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• W1998780829 abstract "Histone deacetylase inhibitors (HDACi) are novel clinical anticancer drugs that inhibit HDAC gene expression and induce cell apoptosis in human cancers. Nevertheless, the detailed mechanism or the downstream HDAC targets by which HDACi mediates apoptosis in human breast cancer cells remains unclear. Here, we show that HDACi reduce tumorigenesis and induce intrinsic apoptosis of human breast cancer cells through the microRNA miR-125a-5p in vivo and in vitro. Intrinsic apoptosis was activated by the caspase 9/3 signaling pathway. In addition, HDACi mediated the expression of miR-125a-5p by activating RUNX3/p300/HDAC5 complex. Subsequently, miR-125a-5p silenced HDAC5 post-transcriptionally in the cells treated with HDACi. Thus, a regulatory loop may exist in human breast cancer cells involving miR-125a-5p and HDAC5 that is controlled by RUNX3 signaling. Silencing of miR-125a-5p and RUNX3 inhibited cancer progression and activated apoptosis, but silencing of HDAC5 had a converse effect. In conclusion, we demonstrate a possible new mechanism by which HDACi influence tumorigenesis and apoptosis via downregulation of miR-125a-5p expression. This study provides clinical implications in cancer chemotherapy using HDACi. Histone deacetylase inhibitors (HDACi) are novel clinical anticancer drugs that inhibit HDAC gene expression and induce cell apoptosis in human cancers. Nevertheless, the detailed mechanism or the downstream HDAC targets by which HDACi mediates apoptosis in human breast cancer cells remains unclear. Here, we show that HDACi reduce tumorigenesis and induce intrinsic apoptosis of human breast cancer cells through the microRNA miR-125a-5p in vivo and in vitro. Intrinsic apoptosis was activated by the caspase 9/3 signaling pathway. In addition, HDACi mediated the expression of miR-125a-5p by activating RUNX3/p300/HDAC5 complex. Subsequently, miR-125a-5p silenced HDAC5 post-transcriptionally in the cells treated with HDACi. Thus, a regulatory loop may exist in human breast cancer cells involving miR-125a-5p and HDAC5 that is controlled by RUNX3 signaling. Silencing of miR-125a-5p and RUNX3 inhibited cancer progression and activated apoptosis, but silencing of HDAC5 had a converse effect. In conclusion, we demonstrate a possible new mechanism by which HDACi influence tumorigenesis and apoptosis via downregulation of miR-125a-5p expression. This study provides clinical implications in cancer chemotherapy using HDACi. Histone deacetylases (HDACs), which target lysine residues, are epigenetic determinants that are essential for transcriptional regulation because they promote chromatin condensation.1Strahl BD Allis CD The language of covalent histone modifications.Nature. 2000; 403: 41-45Crossref PubMed Scopus (6604) Google Scholar Cytoplasmic HDACs (nonhistone HDACs) have been shown to play central roles in mediating processes related to human cancers, including promoting tumor progression and metastases and inhibiting apoptosis.2Glozak MA Sengupta N Zhang X Seto E Acetylation and deacetylation of non-histone proteins.Gene. 2005; 363: 15-23Crossref PubMed Scopus (1287) Google Scholar Consequently, histone deacetylase inhibitors (HDACi) are promising anticancer drugs and several are currently the focus of clinical trials, such as Trichostatin A (TSA) and valproic acid, which decrease HDAC expression.3Johnstone RW Histone-deacetylase inhibitors: novel drugs for the treatment of cancer.Nat Rev Drug Discov. 