Cellular Senescence-inhibited Gene Research Articles

Paradoxical effects of cellular senescence-inhibited gene involved in hepatocellular carcinoma migration and proliferation by ERK pathway and mesenchymal-like markers.

BackgroundCellular senescence-inhibited gene (CSIG) strongly prolongs the progression of replicative senescence. However, roles and mechanisms of CSIG in tumor progression have not been studied widely.MethodsRoles of CSIG in migration and proliferation of SMMC7721 and Huh7 cells were analyzed by transwell or cell viability assays, respectively. Tumorigenicity assays were used to study whether CSIG knockdown could affect SMMC7721 proliferation in vivo. Next, Western blotting and RT-PCR were preformed to evaluate the effects of CSIG on P-ERK cascade and epithelial mesenchymal transformation markers. Then, the location and expression of CSIG protein was detected by immunofluorescence and Western blotting, respectively. Finally, the Cancer Genome Atlas dataset was used to analyze CSIG mRNA levels in hepatocellular carcinoma (HCC) and adjacent non-tumor tissues.ResultsIn this study, we found that CSIG overexpression promoted SMMC7721 cell migration, and CSIG knockdown suppressed tumorigenicity of SMMC7721 cells. In contrast to expectation, CSIG up-regulation could significantly inhibit Huh7 cell growth and migration. CSIG could promote P-ERK activation and levels of mesenchymal-like markers in SMMC7721 cells, whereas CSIG suppressed P-ERK activation and levels of mesenchymal-like markers in Huh7 cells. CSIG protein was located in nucleoli as well as nucleoplasm of SMMC7721 cells, whereas CSIG protein was mainly expressed in the nucleoli rather than nucleoplasm of Huh7 cells. Finally, due to individual differences, raised or down-regulated trends of CSIG in HCC as compared with adjacent non-tumor tissues are different among various patient populations.ConclusionIn summary, these results indicate that CSIG might play different roles in SMMC7721 and Huh7 cells through regulating P-ERK pathway and mesenchymal-like markers. The differential distribution of CSIG might be an important factor that causes its different functions in SMMC7721 and Huh7 cells. CSIG might play different roles in various patient populations.

The Cellular Senescence-Inhibited Gene Is Essential for PPM1A Myristoylation To Modulate Transforming Growth Factor β Signaling.

The cellular senescence-inhibited gene (CSIG) is implicated in important biological processes, including cellular senescence and apoptosis. Our work showed that CSIG is involved in the myristoylation of the serine/threonine protein phosphatase PPM1A. Previous research has shown that myristoylation is necessary for PPM1A to dephosphorylate Smad2 and Smad3. However, the control and the biological significance of the myristoylation remain poorly understood. In this study, we found that CSIG knockdown disturbs PPM1A myristoylation and reduces the dephosphorylation by PPM1A of its substrate Smad2. By regulating PPM1A myristoylation, CSIG is involved in modulating the signaling of transforming growth factor β (TGF-β). Further study of the mechanism indicated that CSIG facilitates the interaction between N-myristoyltransferase 1 (NMT1) and PPM1A. Taking the data together, we found that CSIG is a regulator of PPM1A myristoylation and TGF-β signaling. By promoting the myristoylation of PPM1A, CSIG enhanced the phosphatase activity of PPM1A and further inhibited TGF-β signaling. This work not only extends the biological significance of CSIG but also provides new ideas and a reference for the study of the regulatory mechanism of myristoylation.

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus.

SummaryThe nucleolus is a key organelle that is responsible for the synthesis of rRNA and assembly of ribosomal subunits, which is also the center of metabolic control because of the critical role of ribosomes in protein synthesis. Perturbations of rRNA biogenesis are closely related to cell senescence and tumor progression; however, the underlying molecular mechanisms are not well understood. Here, we report that cellular senescence‐inhibited gene (CSIG) knockdown up‐regulated NOLC1 by stabilizing the 5′UTR of NOLC1 mRNA, and elevated NOLC1 induced the retention of NOG1 in the nucleolus, which is responsible for rRNA processing. Besides, the expression of NOLC1 was negatively correlated with CSIG in the aged mouse tissue and replicative senescent 2BS cells, and the down‐regulation of NOLC1 could rescue CSIG knockdown‐induced 2BS senescence. Additionally, NOLC1 expression was decreased in human hepatocellular carcinoma (HCC) tissue, and the ectopic expression of NOLC1 repressed the proliferation of HCC cells and tumor growth in a HCC xenograft model.

