Huiling Zheng

Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model

A de novo G608G mutation in LMNA gene leads to Hutchinson–Gilford progeria syndrome. Mice lacking the prelamin A-processing metalloprotease, Zmpste24, recapitulate many of the progeroid features of Hutchinson–Gilford progeria syndrome. Here we show that A-type lamins interact with SUV39H1, and prelamin A/progerin exhibits enhanced binding capacity to SUV39H1, protecting it from proteasomal degradation and, consequently, increasing H3K9me3 levels. Depletion of Suv39h1 reduces H3K9me3 levels, restores DNA repair capacity and delays senescence in progeroid cells. Remarkably, loss of Suv39h1 in Zmpste24−/− mice delays body weight loss, increases bone mineral density and extends lifespan by ∼60%. Thus, increased H3K9me3 levels, possibly mediated by enhanced Suv39h1 stability in the presence of prelamin A/progerin, compromise genome maintenance, which in turn contributes to accelerated senescence in laminopathy-based premature aging. Our study provides an explanation for epigenetic alterations in Hutchinson–Gilford progeria syndrome and a potential strategy for intervention by targeting SUV39H1-mediated heterochromatin remodelling.

miR‑107 promotes the erythroid differentiation of leukemia cells via the downregulation of Cacna2d1

microRNAs (miRNAs) have been reported to be involved in various human diseases. They may have uses in diagnosis and as therapeutic targets, thus the discovery of novel miRNAs has the potential to provide clinical tools or shed light on novel mechanisms. In the current study, miR‑107 was revealed to be downregulated in chronic myeloid leukemia cells. Overexpression of miR‑107 in K562 and KCL‑22 chronic myeloid leukemia cells promotes erythroid differentiation, while having no effect on cell proliferation. Further bioinformatics predicted that one target of miR‑107 may be Cacna2d1, a calcium channel protein. A luciferase reporter assay and quantitative polymerase chain reaction were utilized to confirm that Cacna2d1 is a target molecule of miR-107. The effect of miR‑107 on K562 and KCL‑22 cells was mediated through the downregulation of Cacna2d1, as rescued expression of Cacna2d1 reversed the effects of miR‑107. In summary, the current study identified a novel miRNA that is involved in chronic myeloid leukemia cell erythroid differentiation and the associated mechanisms, making it a potential therapeutic target in the treatment of chronic myeloid leukemia.

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression.

Identification of FAM96B as a novel prelamin A binding partner

Prelamin A accumulation causes nuclear abnormalities, impairs nuclear functions, and eventually promotes cellular senescence. However, the underlying mechanism of how prelamin A promotes cellular senescence is still poorly understood. Here we carried out a yeast two-hybrid screen using a human skeletal muscle cDNA library to search for prelamin A binding partners, and identified FAM96B as a prelamin A binding partner. The interaction of FAM96B with prelamin A was confirmed by GST pull-down and co-immunoprecipitation experiments. Furthermore, co-localization experiments by fluorescent confocal microscopy revealed that FAM96B colocalized with prelamin A in HEK-293 cells. Taken together, our data demonstrated the physical interaction between FAM96B and prelamin A, which may provide some clues to the mechanisms of prelamin A in premature aging.

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.

miR‑342‑5p promotes Zmpste24‑deficient mouse embryonic fibroblasts proliferation by suppressing GAS2

Cellular senescence is an irreversible growth arrest of cells that maintain their metabolic activities. Premature senescence can be induced by different stress factors and occurs in mouse embryonic fibroblasts (MEFs) derived from Zmpste24 metalloproteinase-deficient mice, a progeria mouse model of Hutchinson-Gilford Progeria Syndrome. Previous studies have shown that miR-342-5p, an intronic microRNA (miRNA/miR) reportedly involved in ageing associated diseases, is downregulated in Zmpste24−/− MEFs. However, whether miR-342-5p is associated with the premature senescence phenotype of Zmpste24−/− MEFs remains unclear. Thus, the present study investigated the effects of miR-342-5p on cellular senescence and cell proliferation in Zmpste24−/− MEFs. The results showed that miR-342-5p overexpression ameliorated the cellular senescence phenotype to a certain extent, promoted cell proliferation and increased the G2+M cell cycle phase in Zmpste24−/− MEFs. Nonetheless, it was difficult to observe the opposite cell phenotypes in wild-type (WT) MEFs transfected with the miR-342-5p inhibitor. Growth-arrest-specific 2 (GAS2) was identified as a target gene of miR-342-5p in Zmpste24−/− MEFs. In addition, miR-342-5p was identified to be downregulated in WT MEFs during replicative senescence, while Gas2 was upregulated. Taken together, these findings suggest that downregulated miR-342-5p is involved in regulating cell proliferation and cell cycles in Zmpste24−/− MEFs by suppressing GAS2 in vitro.

miR‐194 functions as a novel modulator of cellular senescence in mouse embryonic fibroblasts

MicroRNA‐194 (miR‐194), a typical p53 responsive miRNA, serves as a tumor suppressor similar as p53, and has been demonstrated to play an anti‐proliferation role in various human cancers. In spite of the pivotal role of p53 during aging process, the knowledge of miR‐194s contribution to cellular senescence is limited. We herein sought to explore the role of miR‐194 in the replicative senescence and stress‐induced senescence of mouse embryonic fibroblasts. Our results unraveled that, compared to young cells, miR‐194 is highly expressed in senescent cells, and extra expression of miR‐194 significantly triggers the replicative senescence of MEFs and H2O2‐induced senescence of NIH/3T3 cells, while inhibition of miR‐194 exhibited the opposite effect. We further unveiled that DNMT3A was a direct and authentic target of miR‐194, which has been reported to be closely associated with cellular senescence. Taken together, our data suggest that miR‐194 may significantly promote the development of cellular senescence in mouse embryonic fibroblasts, which potentially occurs through inhibiting the DNMT3A expression.