysis, senescence staining, and tube formation assays using young and senescent microvascular endothelial cells (MVECs) after small RNA treatment or HMGB2 overexpression. HMGB2 expression decreased with age and was regulated at the transcriptional level. siRNA-mediated downregulation inhibited cell proliferation and accelerated cellular senescence. In contrast, ectopic overexpression delayed senescence and maintained relatively higher tube-forming activity. To determine the HMGB2 downregulation mechanism, we screened miRNAs that were significantly upregulated in senescent MVECs and selected HMGB2-targeting miRNAs. Six miRNAs, miR-23a-3p, 23b-3p, -181a-5p, -181b-5p, -221-3p, and -222-3p, were overexpressed in senescent MVECs. Ectopic introduction of miR-23a-3p, -23b-3p, -181a-5p, -181b-5p, and -221-3p, with the exception of miR-222-3p, led to the downregulation of HMGB2, upregulation of senescence-associated markers, and decreased tube formation activity. Inhibition of miR-23a-3p, -181a-5p, -181b-5p, and -221-3p delayed cellular senescence. Restoration of HMGB2 expression using miRNA inhibitors represents a potential strategy to overcome the detrimental effects of cellular senescence in endothelial cells.

Mechanism mediated by a noncoding RNA, nc886, in the cytotoxicity of a DNA-reactive compound.

DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.

Anticancer and radiosensitizing effects of the cyclin-dependent kinase inhibitors, AT7519 and SNS­032, on cervical cancer.

Cyclin-dependent kinases (CDK) are considered to be potential targets of anticancer drugs that can interrupt the uncontrolled division of cancer cells. In this study, we selected two selective CDK inhibitors, AT7519 and SNS­032, from current clinical trials and examined their anticancer and radiosensitizing effects in a cervical cancer model. SNS­032 was found to be more potent than AT7519, with a lower half maximal inhibitory concentration (IC50) value. Both AT7519 and SNS­032 induced the apoptosis, premature senescence and cytostasis of cervical cancer cells, which led to the attenuation of tumor growth in vivo. Moreover, using these CDK inhibitors together with radiation synergistically inhibited tumor growth in a human xenograft tumor model. The concomitant activation of the p53 tumor suppressor and the suppression of cell cycle checkpoint responses mediated by Chk1 led to the cytostasis of cervical cancer cells. Finally, AT7519 and SNS­032 inhibited cancer cell migration, invasion and angiogenesis in vitro, and suppressed lung metastases in a spontaneous metastasis model. On the whole, the findings of this study indicate that the utilization of AT7519 and SNS­032 as part of an adjuvant treatment may help control cervical cancer progression.