MiR-186, miR-216b, miR-337-3p, and miR-760 cooperatively induce cellular senescence by targeting ?? subunit of protein kinase CKII in human colorectal cancer cells
MiR-186, miR-216b, miR-337-3p, and miR-760 cooperatively induce cellular senescence by targeting ?? subunit of protein kinase CKII in human colorectal cancer cellsArticle in
· November 2012 with 24 ReadsDOI: 10.1016/j.bbrc. · Source: We previously demonstrated that downregulation of protein kinase CKII induces cellular senescence in human colon cancer HCT116 cells. To investigate the role of microRNAs (miRNAs) in CKII downregulation during senescence, we employed computational algorithms. Four miRNAs (miR-186, miR-216b, miR-337-3p, and miR-760) were predicted to be miRNAs against CKIIα mRNA. Mimics of all four miRNAs jointly downregulated CKIIα expression in HCT116 cells. Reporter analysis and RT-PCR have suggested that these four miRNAs may stimulate degradation of CKIIα mRNA by targeting its 3' untranslated regions (UTRs). The four miRNA mimics increased senescent-associated β-galactosidase (SA-β-gal) staining, p53 and p21(Cip1/WAF1) expression, and reactive oxygen species (ROS) production. In contrast, concomitant knockdown of the four miRNAs by antisense inhibitors increased the CKIIα protein level and suppressed CKII inhibition-mediated senescence. Finally, CKIIα overexpression antagonized senescence induced by the four miRNA mimics. Therefore, the present results show that miR-186, miR-216b, miR-337-3p, and miR-760 cooperatively promote cellular senescence through the p53-p21(Cip1/WAF1) pathway by CKII downregulation-mediated ROS production in HCT116 cells.One reason that more late-stage patients were collected was that their lesions were clearer and tumor specimen was easier to obtain for miR-216b detection. And with more late-stage cancer tissues, the trend that miR-216b expression decreased with the FIGO stage was more obvious, consistent with reports of other tumors[21,[35][36][37]. Moreover, more latestage specimens can better display the value of miR-216b in cervical cancer development and prognosis as a biomarker. Background
Our previous study showed FOXM1 expression was significantly up-regulated in cervical cancer, and was associated with poor prognosis. To clarify miRNAs-FOXM1 modulation pathways, in this study, we investigated the relationships between miR-216b and FOXM1 and the role of miR-216b in cell proliferation and prognosis of cervical cancer patients. Methods
Western blotting and qPCR were used to determine expression of FOXM1, cell cycle related factors and miR-216b level. MiR-216b overexpression and inhibited cell models were constructed, and siRNA was used for FOXM1 silencing. Cell proliferation was analyzed by MTT and colony formation assay. Dual luciferase reporter assay system was used to clarify the relationships between miR-216b and FOXM1. Kaplan-Meier survival analysis was used to evaluate prognosis. ResultsMiR-216b was down-regulated in cervical cancer cells and tissues, and its ectopic expression could decrease cell proliferation. Western blotting analysis showed miR-216b can inhibit cell proliferation by regulating FOXM1-related cell cycle factors, suppressing cyclinD1, c-myc, LEF1 and p-Rb and enhancing p21 expression. Repressing of miR-216b stimulated cervical cancer cell proliferation, whereas silencing FOXM1 expression could reverse this effect. Western blotting and luciferase assay results proved FOXM1 is a direct target of miR-216b. Survival analysis showed higher level of miR-216b was associated with better prognosis in cervical cancer patients. ConclusionsFOXM1 expression could be suppressed by miR-216b via direct binding to FOXM1 3′-UTR and miR-216b could inhibit cell proliferation by regulating FOXM1 related Wnt/β-catenin signal pathway. MiR-216b level is related to prognosis in cervical cancer patients and may serve as a potential prognostic marker.Article · December 2017This observation might be related to the fact that Klotho expression was higher in huMSCs than in aMSCs. Klotho might promote healing from renal IRI [12] , possibly by inducing MnSOD expression [25] and protecting against a pro-oxidative state, given that Klotho increases the phosphorylation of FOXO3a, a transcription factor that upregulates MnSOD [17, 18]. In fact, the kidneys of animals treated with huMSCs expressed more Klotho and more MnSOD than did those of animals treated with aMSCs. Background
Mesenchymal stromal cells (MSCs) represent an option for the treatment of acute kidney injury (AKI). It is known that young stem cells are better than are aged stem cells at reducing the incidence of the senescent phenotype in the kidneys. The objective of this study was to determine whether AKI leads to premature, stress-induced senescence, as well as whether human umbilical cord-derived MSCs (huMSCs) can prevent ischaemia/reperfusion injury (IRI)-induced renal senescence in rats. Methods