2002; 1: 287-299Crossref PubMed Scopus (1345) Google Scholar TSA and valproic acid stimulate multiple pathways leading to apoptosis, induced cell death, and cell growth arrest in adult patients with solid tumors.4Dokmanovic M Clarke C Marks PA Histone deacetylase inhibitors: overview and perspectives.Mol Cancer Res. 2007; 5: 981-989Crossref PubMed Scopus (954) Google Scholar TSA delays tumor growth and induces apoptosis by mediating expression of Ki-67, matrix metalloprotease 2 (MMP2), caspase 3, and Bcl-xL.5El-Zawahry A Lu P White SJ Voelkel-Johnson C In vitro efficacy of AdTRAIL gene therapy of bladder cancer is enhanced by trichostatin A-mediated restoration of CAR expression and downregulation of cFLIP and Bcl-XL.Cancer Gene Ther. 2006; 13: 281-289Crossref PubMed Scopus (32) Google Scholar,6Medina V Edmonds B Young GP James R Appleton S Zalewski PD Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin A (inhibitors of histone deacetylase): dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway.Cancer Res. 1997; 57: 3697-3707PubMed Google Scholar Furthermore, HDACi have been useful as a chemotherapy strategy to restrain proliferation, dedifferentiation, and self-renewal of cancer stem-like cells.7Jones PA Baylin SB The epigenomics of cancer.Cell. 2007; 128: 683-692Abstract Full Text Full Text PDF PubMed Scopus (3654) Google Scholar However, the specific mechanisms by which HDACi affect these cells are largely unknown. We previously showed that HDACs mediate tumorigenesis8Hsieh TH Tsai CF Hsu CY Kuo PL Hsi E Suen JL et al.n-Butyl benzyl phthalate promotes breast cancer progression by inducing expression of lymphoid enhancer factor 1.PLoS One. 2012; 7: e42750Crossref PubMed Scopus (50) Google Scholar and the epithelial–mesenchymal transition9Hsieh TH Tsai CF Hsu CY Kuo PL Lee JN Chai CY et al.Phthalates stimulate the epithelial to mesenchymal transition through an HDAC6-dependent mechanism in human breast epithelial stem cells.Toxicol Sci. 2012; 128: 365-376Crossref PubMed Scopus (45) Google Scholar in a highly malignant human breast cancer stem-like cells (CSCs) line. These CSCs carry stem-cell markers (CD44+/CD24-, ER+, HER2/neu+) and are capable of self renewal or pluripotent differentiation, making them extremely malignant.10Wang KH Kao AP Chang CC Lee JN Hou MF Long CY et al.Increasing CD44+/CD24(-) tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis.Mol Cancer. 2010; 9: 288Crossref PubMed Scopus (46) Google Scholar HDACi can also rapidly alter microRNA (miRNA) levels11Scott GK Mattie MD Berger CE Benz SC Benz CC Rapid alteration of microRNA levels by histone deacetylase inhibition.Cancer Res. 2006; 66: 1277-1281Crossref PubMed Scopus (439) Google Scholar to induce cell death in thyroid cancer cells12Brest P Lassalle S Hofman V Bordone O Gavric Tanga V Bonnetaud C et al.MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.Endocr Relat Cancer. 2011; 18: 711-719Crossref PubMed Scopus (80) Google Scholar and to inhibit growth via a p53-independent pathway in neuroblastoma SH-SY5Y cells.13Jin H Liang L Liu L Deng W Liu J HDAC inhibitor DWP0016 activates p53 transcription and acetylation to inhibit cell growth in U251 glioblastoma cells.J Cell Biochem. 2013; 114: 1498-1509Crossref PubMed Scopus (24) Google Scholar Nevertheless, the exact mechanisms by which HDACi mediates CSCs apoptosis through miRNA remain unknown. MiRNAs are small (~21–23 nucleotides) noncoding RNAs that reduce the post-transcriptional stability of target mRNAs.14Bartel DP MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15899) Google Scholar MiRNA hybridization with target mRNAs induces mRNA degradation and inhibits translation, thereby regulating a variety of cellular processes, such as proliferation, apoptosis, senescence, differentiation, and death.15Chang TC Mendell JT microRNAs in vertebrate physiology and human disease.Annu Rev Genomics Hum Genet. 2007; 8: 215-239Crossref PubMed Scopus (379) Google Scholar Several specific miRNAs have been shown to act as tumor suppressors or as oncogenes in cancers.16Gandellini P Profumo V Folini M Zaffaroni N MicroRNAs as new therapeutic targets and tools in cancer.Expert Opin Ther Targets. 