Regulation Of Cellular Senescence Research Articles

PA200-Mediated Proteasomal Protein Degradation and Regulation of Cellular Senescence

PA200-Mediated Proteasomal Protein Degradation and Regulation of Cellular Senescence

Abstract 6834: Periostin involvement in cellular senescence in ovarian cancer

Abstract Purpose: Despite improvements in care and advancements in the understanding of ovarian cancer (OC) pathobiology and genetics, survival rates remain disappointingly low compared to other types of cancer. This is due to a lack of diagnosis strategies, high recurrence rates, and the development of chemo-resistant diseases, which require synergy between cancer cells and the tumor microenvironment (TME). We have demonstrated the functional importance of a TME gene, periostin (POSTN), in OC and recurrent OC, using in vitro and in vivo models. Our objectives were to identify differentially expressed genes by POSTN expression in the TME that contribute to the progression of ovarian cancers. Procedures: Gene expression data was generated from an ovarian cancer cell line, HEYA8, cultured in a high POSTN conditioned 3D cell culture environment, using the NovaSeq 6000 system to generate RNA-seq data. The data was analyzed to determine the genes expressed at different levels between the HEYA8 cells grown with or without POSTN-high conditioned medium (CM). The gene functional annotation and KEGG pathway enrichment were further analyzed from 10,115 significant differentially expressed genes between the HEYA8 cells grown in high levels of POSTN (CMPOSTNhigh) or low levels of POSTN (CMCTL) culture conditions, using DAVID bioinformatics. Results: Using a cell culture and co-culture model, we found that OC cells cultured under conditioned media containing CMPOSTNhigh exhibited faster cell migration, more invasiveness (p=0.006), more chemo-resistance (p<0.05), and increased resistance to paclitaxel-induced apoptosis, compared to OC cells cultured with control medium (CMCTL). OC cell lines cultured with CMPOSTNhigh showed increased side population cells relative to CMCTL -cultured cells, suggesting POSTN plays important roles in cancer stem cell enrichment. POSTN-transfected 3T3-L1 cells exhibited more intracellular and extracellular lipids and were linked to increased cancer cell expression of the oncogene fatty acid synthetase. In correspondence to the functions of POSTN in OC cells, the pathways identified to be significantly impacted by POSTN expression included, but were not limited to, the cell cycle, apoptosis, p53 signaling, MAPK signaling, and PI3K-Akt signaling pathways. Interestingly, we also found cellular senescence pathways was one of the most significantly impacted by POSTN suggesting that POSTN may play roles in ovarian cancer via the regulation of cellular senescence, which has not yet been explored. Conclusions: These data emphasize the importance of TME factors in tumor progression and prognosis via various ways in ovarian cancer, establishing promising potential therapeutic targets of OC. Citation Format: Jihua Feng, Ethan Nguyen, Jocelyn Reyes, Zhiqing Huang. Periostin involvement in cellular senescence in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6834.

Identification of Osteoarthritis Inflamm-Aging Biomarkers by Integrating Bioinformatic Analysis and Machine Learning Strategies and the Clinical Validation

To identify inflamm-aging related biomarkers in osteoarthritis (OA). Microarray gene profiles of young and aging OA patients were obtained from the Gene Expression Omnibus (GEO) database and aging-related genes (ARGs) were obtained from the Human Aging Genome Resource (HAGR) database. The differentially expressed genes of young OA and older OA patients were screened and then intersected with ARGs to obtain the aging-related genes of OA. Enrichment analysis was performed to reveal the potential mechanisms of aging-related markers in OA. Three machine learning methods were used to identify core senescence markers of OA and the receiver operating characteristic (ROC) curve was used to assess their diagnostic performance. Peripheral blood mononuclear cells were collected from clinical OA patients to verify the expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. A total of 45 senescence-related markers were obtained, which were mainly involved in the regulation of cellular senescence, the cell cycle, inflammatory response, etc. Through the screening with the three machine learning methods, 5 core senescence biomarkers, including FOXO3, MCL1, SIRT3, STAG1, and S100A13, were obtained. A total of 20 cases of normal controls and 40 cases of OA patients, including 20 cases in the young patient group and 20 in the elderly patient group, were enrolled. Compared with those of the young patient group, C-reactive protein (CRP), interleukin (IL)-6, and IL-1β levels increased and IL-4 levels decreased in the elderly OA patient group (P<0.01); FOXO3, MCL1, and SIRT3 mRNA expression decreased and STAG1 and S100A13 mRNA expression increased (P<0.01). Pearson correlation analysis demonstrated that the selected markers were associated with some indicators, including erythrocyte sedimentation rate (ESR), IL-1β, IL-4, CRP, and IL-6. The area under the ROC curve of the 5 core aging genes was always greater than 0.8 and the C-index of the calibration curve in the nomogram prediction model was 0.755, which suggested the good calibration ability of the model. FOXO3, MCL1, SIRT3, STAG1, and S100A13 may serve as novel diagnostic biomolecular markers and potential therapeutic targets for OA inflamm-aging.