By clamping both renal arteries for 45 min, we induced IRI in male rats. Six hours later, some rats received 1 × 106 huMSCs or human adipose-derived MSCs (aMSCs) intraperitoneally. Rats were euthanised and studied on post-IRI days 2, 7 and 49. ResultsOn post-IRI day 2, the kidneys of huMSC-treated rats showed improved glomerular filtration, better tubular function and higher expression of aquaporin 2, as well as less macrophage infiltration. Senescence-related proteins (β-galactosidase, p21Waf1/Cip1, p16INK4a and transforming growth factor beta 1) and microRNAs (miR-29a and miR-34a) were overexpressed after IRI and subsequently downregulated by the treatment. The IRI-induced pro-oxidative state and reduction in Klotho expression were both reversed by the treatment. In comparison with huMSC treatment, the treatment with aMSCs improved renal function to a lesser degree, as well as resulting in a less pronounced increase in the renal expression of Klotho and manganese superoxide dismutase. Treatment with huMSCs ameliorated long-term kidney function after IRI, minimised renal fibrosis, decreased β-galactosidase expression and increased the expression of Klotho. Conclusions
Our data demonstrate that huMSCs attenuate the inflammatory and oxidative stress responses occurring in AKI, as well as reducing the expression of senescence-related proteins and microRNAs. Our findings broaden perspectives for the treatment of AKI.Article · December 2017As shown in Table 6 for five case studies, CSmiRTar can successfully identify the experimentally validated common target genes of multiple miRNAs. For example, it is known that human miR186-5p, miR-216b-5p, miR-337-3p, and miR-760 cooperatively induce cellular senescence by targeting the gene CSNK2A1 in human colorectal cancer cells [35]. By considering the predicted target genes supported by three existing miRNA target prediction databases (i.e. MicroRNAs (miRNAs) are functional RNA molecules which play important roles in the posttranscriptional regulation. miRNAs regulate their target genes by repressing translation or inducing degradation of the target genes' mRNAs. Many databases have been constructed to provide computationally predicted miRNA targets. However, they cannot provide the miRNA targets expressed in a specific tissue and related to a specific disease at the same time. Moreover, they cannot provide the common targets of multiple miRNAs and the common miRNAs of multiple genes at the same time. To solve these two problems, we construct a database called CSmiRTar (Condition-Specific miRNA Targets). CSmiRTar collects computationally predicted targets of 2588 human miRNAs and 1945 mouse miRNAs from four most widely used miRNA target prediction databases (miRDB, TargetScan, microRNA.org and DIANA-microT) and implements functional filters which allows users to search (i) a miRNA's targets expressed in a specific tissue or/and related to a specific disease, (ii) multiple miRNAs' common targets expressed in a specific tissue or/and related to a specific disease, (iii) a gene's miRNAs related to a specific disease, and (iv) multiple genes' common miRNAs related to a specific disease. We believe that CSmiRTar will be a useful database for biologists to study the molecular mechanisms of post-Transcriptional regulation in human or mouse. CSmiRTar is available at http://cosbi.ee.ncku.edu.tw/CSmiRTar/ or http://cosbi4. ee.ncku.edu.tw/CSmiRTar/. (C) 2017 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Article · July 201736 In colorectal cancer, miR-337-enhanced cellular senescence through negative regulation of α subunit of protein kinase CKII. 37 In gastric cancer, miR-337 expression was lower and significantly correlated with LNM. Upregulation of miR-337 inhibited the cell growth, invasion, metastasis, and angiogenesis in gastric cancer in vitro and in vivo through downregulating myeloid zinc finger 1-facilitated expression of matrix metalloproteinase 14. 38 In pancreatic ductal adenocarcinoma, miR-337 was downregulated in tumor tissues and strongly associated with TNM (tumor, node, and metastasis) stage and lymph node status. Cervical cancer is the fourth most commonly occurring malignancy in females worldwide. Accumulated studies have demonstrated that the aberrant expression of microRNAs plays important roles in tumorigenesis and tumor development and potentially serves as therapeutic targets in various cancers including cervical cancer. Therefore, the identification of specific microRNAs contributed to cervical cancer formation and progression would provide critical clues for the treatments for patients with this disease. In this study, we aimed to detect microRNA-337 expression pattern and investigate the biological roles of microRNA-337 