2011; 15: 265-279Crossref PubMed Scopus (81) Google Scholar In the present work, we demonstrate that HDACi mediate apoptosis through miRNA in human breast cancer cells and we identify possible downstream target genes. This study revealed a novel pathway by which HDACi promotes apoptosis. To determine whether miRNAs were induced by HDACi in human breast cancer cells, the Human 384 SeraMir miRNA profiler was used to assess two breast cancer cell lines treated with the HDACi TSA, a breast cancer stem cell line (R2N1d) and a metastasis-type breast cancer cell line (MDA-MB-231) (Figure 1a). To compare treatment with control cells, 380 miRNAs were measured and the top miRNA most significantly (>10-fold) increased/decreased miRNAs in the TSA-treated cells relative to control cells are shown in Figure 1b and Supplementary Table S1. miR-125a-5p, miR-150, miR-362-3p, miR-503, miR-133a, let-7c, miR-548b-5p, let-7b, miR-149, miR-512-5p, miR-29c, miR-513c, and miR-187 were induced by TSA, but miR-331-5p, miR-33b, miR-192, miR-195, let-7i, miR-541, miR-200b, miR-146-3p, Hsa-miR-200a, miR-193a-5p, miR-99b, miR-34b, and miR-373 were decreased by TSA in both R2N1d and MDA-MB-231 cells. MiR-125a-5p was the most highly induced by TSA treatment in both human breast cancer lines. We further confirmed that HDACi can induce miR-125a-5p expression with qRT-PCR of breast cancer cells treated with six different HDACi (TSA, valproic acid, sodium butyrate, splitomycin, apicidin, and M344). We found that miR-125a-5p increased in a concentration-dependent manner with each of the HDACi (Figure 1c). Previous studies have reported that HDACi induce apoptosis through the intrinsic pathway in cancer cells. Therefore, we examined whether miR-125a-5p regulates apoptosis in the two breast cancer cell lines. First, we examined miR-125a-5p expression with a dose-dependent manner of miR-125a-5p plasmid by q-PCR. We found that miR-125a-5p expression was positively correlated with indicated doses in both R2N1d (Figure 2a) and MDA-MB-231 (Supplementary Figure S1a) cells. In addition, we investigated cell cycle progression by flow cytometry and found that overexpression of miR-125a-5p caused a significant increase in cell cycle arrest at the sub-G1 phase and a decrease in the proportion of cells in G1 phase in both R2N1d (Figure 2b) and MDA-MB-231 (Supplementary Figure S1b) cells. Cell cycle arrest at the sub-G1 phase frequently leads to cell death through apoptosis.17Darzynkiewicz Z Juan G Li X Gorczyca W Murakami T Traganos F Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).Cytometry. 1997; 27: 1-20Crossref PubMed Scopus (1033) Google Scholar Consistent with this, TUNEL assays showed that miR-125a-5p overexpression significantly enhanced apoptosis in both R2N1d (Figure 2c) and MDA-MB-231 (Supplementary Figure S1c) cells compared with the control cells. Next, a caspase activity assay showed that miR-125a-5p overexpression activated caspase 3 and 9 activity in both R2N1d (Figure 2d) and MDA-MB-231 (Supplementary Figure S2a) cells. We used specific inhibitors of caspases 2, 3, 8, and 9 to further analyze the apoptosis signaling pathway. The caspase 9 inhibitor blocked the miR-125a-5p–mediated increase in caspase 3 activation (Supplementary Figure S2c), but the caspase 3 inhibitor did not affect caspase 9 activation (Supplementary Figure S2e). Furthermore, caspase 2 and 8 were not activated by overexpression of miR-125a-5p in the breast cancer cell lines (Supplementary Figure S2b,d). To further confirm that miR-125a-5p promotes apoptosis, we examined the levels of caspase 3, and Bcl-xL by immunoblotting. We found that active caspase 3 was induced, but Bcl-xL decreased in R2N1d (Figure 2e,f) and MDA-MB-231 (Supplementary Figure S2f,g) cells overexpressing miR-125a-5p. These findings suggested that miR-125a-5p overexpression functions in the same way as HDACi to induce the intrinsic apoptosis pathway through caspases 9 and 3. We next used RepTar and RNAhybrid,18Elefant N Berger A Shein H Hofree M Margalit H Altuvia Y RepTar: a database of predicted cellular targets of host and viral miRNAs.Nucleic Acids Res. 2011; 39: D188-D194Crossref PubMed Scopus (43) Google Scholar,19Krüger J Rehmsmeier