CLCA2: A Potential Guardian against Premature Senescence and Skin Aging.

Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.

P21 as a Predictor and Prognostic Indicator of Clinical Outcome in Rectal Cancer Patients.

The cell cycle plays a key and complex role in the development of human cancers. p21 is a potent cyclin-dependent kinase inhibitor (CDKI) involved in the promotion of cell cycle arrest and the regulation of cellular senescence. Altered p21 expression in rectal cancer cells may affect tumor cells' behavior and resistance to neoadjuvant and adjuvant therapy. Our study aimed to ascertain the relationship between the differential expression of p21 in rectal cancer and patient survival outcomes. Using tissue microarrays, 266 rectal cancer specimens were immunohistochemically stained for p21. The expression patterns were scored separately in cancer cells retrieved from the center and the periphery of the tumor; compared with clinicopathological data, tumor regression grade (TRG), disease-free, and overall survival. Negative p21 expression in tumor periphery cells was significantly associated with longer overall survival upon the univariate (p = 0.001) and multivariable analysis (p = 0.003, HR = 2.068). Negative p21 expression in tumor periphery cells was also associated with longer disease-free survival in the multivariable analysis (p = 0.040, HR = 1.769). Longer overall survival times also correlated with lower tumor grades (p= 0.011), the absence of vascular and perineural invasion (p = 0.001; p < 0.005), the absence of metastases (p < 0.005), and adjuvant treatment (p = 0.009). p21 expression is a potential predictive and prognostic biomarker for clinical outcomes in rectal cancer patients. Negative p21 expression in tumor periphery cells demonstrated significant association with longer overall survival and disease-free survival. Larger prospective studies are warranted to investigate the ability of p21 to identify rectal cancer patients who will benefit from neoadjuvant and adjuvant therapy.

Cellular Senescence in Non-Small Cell Lung Cancer.

Lung cancer has the highest mortality rate amongst all malignancies worldwide, and is the second-highest incidence of cancer in women. Non-small cell lung cancer (NSCLC) is responsible for approximately 80% of lung cancer cases. Recent studies indicate that cellular senescence may be a promising cancer biomarker. However, the regulation of cellular senescence and its underlying mechanisms in NSCLC are not yet fully understood. Here, we present a comprehensive analysis of the genes linked to cellular senescence in NSCLC. We also describe the secretory phenotype associated with NSCLC and examine its immune profile and prognostic potential. Our findings offer novel insights into the development of effective NSCLC treatments.

The chemotherapeutic agent doxorubicin induces brain senescence, with modulation by APOE genotype

Many cancer patients experience serious cognitive problems related to their treatment, which can greatly affect their quality of life. The molecular mechanisms of this cancer chemotherapy-induced cognitive impairment (CICI) are unknown, thus slowing the development of preventative approaches. We hypothesized that cancer chemotherapies could induce cellular senescence in the brain, creating a pro-inflammatory environment and damaging normal brain communication. We tested this hypothesis using the common chemotherapeutic agent doxorubicin in two independent mouse models. In the first model, we used mice that express tdTomato under the pdkn2a (p16) promoter; p16 is a regulator of cellular senescence, and its upregulation is denoted by the presence of fluorescently tagged cells. Two weeks after exposure to three doses of 5 mg/kg doxorubicin, the number of tdTomato positive cells were increased nearly three-fold in both the cerebral cortex and the hippocampus. tdTomato staining co-localized with neurons, microglia, oligodendrocyte precursor cells, and endothelial cells, but not astrocytes. In the second model, we used APOE knock-in mice, since the APOE4 allele is a risk factor for CICI in humans and mouse models. We isolated RNA from the cerebral cortex of APOE3 and APOE4 mice from one to 21 days after a single dose of 10 mg/kg doxorubicin. Using NanoString analysis of over 700 genes related to neuroinflammation and RT-qPCR analysis of cerebral cortex transcripts, we found two-fold induction of four senescence-related genes at three weeks in the APOE4 mice compared to the APOE3 control mice: p21(cdkn1a), p16, Gadd45a, and Egr1. We conclude that doxorubicin promotes cellular senescence pathways in the brain, supporting the hypothesis that drugs to eliminate senescent cells could be useful in preventing CICI.