in the regulation of the malignant phenotypes of cervical cancer and its underlying mechanisms. We found that microRNA-337 expression was significantly downregulated in cervical cancer tissues and cell lines. In addition, its aberrant expression levels were positively correlated with tumor size, International Federation of Gynecology and Obstetrics stage, and lymph node metastasis of cervical cancer. The ectopic expression of microRNA-337 suppressed cell proliferation and invasion of cervical cancer in vitro. Furthermore, specificity protein 1 was identified as a direct target of microRNA-337 in cervical cancer. The expression of specificity protein 1 increased in cervical cancer tissues and negatively correlated with microRNA-337 expression level. Moreover, rescue experiments revealed that upregulation of specificity protein 1 could rescue the effects of microRNA-337 on cervical cancer cells. Taken together, these findings collectively demonstrate that microRNA-337 exerts its tumor-suppressing roles in cervical cancer by directly targeting specificity protein 1, thereby indicating a potential novel potential therapeutic target for patients with cervical cancer.Article · June 2017How does CK2 activity decrease with advancing age in C. elegans? Because our previous studies demonstrated that DNA methylation and microRNAs (miRNAs) are primarilyinvolved in CK2α gene silencing during replicative senescence of human cells[28][29][30], we speculate that the underlying cause of age-dependent CK2 downregulation in worms may be related to DNA methylation or miRNAs. Components of IIS, such as PI3K and AKT, have been shown to modulate longevity in yeast, worms, and flies[1][2][3][4]. Studies show that a decrease in protein kinase CK2 (CK2) activity is associated with cellular senescence. However, the role of CK2 in organism aging is still poorly understood. Here, we investigated whether protein kinase CK2 (CK2) modulated longevity in Caenorhabditis elegans. CK2 activity decreased with advancing age in the worms. Knockdown of kin-10 (the ortholog of CK2β) led to a short lifespan phenotype and induced age-related biomarkers, including retardation of locomotion, decreased pharyngeal pumping rate, increased lipofuscin accumulation, and reduced resistance to heat and oxidative stress. The long lifespan of age-1 and akt-1 mutants was significantly suppressed by kin-10 RNAi, suggesting that CK2 acts downstream of AGE-1 and AKT-1. Kin-10 knockdown did not further shorten the short lifespan of daf-16 mutant worms but either decreased or increased the transcriptional activity of DAF-16 depending on the promoters of the target genes, indicating that CK2 is an upstream regulator of DAF-16 in C. elegans. Kin-10 knockdown increased production of reactive oxygen species (ROS) in the worms. Finally, the ROS scavenger N-acetyl-L-cysteine significantly counteracts the lifespan shortening and lipofuscin accumulation induced by kin-10 knockdown. Therefore, the present results suggest that age-dependent CK2 downregulation reduces longevity by associating with both ROS generation and the AGE-1-AKT-1-DAF-16 pathway in C. elegans.Article · April 2017Functional analyses indicated that these miRNAs, which function as oncogenes or tumor suppressors, have indeed been implicated in the carcinogenesis of LSCC. Recently, studies have shown that synergistic expression of certain miRNAs could jointly effect tumor behavior (18,19 ). To validate the function of the coordination among miRNAs, the present study aimed to investigate whether the combined expression of miRNAs have superior tumor-suppression effects to those of a single miRNA. MicroRNAs (miRNAs) are reported to be important regulators of cancer-related processes, and function either as oncogenes or as tumor-suppressor genes. It was found that miR-375 was downregulated in samples of laryngeal squamous cell carcinomas (LSCCs) as compared to the level noted in adjacent non-tumor tissues, and it was inversely correlated with T grade, lymph node metastases and clinical tumor stage. Overexpression of miR-375 led to a decreased protein level of Krüppel-like factor 4 (KLF4) and marked suppression of the proliferation and invasion, and induced apoptosis of LSCC cell line Hep-2 using Cell Counting Kit-8, Transwell chamber and cell cycle assays. In addition, we examined the influence of the upregulation of miR-206 alone and upregulation of both miR-375 and miR-206 on the expression of KLF4 and Hep-2 cell behavior. The results showed that compared with the function of miR-375 in tumor suppression by regulating KLF4, co-transfection of miR-375 and miR-206 exhibited a less effective inhibitory effect not only on tumor cell proliferation and invasion, but also on tumor cell apoptosis. Taken together, miR-375 is possibly a tumor suppressor in LSCC by regulating KLF4. In