M RNAhybrid: microRNA target prediction easy, fast and flexible.Nucleic Acids Res. 2006; 34: W451-W454Crossref PubMed Scopus (1219) Google Scholar a computational motif prediction method, to identify miR-125a-5p target genes in the human genome (Supplementary Table S2). RNAhybrid calculates the binding thermodynamic between miRNA sequences and the 3′-UTRs of putative target genes. RepTar and RNAhybrid identified HDAC5 as a possible target, with two free energy value for binding in the 3′-UTR of −22.3 (Supplementary Figure S3a) and −32.9 kcal/mol (Figure 3a). RepTar prediction software found that 3′UTR of HDAC5 has two miR-125a-5p targeting site located at ~425 and 710 bp downstream of the HDAC5 3′ start (Supplementary Figure S3a). Therefore, the full length, 427~449 bp and 708~734 bp fragment of 3′-UTR was cloned into a luciferase reporter vector and transfecting into HEK-293T cells. The results found that full length and 708~734 bp fragment of HDAC5 3′-UTR was decreased by miR-125a-5p, but 427~449 bp signal was not (Supplementary Figure S3b). We next examined whether miR-125a-5p could directly silence the biological function of HDAC5 through binding the 3′-UTR (708~734 bp fragment) by cloning the wild-type (WT) and mutated (MT) 3′-UTR into a luciferase reporter vector and transfecting these plasmids, along with different concentrations of the miR-125a-5p expression plasmid into HEK-293T cells. We found that the HDAC5 WT luciferase signal was decreased by miR-125a-5p in a concentration-dependent manner, but the HDAC5 MT signal was not (Figure 3b). To verify these results, we investigated whether miR-125a-5p affected HDAC5 protein levels. The levels of HDAC5 were lower in miR-125a-5p–overexpressing cells compared with control cells, whereas the levels of other HDACs (HDAC7, HDAC10) were not affected by miR-125a-5p (Figure 3c). We also analyzed the biological functions of HDAC5 in R2N1d and MDA-MB-231 cells. Silencing of HDAC5 with siRNA-1/2 resulted in decreased cell growth, wound healing and invasion (Figure 3e,g,h and Supplemetary Figure S4a,c,d), but increased apoptosis (Figure 3f and Supplementary Figure S4b). Immunoblotting also showed that Ki-67, active MMP2, and Bcl-xL levels decreased, but active caspase 3 levels increased when HDAC5 was silenced in human breast cancer cells (Figure 3d). Hence, these data indicate that HDAC5 levels are controlled by miR-125a-5p and that silencing of HDAC5 has the same biological effect as increasing miR-125a-5p in human breast cancer cells. To further analyze the underlying mechanism by which HDACi affect miR-125a-5p, the sequence of the miR-125a-5p promoter region was examined with the TFSEARCH program for potential transcription factor binding sites. We identified a potential p300 binding motif (TGACTCCCTCTTATT) in the proximal region of the promoter (Figure 4a). Pre-miR-125a was located at chr19: 51693254-339 and has two clustered miRNAs in the upstream sequence including pre-mir-99b (chr19: 51692612-81) and pre-let-7e (chr19: 51692786-864). We found that pre-miR-125a and pre-mir-99b-let-7e may have independent promoters and only one P300 putative binding site located at ~160 bp upstream of the pre-miR-125a 5′ end (Supplementary Figure S5). Interestingly, a previous study demonstrated that HDACi increase acetylated RUNX3 through inhibition of HDAC5, which in turn enhances binding of the RUNX3-p300 complex to target promoters.20Jin YH Jeon EJ Li QL Lee YH Choi JK Kim WJ et al.Transforming growth factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation.J Biol Chem. 2004; 279: 29409-29417Crossref PubMed Scopus (181) Google Scholar Therefore, we hypothesized that HDACi induce miR-125a-5p expression by decreasing HDAC5 expression and acetylating RUNX3, leading to enhanced binding of the RUNX3/p300 complex to the miR-125a-5p promoter in human breast cancer. To test this hypothesis, RUNX3, p300, and HDAC5 were silenced with siRNA in the presence and absence of TSA, and the