The Function and Molecular Mechanism of Cellular Senescence-Inhibited Gene

To explore the genetic mechanism of replicative senescence, we identified and cloned a Cellular Senescence-Inhibited Gene/ribosomal L1 domain containing 1 (CSIG/RSL1D1) through suppressive subtractive hybridization. CSIG expression is high in earlypassaged human diploid fibroblast cells but decreased during senescence. Our results demonstrated that CSIG inhibited the senescence progression and mediated the apoptosis signaling pathway under ultraviolet (UV) irradiation. Moreover, emerging evidences have indicated that CSIG is involved in other processes including cell cycle regulation and tumor development. This is a brief overview to introduce the studies on the function and molecular mechanism of CSIG

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. The data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence.

Editorial: Transcriptional Regulation in Cancers and Metabolic Diseases.

I am privileged to edit this Research Topic, Transcriptional Regulation in Cancers and Metabolic Diseases under the guidance of Dr. Carol Prives. We hereby thank Drs. Antonino Belfiore and Claire Perks for giving us the opportunity of editing the Research Topic. Given the large number of recent publications on transcriptional regulation, this Research Topic is timely needed. It covers many research hotspots, including p53’s gain-of-function mutation, p63’s role in epithelial cells, mitosis- or senescence-related transcription, and cancer-specific exosome. Overall, this Research Topic reviews and updates the current trends in transcriptional regulation. Two Mini Reviews summarize the roles of heat shock proteins in transcriptional regulation (1, 2). In the first Mini Review, Alexandrova and Marchenko focused on a heat shock protein HSF1 in mutant p53 (mutp53)-mediated oncogenic activation. They highlighted a novel connection between HSF1 and mutp53 and hypothesized that pharmaceutically disrupting HSF1-mutp53 cooperation might be beneficial to cancer patients. In the second Mini Review, Khurana and Bhattacharyya outlined mechanisms through which another heat shock protein HSP90 activates gene expression. That is, HSP90 co-activates transcription factors, interacts with chromatin remodeling factors, and evicts histones from certain gene promoters. Three Reviews explain transcriptional regulation of fate-determining transcription factors or cofactors (3–5). In the first Review, Yoh and Prywes illustrated the regulatory network that impinges upon the key epithelial transcription factor p63. In the second Review, Yoo and colleagues focused on a ubiquitin-fold modifier, UFM1 in breast cancer. In the third Review, Sun and colleagues categorized lysine acetyltransferases in normal and abnormal development of blood cells. Notably, Sun et al. reported current drug developments in lysine acetyltransferase inhibitors. Two Original research articles evaluate transcriptional regulation in uveal melanoma and aging, respectively (6, 7). In the first Original research article, Huffman, Carstens, and Martinez profiled the expression levels of 48 human NRs across a panel of cell lines from uveal melanoma, cutaneous melanoma and melanocytes. In addition, the NR-to-NR and NR-to-genome expression correlation analyses identified RXRγ as a potential driver for melanoma-specific signaling, and ERRα as the uveal-melanoma-specific NR. In the second Original research article, Ma and colleagues analyzed microarray data of differentially expressed genes after the knockdown of the cellular senescence-inhibited gene (CSIG). CSIG, originally identified by this research group, is critical in regulating cell senescence, cell cycle progression, stress response, and tumor metastasis. In this article, they discovered novel CSIG targets that correlate with senescence. Interestingly, they inferred that CSIG regulates the stability of certain target-gene transcripts. Nath and colleagues presented an interesting Review on mitotic proteins in cancer development (8). As the authors pointed out, deregulated transcriptional regulation of mitotic genes are common in cancers, yet mutations are rarely observed for mitotic genes. Cell cycle-related transcription program is interesting and warrants further investigation. One Review and one Opinion article appraised the possible use of exosome in early cancer diagnosis (9, 10). In the Review article, Qin and colleagues summarized the recent progress on the identification of miRNAs from tumor samples. They grouped different miRNAs based on their location as the cytosolic, body-fluid or exosomal miRNAs. The authors proposed exosomal miRNAs as diagnostic biomarkers for lung tumors. In addition, they described the current methods for the isolation and detection of these RNAs in the tumor samples. Moreover, the authors hypothesized how miRNAs have been transported/released into body fluids from the tumor cells. In accompanying Opinion article, Oltra supported cancer-associated exosome in cancer diagnosis and prognosis. In the end of this Research Topic, Davis and colleagues presented a very interesting Review on Nanotetrac in treating cancers (11, 12). In many cancers, Thyroid hormones, T3 and T4 have pro-angiogenic effects, which might be mediated by αVβ3 integrin. Nanotetrac is a nanoparticulate preparation of a T4 substitute, tetrac. Nanotetrac blocks T4-triggered αVβ3-mediated transcriptional regulation. Therefore, Nanotetrac might be useful in treating cancers. By compiling all these excellent manuscripts into one Research Topic, we hope that our readers will find this Research Topic enlightening. We owe our thanks to the staff of Frontiers Endocrinology Office, for their work in the completion of this Research Topic. We are particularly thankful to Davor Vidic, Shaun Evans, Caroline Drage and Byron Bitanihirwe for help in communicating with authors during initiation and completion of the Research Topic. All authors in this Research Topic have provided their broad perspectives on transcriptional regulation, whose insightful thoughts will both benefit the field and be appreciated by fellow researchers. We are indebted to our reviewers/review editors for their contribution to this work.