addition, simple overexpression of several miRNAs did not entail higher efficacy than a single miRNA, similar to co-transfecions of miR-375 and miR-206.Article · June 2016Deng et al. [21] found that miRNA-216b was downregulated in NPC cell lines and specimens and played a tumor suppressive role in NPC by targeting KRAS. In cooperation with other three miRNAs, miRNA-216b could induce cellular senescence through the p53-p21 Cip1/WAF1 pathway by protein kinase CKII downregulation-mediated ROS production in human colorectal cancer cells [31] . In HCC, miRNA- 216b could function as a tumor suppressor by targeting IGF2BP2 and subsequently suppressing the downstream IGF2 [22]. Esophageal squamous cell carcinoma (ESCC) is a common human malignancy with poor survival, which was usually diagnosed at an advanced stage. MicroRNAs (miRNAs), a class of single stranded noncoding RNAs with only 17–25 ribonucleotides, were demonstrated to play an important role in lots of cancers. In the recent years, increasing evidence revealed that circulating miRNAs exhibited great potential in the diagnosis of various types of cancers. The present study was designed to evaluate the diagnostic value of plasma miRNA-216a/b for ESCC. Our results showed that the expression level of plasma miRNA-216a/b was significantly lower in ESCC patients compared with that of healthy controls. The receiver operating characteristic (ROC) curve analysis yielded an area under the ROC curve (AUC) value of 0.877 [95% CI (confidence interval): 0.818–0.922] for miRNA-216a and 0.756 (95% CI: 0.685–0.819) for miRNA-216b. Clinical data indicated that plasma miRNA-216a/b were inversely correlated with lymph node metastasis and TNM stage. Additionally, the plasma miRNA-216b expression level was significantly upregulated in postoperative samples compared to preoperative samples. Our study, for the first time, demonstrated that plasma miRNA-216a/b might serve as potential biomarkers for the diagnosis of ESCC and dysregulation of miRNA-216a/b might be involved in the progression of ESCC.Article · February 2016In non-small cell lung carcinoma, miR-186 could inhibit the proliferation by inducing G(1)-S checkpoint arrest. In human colon cancer HCT116 cells, miR-186 also acted as a tumor suppressor to promote the cellular senescence through p53–p21 Cip1/WAF1 pathway[9][10][11]. However, the expression and function of miR-186 in gliomas still remain unclear. The long non-coding RNA Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that activated early in colorectal neoplasia, but it is also up-regulated in many other solid tumors. Herein, the function and underlying mechanism of CRNDE in regulating glioma stem cells (GSCs) were investigated. We found that CRNDE expression was up-regulated while miR-186 expression was down-regulated in GSCs. Overexpression of CRNDE could promote the cellular proliferation, migration, invasion and inhibit the apoptosis in GSCs. Overexpression of miR-186 exerted functions of inhibiting the proliferation, migration and invasion of GSCs and promoting apoptosis. And CRNDE decreased the expression levels of XIAP and PAK7 by binding to miR-186 and negatively regulating it. In addition, miR-186 binded to XIAP and PAK7 3'UTR region, and decrease the expression of them, thus regulating the expression levels of downstream target proteins such as caspase 3, BAD, cyclin D1 and MARK2. The in vivo effect of CRNDE and miR-186 showed that the tumor formation rate was minimum in tumor-bearing nude mice with the knockdown of CRNDE and the overexpression of miR-186. In conclusion, CRNDE played an oncogenic role of GSCs through the negative regulation of miR-186. Both CRNDE and miR-186 could be regarded as potential targets in the glioma therapy.Article · July 2015In prostate cancer, miR-186 often exhibits diminished expression, and its down-regulation correlates with the elevated expression of several prostate cancer-associated genes, such as alpha-methylacyl-CoA racemase and prostate-specific membrane antigen gene [28] . In colorectal cancer cells, miR-186 cooperates with other miRNAs to promote cellular senescence through the p53–p21 Cip1/WAF1 path- way [29]. The difference in the carcinogenic effects of miR-186 among these cancers highlights that a specific pathogenesis underlies various cancers. MicroRNAs can function as key tumor suppressors or oncogenes and act as biomarkers for cancer diagnosis or prognosis. Although high-throughput assays have revealed many miRNA biomarkers for pancreatic ductal adenocarcinoma (PDAC), only a few have been validated in independent populations or investigated for functional significance in PDAC pathogenesis. In this study, we correlated the expression of 36 potentially prognostic miRNAs within PDAC tissue with clinico-pathological features and survival in 151 Chinese patients. We then