association of the RUNX3/p300/HDAC5 complex with miR-125a-5p was examined. MiR-125a-5p expression was essentially blocked by different concentrations of RUNX3 siRNA (Supplementary Figure S6a) and p300 siRNA (Supplementary Figure S6b). On the other hand, HDAC5 siRNA (Supplementary Figure S6c) promoted miR-125a-5p expression when cells were treated with TSA (Figure 4b). Immunoprecipitation assays showed that acetylated RUNX3 increased in the presence of TSA and HDAC5 siRNA (Figure 4c). Additionally, ChIP showed that RUNX3, but not HDAC5, was required for p300 to bind the miR-125a-5p promoter (Figure 4d). These results suggested that RUNX3 may play an important role in tumor suppression through miR-125a-5p. Indeed, upon RUNX3 silencing, levels of Ki-67, active MMP2, and Bcl-xL increased, whereas active caspase 3 decreased in both R2N1d and MDA-MB-231 cells (Figure 4e), and this was accompanied by increased cell growth, wound healing, and invasion (Figure 4f–h). HDAC5 appears to act upstream of miR-125a-5p in the apoptosis-induction pathway and may mediate miR-125a-5p expression in human breast cancer. We hypothesized that HDAC5 may participate in a positive feedback loop mediated through miR-125a-5p. To test this hypothesis, we analyzed miR-125a-5p expression following overexpression of HDAC5 in R2N1d cells by qRT-PCR. We found that miR-125a-5p expression decreased in a HDAC5 concentration-dependent manner (Figure 5a,b). Converse, miR-125a-5p expression was induced by silencing of HDAC5 in a dose-dependent manner (Figure 5c,d). Additionally, cell growth increased with overexpression of HDAC5 in both cell lines (Figure 5e). We also found that overexpression of HDAC5 abolished miR-125a-5p-induced apoptosis (Figure 5f) and increased migration (Figure 5g) in R2N1d. Thus, knocking down HDAC5 will mimic the effect of TSA on miR-125a expression. HDAC5 and miR-125a-5p appear to operate through a positive feedback mechanism via the RUNX3/p300 complex to affect tumor growth, metastasis and apoptosis in human breast cancer. We next examined whether HDACi inhibit cancer stem-like cell tumorigenesis via miR-125a-5p targeting of HDAC5 in vivo. R2N1d-YFP and R2N1d-GFP-mir-125a-5p cells were implanted into immunodeficient nude mice and tumor growth was monitored, respectively. After 1 week, the mice were intratumorally injected with TSA (500 μg/kg) or normal saline once every 2 days for 30 days. Mir-125a-5p and TSA inhibited R2N1d cell tumorigenesis as indicated by a decrease in the fluorescence signal by in vivo imaging (Figure 6a) and tumor photon flux (Figure 6b). In addition, fluorescence microscopy, TUNEL assay, in situ miR-125a-5p hybridization, and HDAC5 immunochemistry of the tumor tissues, showed similar results (Figure 6c) to those observed in vitro. Specifically, the R2N1d-YFP fluorescence signal in xenograft tumor sections remained constant, whereas the apoptosis-inducing ability or the expression of miR-125a-5p increased, but expression of HDAC5 decreased with TSA treatment. In this study, we used a commercial human miRNA profiler, to identify miRNAs associated with HDACi treatment in human breast cancer stem-like cells. We identified and validated the induction of miR-125a-5p by HDACi, which activated the RUNX3/p300/HDAC5 complex and mediated cell apoptosis through a feedback loop with the target gene HDAC5 (Figure 6d). We further showed that miR-125a-5p and HDAC5 modulate the tumorigenesis of cancer stem-like cells in vivo/vitro and may be appropriate clinical indicators to monitor the success of TSA treatment in breast cancer patients. Among the differentially expressed miRNAs identified by the profiler, miR-125a-5p, miR-150,21Ma Y Zhang P Wang F Zhang H Yang J Peng J et al.miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer.Gut. 2012; 61: 1447-1453Crossref PubMed Scopus (163) Google Scholar miR-362-3p,22Christensen LL Tobiasen H Holm A Schepeler T Ostenfeld MS Thorsen K COLOFOL steering group et al.MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.Int J Cancer. 