Global Characteristics of CSIG-Associated Gene Expression Changes in Human HEK293 Cells and the Implications for CSIG Regulating Cell Proliferation and Senescence.

Cellular senescence-inhibited gene (CSIG), also named as ribosomal_L1 domain-containing 1 (RSL1D1), is implicated in various processes including cell cycle regulation, cellular senescence, apoptosis, and tumor metastasis. However, little is known about the regulatory mechanism underlying its functions. To screen important targets and signaling pathways modulated by CSIG, we compared the gene expression profiles in CSIG-silencing and control HEK293 cells using Affymetrix microarray Human Genome U133 Plus 2.0 GeneChips. A total of 590 genes displayed statistically significant expression changes, with 279 genes up-regulated and 311 down-regulated, respectively. These genes are involved in a broad array of biological processes, mainly in transcriptional regulation, cell cycle, signal transduction, oxidation reduction, development, and cell adhesion. The differential expression of genes such as ZNF616, KPNA5, and MAP3K3 was further validated by real-time PCR and western blot analysis. Furthermore, we investigated the correlated expression patterns of Cdc14B, ESCO1, KPNA5, MAP3K3, and CSIG during cell cycle and senescence progression, which imply the important pathways CSIG regulating cell cycle and senescence. The mechanism study showed that CSIG modulated the mRNA half-life of Cdc14B, CASP7, and CREBL2. This study shows that expression profiling can be used to identify genes that are transcriptionally or post-transcriptionally modified following CSIG knockdown and to reveal the molecular mechanism of cell proliferation and senescence regulated by CSIG.