analyzed the functional roles and target genes of two miRNAs in PDAC development. We found that high expression of miR-186 and miR-326 predict poor and improved survival, respectively. miR-186 was over-expressed in PDAC patients compared with controls, especially in patients with large tumors (&2 cm), lymph node metastasis, or short-term survival (& 24 months). In contrast, miR-326 was down-regulated in patients compared with controls and displayed relatively increased expression in the patients with long-term survival or without venous invasion. Functional experiments revealed that PDAC cell proliferation and migration was decreased following inhibition and enhanced following over-expression of miR-186. In contrast, it was enhanced following inhibition and decreased after over-expression of miR-326. A luciferase assay indicated that miR-186 can bind directly to the 3'-UTR of NR5A2 to repress gene expression. These findings suggest that miR-186 over-expression contributes to the invasive potential of PDAC, likely via suppression of NR5A2, thereby leading high miR-326 expression prolongs survival likely via the decreasing invasive potential of PDAC cells. These two miRNAs can be used as markers for clinical diagnosis and prognosis, and they represent therapeutic targets for PDAC.Article · March 2015In humans, miRNAs play important roles in cellular physiology, development, and disease by negatively regulating gene expression through translational repression or post-transcriptional degradation [28]. miR-337 has been reported to be expressed abnormally in many cancers [29,30] such as breast cancer [31], esophageal squamous cell carcinoma [32], and lung cancer [33]. Its expression was found to be related to the survival of patients with ovarian cancer [34] and gastric cancer [35]. BackgroundmiRNAs are involved in coordinating a variety of cellular processes by regulating their target genes. Aberrant expression of miRNAs is correlated with various cancers. Previous studies have shown that miR-337 is significantly down-regulated in pancreatic ductal adenocarcinoma (PDAC) and that its expression is negatively correlated to the expression of HOXB7. Both miR-337 and HOXB7 are associated with the prognosis of PDAC patients. The purpose of this study was to identify the molecular mechanisms by which miR-337 acts as a tumor suppressor in PDAC.Methods
Synthetic miR-337 mimics were transfected into PANC-1 and As-PC-1 cells using LipofectamineTM 2000. The expression of HOXB7 protein was analyzed by Western blot. Luciferase reporter plasmids were constructed to confirm that HOXB7 3?UTR was the target of miR-337. The effect of miR-337 on cell proliferation was evaluated by CCK8 assay and colony formation assay, and cell invasion was evaluated by wound healing assay and transwell assay.ResultsWestern blot and luciferase activity assays identified HOXB7 as the target of miR-337. A CCK-8 assay showed the absorbance of cells transfected with miR-337 mimics to be less than that of control cells, and that the number of cell clones was significantly decreased by miR-337 expression. A wound healing assay showed the invasion rate of cells transfected with miR-337 mimics at 36 h to be markedly lower than in controls. The average number of cells penetrating the Matrigel was significantly lower than the controls.Conclusion
These findings suggest that miR-337 targets HOXB7 and effects significant suppression of PDAC cell proliferation and invasion.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/_171.Article · September 2014Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.Article · March 1997Cellular senescence is thought to be an important tumor suppression process in vivo. We have previously shown that p53 activation is necessary for CKII inhibition-mediated cellular senescence. Here, CKII inhibition induced acetylation of p53 at K382 in HCT116 and HEK293 cells. This acetylation event was suppressed by SIRT1 activation. CKIIα and CKIIβ were co-immunoprecipitated with SIRT1 in a p53-independent manner. Maltose binding protein pull-down and yeast two-hybrid indicated that SIRT1 bound to CKIIβ, but not to CKIIα. CKII inhibition reduced SIRT1 activity in cells. CKII phosphorylated and activated human SIRT1 in vitro. Finally, SIRT1 overexpression antagonized CKII inhibition-mediated cellular senescence. These results reveal that CKII downregulation induces p53 stabilization by negatively regulating SIRT1 deacetylase activity during senescence.Article · November 2011MicroRNAs (miRNAs) are small noncoding RNAs that are involved in various diseases, including cancer. In the present study, we found that miR-216b was downregulated in nasopharyngeal carcinoma (NPC) cell lines and specimens. Decreased expression of miR-216b was directly related to advanced clinical stage and lymph node metastasis. miR-216b