The Role and Application of the AMPK-Sirtuins Network in Cellular Senescence.

Aging and related diseases significantly affect the health and happiness index around the world. Cellular senescence is the basis of physiological aging and is closely related to various senile diseases. AMP-activated protein kinase (AMPK) is associated with both the regulation of cellular energy metabolism and the regulation of cellular senescence. Another set of proteins, sirtuins, has also been demonstrated to play an important role in cell senescence. However, it is not clear how AMPK and sirtuins coordinate to regulate cellular senescence. Herein, we summarized the role of AMPK and sirtuins in regulating metabolism, repairing DNA damage, and even prolonging human life. We have provided a detailed explanation of the clinical trials relating to the AMPK and sirtuins involved in aging. Systematically analyzing individual senescence genes and developing functional reference notes will aid in understanding the potential mechanisms underlying aging and identify therapeutic targets for both anti-aging interventions and age-related illnesses.

Metabolic regulation of endothelial senescence.

Endothelial cell (EC) senescence is increasingly recognized as a significant contributor to the development of vascular dysfunction and age-related disorders and diseases, including cancer and cardiovascular diseases (CVD). The regulation of cellular senescence is known to be influenced by cellular metabolism. While extensive research has been conducted on the metabolic regulation of senescence in other cells such as cancer cells and fibroblasts, our understanding of the metabolic regulation of EC senescence remains limited. The specific metabolic changes that drive EC senescence are yet to be fully elucidated. The objective of this review is to provide an overview of the intricate interplay between cellular metabolism and senescence, with a particular emphasis on recent advancements in understanding the metabolic changes preceding cellular senescence. I will summarize the current knowledge on the metabolic regulation of EC senescence, aiming to offer insights into the underlying mechanisms and future research directions.

C-Jun N-terminal kinase signaling in cellular senescence.

Cellular senescence leads to decreased tissue regeneration and inflammation and is associated with diabetes, neurodegenerative diseases, and tumorigenesis. However, the mechanisms of cellular senescence are not fully understood. Emerging evidence has indicated that c-Jun N-terminal kinase (JNK) signaling is involved in the regulation of cellular senescence. JNK can downregulate hypoxia inducible factor-1α to accelerate hypoxia-induced neuronal cell senescence. The activation of JNK inhibits mTOR activity and triggers autophagy, which promotes cellular senescence. JNK can upregulate the expression of p53 and Bcl-2 and accelerates cancer cell senescence; however, this signaling also mediates the expression of amphiregulin and PD-LI to achieve cancer cell immune evasion and prevents their senescence. The activation of JNK further triggers forkhead box O expression and its target gene Jafrac1 to extend the lifespan of Drosophila. JNK can also upregulate the expression of DNA repair protein poly ADP-ribose polymerase 1 and heat shock protein to delay cellular senescence. This review discusses recent advances in understanding the function of JNK signaling in cellular senescence and includes a comprehensive analysis of the molecular mechanisms underlying JNK-mediated senescence evasion and oncogene-induced cellular senescence. We also summarize the research progress in anti-aging agents that target JNK signaling. This study will contribute to a better understanding of the molecular targets of cellular senescence and provides insights into anti-aging, which may be used to develop drugs for the treatment of aging-related diseases.

Exploring the Molecular Mechanism of lncRNA NEAT1 Regulation of Liver Fibrosis Based on ceRNA Network