The Retinoblastoma Protein Selectively Represses E2F1 Targets via a TAAC DNA Element during Cellular Senescence

The retinoblastoma (Rb) protein mediates heterochromatin formation at the promoters of E2 transcription factor 1 (E2F1) target genes, such as proliferating cell nuclear antigen and cyclin A2 (CCNA2), and represses these genes during cellular senescence. However, the selectivity of Rb recruitment is still not well understood. Here, we demonstrate that a senescence-associated gene is a direct target of E2F1 and is also repressed by heterochromatin in senescent cells. In contrast, ARF and p27(KIP1), which are also E2F1 targets, are not repressed by Rb and heterochromatin formation. By comparing the promoter sequences of these genes, we found a novel TAAC element that is present in the cellular senescence-inhibited gene, proliferating cell nuclear antigen, and CCNA2 promoters but absent from the ARF and p27(KIP1) promoters. This TAAC element associates with Rb and is required for Rb recruitment. We further determined that TAAC element-mediated Rb association requires the E2F1 binding site, but not E2F1 protein. These results provide a novel molecular mechanism for the different expression patterns of E2F1 targets and afford new mechanistic insight regarding the selectivity of Rb-mediated heterochromatin formation and gene repression during cellular senescence.

Nucleolar protein CSIG is required for p33ING1 function in UV-induced apoptosis

Cellular senescence-inhibited gene (CSIG) protein, a nucleolar protein with a ribosomal L1 domain in its N-terminus, can exert non-ribosomal functions to regulate biological processes, such as cellular senescence. Here, we describe a previously unknown function for CSIG: promotion of apoptosis in response to ultraviolet (UV) irradiation-induced CSIG upregulation. We identified p33ING1 as a binding partner that interacts with CSIG. After UV irradiation, p33ING1 increases its protein expression, translocates into the nucleolus and binds CSIG. p33ING1 requires its nucleolar targeting sequence region to interact with CSIG and enhance CSIG protein stability, which is essential for activation of downstream effectors, Bcl-2-associated X protein, to promote apoptosis. Thus, our data imply that p33ING1–CSIG axis functions as a novel pro-apoptotic regulator in response to DNA damage.

Decreased proliferation of aging tenocytes is associated with down‐regulation of cellular senescence‐inhibited gene and up‐regulation of p27

Symptomatic tendinopathy tends to be age-related. However, the molecular mechanisms of ageing and its effects on tenocyte proliferation and cell cycle progression are unknown. We examined tenocytes from Achilles tendons in rats from three age groups (young, 2 months; middle-aged, 12 months, and near senescence, 24 months). Tenocyte proliferation was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Senescence-associated β-galactosidase (SA β-gal) staining was performed in all groups of tenocytes. mRNA and protein expression of cellular senescence-inhibited gene (CSIG) and p27 was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results of MTT assay revealed that tenocyte proliferation decreased with age (p < 0.05). Cell cycle progression was arrested at G0/G1 phase in senescent tenocytes. More senescent tenocytes expressed SA β-gal than young tenocytes did. By RT-PCR and Western blot analysis, the gene and protein expression of CSIG was found to be down-regulated, whereas that of p27 was up-regulated with age. In conclusion, the proliferation of tenocytes declines with age and is associated with the down-regulation of CSIG and up-regulation of p27.

CSIG Inhibits PTEN Translation in Replicative Senescence

Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) that was abundant in young human diploid fibroblast cells but declined upon replicative senescence. Overexpression or knockdown of CSIG did not influence p21(Cip1) and p16(INK4a) expressions. Instead, CSIG negatively regulated PTEN and p27(Kip1) expressions, in turn promoting cell proliferation. In PTEN-silenced HEK 293 cells and PTEN-deficient human glioblastoma U87MG cells, the effect of CSIG on p27(Kip1) expression and cell division was abolished, suggesting that PTEN was required for the role of CSIG on p27(Kip1) regulation and cell cycle progression. Investigation into the underlying mechanism revealed that the regulation of PTEN by CSIG was achieved through a translational suppression mechanism. Further study showed that CSIG interacted with PTEN mRNA in the 5' untranslated region (UTR) and that knockdown of CSIG led to increased luciferase activity of a PTEN 5' UTR-luciferase reporter. Moreover, overexpression of CSIG significantly delayed the progression of replicative senescence, while knockdown of CSIG expression accelerated replicative senescence. Knockdown of PTEN diminished the effect of CSIG on cellular senescence. Our findings indicate that CSIG acts as a novel regulatory component of replicative senescence, which requires PTEN as a mediator and involves in a translational regulatory mechanism.