levels correlated inversely with levels of KRAS protein during nasopharyngeal tumorigenesis. Furthermore, we demonstrated that miR-216b can bind to the 3' untranslated region (UTR) of KRAS and inhibit expression of the KRAS protein. Both in vitro and in vivo assays revealed that miR-216b attenuated NPC cell proliferation, invasion and tumor growth in nude mice. miR-216b exerts its tumor suppressor function through inhibition of the KRAS-related AKT and ERK pathways. Our findings provide, for the first time, significant clues regarding the role of miR-216b as a tumor suppressor by targeting KRAS in NPC.Article · September 2011Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.Article · April 2011Moens and colleagues express concern that our report of phosphorylation of p38-regulated/activated protein kinase (PRAK) at Ser(93) in senescent cells may be incorrect as a result of their inability to detect phosphorylated PRAK with the same antibody. We provide additional characterization of the antibody that we used and show that there is a 42-kilodalton phosphoprotein detected by this antibody, but that this phosphoprotein may not be PRAK.Article · July 2010We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIalpha small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22(phox) siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII.Article · July 2010MicroRNAs (miRNAs) are short non-coding RNAs that regulate diverse biological processes by controlling the pattern of expressed proteins. In mammalian cells, miRNAs partially complement their target sequences leading to mRNA degradation and/or decreased mRNA translation. Here, we have analyzed transcriptome-wide changes in miRNAs in senescent relative to early-passage WI-38 human diploid fibroblasts (HDFs). Among the miRNAs downregulated with senescence were members of the let-7 family, while upregulated miRNAs included miR-1204, miR-663 and miR-519. miR-519 was recently found to reduce tumor growth at least in part by lowering the abundance of the RNA-binding protein HuR. Overexpression of miR-519a in either WI-38 or human cervical carcinoma HeLa cells triggered senescence, as measured by monitoring beta-galactosidase activity and other senescence markers. These data suggest that miR-519 can suppress tumor growth by triggering senescence and that miR-519 elicits these actions by repressing HuR expression.Article · June 2010Recent evidence supports a role for microRNAs (miRNAs) in regulating the life span of model organisms. However, little is known about how these small RNAs contribute to human aging. Here, we profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.Article · May 2010Luminal-like breast tumor cells express estrogen receptor alpha (ERalpha), a member of the nuclear receptor family of ligand-activated transcription factors that controls their proliferation, survival, and functional status. To identify the molecular determinants of this hormone-responsive tumor phenotype, a comprehensive genome-wide analysis was performed in estrogen stimulated MCF-7 and ZR-75.1 cells by integrating time-course mRNA expression profiling with global mapping of genomic ERalpha binding sites by chromatin immunoprecipitation coupled to massively parallel sequencing, microRNA expression profiling, and in silico analysis of transcription units and receptor binding regions identified. All 1270 genes that were found to respond to 17beta-estradiol in both cell lines cluster in 33 highly concordant groups, each of which showed defined kinetics of RNA changes. This hormone-responsive gene set includes several direct targets of ERalpha and is organized in a gene regulation cascade, stemming from ligand-activated receptor and reaching a large number of downstream targets via AP-2gamma, B-cell activating transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and RXRalpha transcription factors. MicroRNAs are also integral components of this gene regulation network because miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated by 17beta-estradiol along with other microRNAs that can target a significant number of transcripts belonging to one or more estrogen-responsive gene clusters.Article · March 2010Three dimensional quantitative structure activity relationship between diazabicyclo[4.2.0]octanes and nicotinic acetylcholine receptor (halpha4beta2 and halpha3beta4) agonists was studied using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). From 11 CoMFA and CoMSIA models, CoMSIA with steric and electrostatic fields gave the best predictive models (q(2)=0.926 and 0.945, r(2) (ncv)=0.983 and 0.988). This study can be used to develop potent halpha4beta2 receptor agonists with low activity on halpha3beta4 subtype.Article · February 2009Article · August 2014Article · March 2013Article · July 2014Article · December 2013