Objective: To analyze the molecular mechanism of lncRNA NEAT1 regulation of liver fibrosis with the help of bioinformatics and experimental validation, hoping to provide new ideas for the prevention and treatment of liver fibrosis and drug development. Methods: Screening lncRNA NEAT1 target miRNAs with the help of Starbase database; screening miRNA target mRNAs using miRDB database; obtaining liver fibrosis-related targets through OMIM, CTD and GeneCards databases; interacting lncRNA NEAT1 regulatory targets with liver fibrosis targets, and performing key target Enrichment analysis was performed. Hepatic stellate cells (HSC) were activated with LPS and divided into blank and model groups. HSC were transfected with siNEAT1 and negative control and divided into siNEAT1 NC+LPS and siNEAT1+LPS. MTT method was used to detect the proliferation of HSC and flow cytometry was used to detect the apoptosis rate of HSC; molecular biology was used to detect α-SMA and Collagen I expression. Results: lncRNA NEAT1 targeted 26 miRNA molecules, miRNA targeted 564 mRNAs, 617 liver fibrosis-related targets, lncRNA NEAT1 regulated 20 key targets of liver fibrosis, lncRNA NEAT1 involvement in liver fibrosis may be closely related to its regulation of cellular senescence. lncRNA NEAT1 expression increased significantly upon HSC activation. The proliferation rate of HSC cells was significantly decreased and apoptosis rate was significantly increased after NEAT1 silencing, and the expression of α-SMA and Collagen I was also significantly decreased. Conclusion: NEAT1 is involved in the development of liver fibrosis by regulating HSC proliferation and apoptosis, and may be closely related to its involvement in the ceRNA network that regulates cellular senescence.

Schisandrin B promotes senescence of activated hepatic stellate cell via NCOA4-mediated ferritinophagy

Context Schisandrin B (Sch B), an active ingredient from Schisandrae chinensis (Turcz.) Baill. (Schisandraceae) Fructus, possesses diverse pharmacological activities including antitumor, anti-inflammation, and hepatoprotection. Objective To explore the effect of Sch B on activated HSCs senescence in hepatic fibrosis and the mechanisms implicated. Materials and methods ICR mice with CCl4-induced hepatic fibrosis were supplemented with Sch B (40 mg/kg) for 30 d and LX2 cells were treated with Sch B (5, 10 and 20 μM) for 24 h. Cellular senescence was assessed by senescence-related indicators senescence-associated β-galactosidase (SA-β-gal) activity and the expression of p16, p21, p53, γ-H2AX, H3K9me3, TERT, TRF1, and TRF2. Ferric ammonium citrate (FAC) and NCOA4 siRNA were used to evaluate the mechanisms underlying Sch B’s regulation of cellular senescence. Results Sch B (40 mg/kg) reduced serum levels of AST and ALT (53.2% and 63.6%), alleviated hepatic collagen deposition, and promoted activated HSCs senescence in mice. Treatment with Sch B (20 μM) decreased cell viability to 80.38 ± 4.87% and elevated SA-β-gal activity, with the levels of p16, p21 and p53 increased by 4.5-, 2.9-, and 3.5-fold and the levels of TERT, TRF1 and TRF2 decreased by 2.4-, 2.7-, and 2.6-fold in LX2 cells. FAC (400 μM) enhanced Sch B’s effect mentioned above. NCOA4 siRNA weakened the effects of Sch B on iron deposition and HSCs senescence. Conclusions Sch B could ameliorate hepatic fibrosis through the promotion of activated HSCs senescence, which might be attributed to its induction of NCOA4-mediated ferritinophagy and subsequent iron overload.

Regulation of Cellular Senescence in Type 2 Diabetes Mellitus: From Mechanisms to Clinical Applications.

Cellular senescence is accelerated by hyperglycemia through multiple pathways. Therefore, senescence is an important cellular mechanism to consider in the pathophysiology of type 2 diabetes mellitus (T2DM) and an additional therapeutic target. The use of drugs that remove senescent cells has led to improvements in blood glucose levels and diabetic complications in animal studies. Although the removal of senescent cells is a promising approach for the treatment of T2DM, two main challenges limit its clinical application: the molecular basis of cellular senescence in each organ is yet to be understood, and the specific effect of removing senescent cells in each organ has to be determined. This review aims to discuss future applications of targeting senescence as a therapeutic option in T2DM and elucidate the characteristics of cellular senescence and senescence-associated secretory phenotype in the tissues important for regulating glucose levels: pancreas, liver, adipocytes, and skeletal muscle.

HCINAP alleviates senescence by regulating MDM2 via p14ARF and the HDAC1/CoREST complex.