2013; 133: 67-78Crossref PubMed Scopus (86) Google Scholar miR-503,23Forrest AR Kanamori-Katayama M Tomaru Y Lassmann T Ninomiya N Takahashi Y et al.Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation.Leukemia. 2010; 24: 460-466Crossref PubMed Scopus (207) Google Scholar miR-133a,24Kano M Seki N Kikkawa N Fujimura L Hoshino I Akutsu Y et al.miR-145, miR-133a and miR-133b: Tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma.Int J Cancer. 2010; 127: 2804-2814Crossref PubMed Scopus (437) Google Scholar let-7c,25Esquela-Kerscher A Trang P Wiggins JF Patrawala L Cheng A Ford L et al.The let-7 microRNA reduces tumor growth in mouse models of lung cancer.Cell Cycle. 2008; 7: 759-764Crossref PubMed Scopus (565) Google Scholar miR-548b-5p, let-7b,25Esquela-Kerscher A Trang P Wiggins JF Patrawala L Cheng A Ford L et al.The let-7 microRNA reduces tumor growth in mouse models of lung cancer.Cell Cycle. 2008; 7: 759-764Crossref PubMed Scopus (565) Google Scholar miR-149,26Lin RJ Lin YC Yu AL miR-149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells.Mol Carcinog. 2010; 49: 719-727PubMed Google Scholar miR-512-5p, miR-29c,27Pass HI Goparaju C Ivanov S Donington J Carbone M Hoshen M et al.hsa-miR-29c* is linked to the prognosis of malignant pleural mesothelioma.Cancer Res. 2010; 70: 1916-1924Crossref PubMed Scopus (131) Google Scholar miR-513c, and miR-187 emerged as the most consistently increased, suggesting that these miRNA may potentially act as tumor suppressors. HDACi have been evaluated in clinical trials as potential anticancer drugs,28Kim HJ Bae SC Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs.Am J Transl Res. 2011; 3: 166-179PubMed Google Scholar and TSA has shown great promise as a clinical therapy for human breast cancer.29Vigushin DM Ali S Pace PE Mirsaidi N Ito K Adcock I et al.Trichostatin A is a histone deacetylase inhibitor with potent antitumor activity against breast cancer in vivo.Clin Cancer Res. 2001; 7: 971-976PubMed Google Scholar,30Alao JP Stavropoulou AV Lam EW Coombes RC Vigushin DM Histone deacetylase inhibitor, trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells.Mol Cancer. 2006; 5: 8Crossref PubMed Scopus (68) Google Scholar HDACi decrease tumorigenesis and induce apoptosis through the intrinsic apoptosis pathway in different cancer types.31Bolden JE Peart MJ Johnstone RW Anticancer activities of histone deacetylase inhibitors.Nat Rev Drug Discov. 2006; 5: 769-784Crossref PubMed Scopus (2521) Google Scholar Interestingly, we found that miR-125a-5p mediated the intrinsic apoptosis pathway through caspases 9 and 3, providing a potential mechanism for the induction of the intrinsic apoptosis pathway by HDACi and identifying miR-125a-5p as a potential therapeutic target for HDACi. Previous studies also have showed that class I HDAC inhibitor, entinostat induced apoptosis through miR-125a, miR-125b, and miR-205 in erbB2-overexpressing breast cancer cells.32Wang S Huang J Lyu H Lee CK Tan J Wang J et al.Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells.Cell Death Dis. 2013; 4: e556Crossref PubMed Scopus (107) Google Scholar Therefore, HDACi positively have the ability to induce miR-125a-5p expression and mediate apoptosis. MiR-125a-5p has been reported to be downregulated in non-small cell lung cancer tissues, and to decrease migration and invasion of human lung cancer cell lines.33Jiang L Huang Q Zhang S Zhang Q Chang J Qiu X et al.Hsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancer and have inverse effects on invasion and migration of lung cancer cells.BMC Cancer. 