Cellular senescence is a major process affected by multiple signals and coordinated by a complex signal response network. Identification of novel regulators of cellular senescence and elucidation of their molecular mechanisms will aid the discovery of new treatment strategies for aging-related diseases. In the present study, we identified human coilin-interacting nuclear ATPase protein (hCINAP) as a negative regulator of aging. Depletion of cCINAP significantly shortened the lifespan of Caenorhabditis elegans and accelerated primary cell aging. Moreover, mCINAP deletion markedly promoted organismal aging and stimulated senescence-associated secretory phenotype in the skeletal muscle and liver from mouse models of radiation-induced senescence. Mechanistically, hCINAP functions through regulating MDM2 status by distinct mechanisms. On one hand, hCINAP decreases p53 stability by attenuating the interaction between p14ARF and MDM2; on the other hand, hCINAP promotes MDM2 transcription via inhibiting the deacetylation of H3K9ac in the MDM2 promoter by hindering HDAC1/CoREST complex integrity. Collectively, our data demonstrate that hCINAP is a negative regulator of aging and provide insight into the molecular mechanisms underlying the aging process.

Induction of filamin-C and its involvement in the regulation of cellular senescence and apoptosis in Huh-7 hepatoma cells during arsenic trioxide exposure

Arsenic trioxide (ATO) is one of the most toxic inorganic arsenic compounds. In this study, we examined the effects of long-term (7 days) exposure to low dose (5 μM) ATO on a human hepatocellular carcinoma cell line, Huh-7. Along with apoptosis accompanied by secondary necrosis though GSDME cleavage, we observed enlarged and flattened cells adhering to the culture dish and surviving even after exposure to ATO. An increase in cyclin-dependent kinase inhibitor p21 levels as well as positive staining for senescence-associated β-galactosidase activity were observed in ATO-treated cells, indicating cellular senescence. Screening for both ATO-inducible proteins by MALDI-TOF-MS analysis and ATO-inducible genes by DNA microarray analysis showed a marked increase in filamin-C (FLNC), an actin cross-linking protein. Interestingly, the increase in FLNC was observed in both dead and surviving cells, suggesting that the upregulation of FLNC by ATO occurs in both apoptotic and senescent cells. Small interference RNA-mediated knock down of FLNC resulted in not only a reduction of senescence-associated enlarged morphology of the cells, but also an exacerbation of cell death. Taken together, these results suggest a regulatory role of FLNC in the execution of senescence as well as apoptosis during ATO exposure.

Regulation and role of calcium in cellular senescence

Cellular senescence is a state of stable cell proliferation arrest accompanied by a distinct secretory program impacting the senescent cell microenvironment. This phenotype can be induced by many stresses, including telomere shortening, oncogene activation, oxidative or genotoxic stress. Cellular senescence plays a key role in the organism throughout life, with beneficial effects at a young age for instance in embryonic development and wound healing, and deleterious effects during aging and in aging-related diseases. In the last decade calcium and calcium signaling have been established as critical factors in the implementation and regulation of cellular senescence. In this review we will present and discuss the main discoveries in this field, from the observation of an increased intracellular calcium concentration in senescent cells to the identification of calcium-binding proteins, calcium channels (TRP, ITPR, …) and MERCs (mitochondria-endoplasmic reticulum contact sites) as key players in this context.

DNA damage-induced cellular senescence is regulated by 53BP1 accumulation in the nuclear foci and phase separation.

Cellular senescence is linked to a wide range of age-related diseases and can be triggered by a variety of stresses, including DNA damage. A variety of genotoxic stressors, such as anti-cancer drugs, cause DNA double-strand breaks (DSBs), which trigger the accumulation of the tumour suppressor protein p53 in the nucleus. Cellular stresses stabilize and activate the p53 signalling pathway, which regulates various cellular processes, such as apoptosis, DNA repair, and senescence. Although p53 signalling is a well-known tumour suppressor pathway, it remains unclear how it is regulated during cellular senescence. Here, we show that p53-binding protein 1 (53BP1) accumulation in the nuclear foci is required for DNA damage-induced cellular senescence via p53 activation. In human immortalized fibroblast, shRNA-mediated 53BP1 depletion decreased not only the expression of p53-target genes but also the cellular senescence induced by adriamycin treatment. Furthermore, we confirmed that DSBs trigger the hyperaccumulation of 53BP1 in the nuclear foci, which plays a key role in the regulation of cellular senescence. To prevent the accumulation of 53BP1 in the nuclear foci, we used phase separation inhibitors, and siRNA against RNF168, which accumulates at DSB loci and forms complexes with 53BP1. This blocks the formation of 53BP1 nuclear foci and DNA damage-induced cellular senescence by activating the p53 signaling pathway. In conclusion, we demonstrated that increased accumulation of 53BP1 in the nuclear foci following DNA damage activates p53 and governs cellular senescence via a liquid-liquid phase separation mechanism.

Regulation of cellular senescence by innate immunity.

During the COVID-19 pandemic, the interplay between the processes of immunity and senescence is drawing more and more intensive attention. SARS-CoV-2 infection induces senescence in lung cells, failure to clear infected cells and increased presence of inflammatory factors could lead to a cytokine storm and acute respiratory disease syndrome (ARDS), which together with aging and age-associated disease lead to 70% of COVID-19-related deaths. Studies on how senescence initiates upon viral infection and how to restrict excessive accumulation of senescent cells to avoid harmful inflammation are crucially important. Senescence can induce innate immune signaling, and innate immunity can engage cell senescence. Here, we mainly review the innate immune pathways, such as cGAS-STING, TLRs, NF-κB, and NLRP3 inflammasome, participating in the senescence process. In these pathways, IFN-I and inflammatory factors play key roles. At the end of the review, we propose the strategies by which we can improve the immune function and reduce inflammation based on these findings.

PS-B01-3: IS IT POSSIBLE TO ACCELERATE SENESCENCE IN THE VASCULAR ENDOTHELIAL CELL BY MODULATING SEVERAL MICRORNAS?

Backgrounds: Many factors are involved in cellular aging, and senescence induction requires complex regulation of various signaling networks and processes. Specifically, in the area of aging-related vascular cognitive impairment, laboratory-based findings have not yet yielded agents of practical use for clinical settings. One possible reason is that the physiologic elements of aging have been insufficiently considered such as in a drug screening experiments (particular in the vitro-blood brain barrier model). It is difficult to age primary human cerebral vascular endothelial cells for the experiments using standard methods because of their limited ability to survive in vitro. To establish techniques to mimic cellular aging by modulation of microRNAs, we aimed to identify key microRNAs capable of fine-tuning aging-associated genes, and thereby regulating the senescence of vascular endothelial cells. Methods: To identify candidate microRNAs, expression microRNA array was performed between control and senescent vascular endothelial cells. Thereafter, bioinformatic analysis was used to select key microRNAs. These candidates were then modulated in vitro using microRNA mimics and inhibitors in vascular endothelial cells, and senescence-associated gene expression patterns were evaluated by qPCR. Results: Seventeen microRNAs were significantly increased more than 2-fold in senescent cells. Of those, bioinformatic analysis concluded that miR-181a-5p, miR-30a-5p, miR-30a-3p, miR-100-5p, miR-21-5p, and miR-382-5p were likely associated with regulation of cellular senescence. We evaluated the potential targets of these six microRNAs by comparing them with cell-cycling and apoptosis-related genes from published mRNA transcriptional array data from aged tissues, and found that miR-181a-5p, miR-30a-5p and miR-30a-3p were enriched in overlapping targets compared with the other candidates. When modulating and comparing these microRNAs in vascular endothelial cells with several patterns, we noticed that over-expression of miR-30a-5p, and inhibition of both miR-30a-3p and miR-181a-5p, induce senescence. Conclusion: Simultaneous upregulation of miR-30a-5p, and downregulation of both miR-30a-3p and miR-181a-5p, mimic cellular senescence in vascular endothelial cells. This single step induction, as opposed to the time-consuming process of replicative passage senescence, would be useful for mimicking cell senescence vitro model.

RNAa-mediated epigenetic attenuation of the cell senescence via locus specific induction of endogenous SIRT1

SIRT1, a known regulator of cellular senescence, is a therapeutic target for age related disorders and its upregulation is a strategy to improve the cell therapeutic potentials of human mesenchymal stem cell (MSCs). Knockdown of natural antisense transcripts via small activating RNAs (RNAa) is an emerging approach for safe and locus specific gene regulation. We have recently identified a natural antisense transcript at human SIRT1 locus (SIRT1-NAT), the expression of which shows a negative correlation with that of SIRT1. To test the hypothetic upregulation of SIRT1 via knockdown of SIRT1-NAT, in this study we designed a single stranded oligonucleotide (SIRT1-antagoNAT) against the antisense transcript, transfection of which efficiently knocked down the SIRT1-NAT and induced SIRT1 transcription in human MSCs. In addition, activation of SIRT1 transfection via knockdown of SIRT1-NAT in human MSCs enhanced their proliferation and differentiation potentials, reduced senescence associated β-galactosidase activity and reversed the senescence associated molecular alterations. Our findings introduce an RNAa mediated approach for epigenetic induction of endogenous SIRT1 and the consequent attenuation of senescence. Further studies should evaluate the therapeutic potentials of this approach against various age related disorders.