2010; 10: 318Crossref PubMed Scopus (197) Google Scholar MiR-125a-5p interacts with hepatitis B virus surface antigen and directly suppresses its activity.34Potenza N Papa U Mosca N Zerbini F Nobile V Russo A Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen.Nucleic Acids Res. 2011; 39: 5157-5163Crossref PubMed Scopus (145) Google Scholar In addition, miR-125a-5p decreases cell growth more potently when combined with trastuzumab in the treatment of gastric cancer,35Nishida N Mimori K Fabbri M Yokobori T Sudo T Tanaka F et al.MicroRNA-125a-5p is an independent prognostic factor in gastric cancer and inhibits the proliferation of human gastric cancer cells in combination with trastuzumab.Clin Cancer Res. 2011; 17: 2725-2733Crossref PubMed Scopus (225) Google Scholar and mediates apoptosis of human lung cancer cells through a p53-dependent pathway.36Jiang L Huang Q Chang J Wang E Qiu X MicroRNA HSA-miR-125a-5p induces apoptosis by activating p53 in lung cancer cells.Exp Lung Res. 2011; 37: 387-398Crossref PubMed Scopus (72) Google Scholar These previous findings are similar to our observation that miR-125a-5p promotes apoptosis in human breast cancer stem cells. Therefore, miR-125a-5p appears to play an important role in promoting cell apoptosis by targeting apoptosis-related genes in multiple cancer types. Furthermore, an early report found that miR-125a-5p targets proapoptotic protein, BAK1 to suppress apoptosis in immature hematopoietic stem cells37Guo S Lu J Schlanger R Zhang H Wang JY Fox MC et al.MicroRNA miR-125a controls hematopoietic stem cell number.Proc Natl Acad Sci USA. 2010; 107: 14229-14234Crossref PubMed Scopus (273) Google Scholar and thus revealing that miR-125a-5p biology can assume varied roles. HDAC5 is a member of the class II HDAC family (HDAC4, 5, 6, 9, and 11) and localizes in both the nucleus and cytoplasm. HDAC4 and HDAC5 are highly similar enzymes, with an overall sequence identity of ~70%.38de Ruijter AJ van Gennip AH Caron HN Kemp S van Kuilenburg AB Histone deacetylases (HDACs): characterization of the classical HDAC family.Biochem J. 2003; 370: 737-749Crossref PubMed Scopus (2462) Google Scholar Early studies indicated that miR-2861 represses HDAC5 expression to enhance bone morphogenetic protein 2-induced osteoblastogenesis.39Li H Xie H Liu W Hu R Huang B Tan YF et al.A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.J Clin Invest. 2009; 119: 3666-3677Crossref PubMed Scopus (407) Google Scholar Interesting, we found that in addition to directly targeting HDAC5, miR-125a-5p can also directly target HDAC4 and inhibit its protein expression (data not shown). This result reveals that, not only do HDACs regulate miRNA expression, but miRNAs can also reciprocally control HDAC activity. In addition, high levels of HDAC5 are significantly associated with poor survival in human brain cancer patients and knockdown of HDAC5 enhances apoptosis through caspase 3 (ref. 40Milde T Oehme I Korshunov A Kopp-Schneider A Remke M Northcott P et al.HDAC5 and HDAC9 in medulloblastoma: novel markers for risk stratification and role in tumor cell growth.Clin Cancer Res. 2010; 16: 3240-3252Crossref PubMed Scopus (157) Google Scholar). Consistent with these results, we found that silencing of HDAC5 inhibits cell growth, migration, and invasion and increases apoptosis in human breast cancer stem-like cells. RUNX3 is a tumor suppressor41Bae SC Choi JK Tumor suppressor activity of RUNX3.Oncogene. 2004; 23: 4336-4340Crossref PubMed Scopus (119) Google Scholar that controls gene expression by interacting with p300 and HDAC5 (ref. 20Jin YH Jeon EJ Li QL Lee YH Choi JK Kim WJ et al.Transforming growth factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation.J Biol Chem. 2004; 279: 29409-29417Crossref PubMed Scopus (181) Google Scholar). RUNX3 promotes apoptosis in K-Ras-activated lung cancer cells42Lee YS Lee JW Jang JW Chi XZ Kim JH Li YH et al.Runx3 inactivation is a crucial early event in the development of lung adenocarcinoma.Cancer Cell. 2013; 24: 603-616Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar and transcriptionally activates the proapoptotic gene Bim in transforming growth factor-β-induced apoptosis.43Yano T Ito K Fukamachi H Chi XZ Wee HJ Inoue K et al.The RUNX3 tumor suppressor upregulates Bim in gastric epithelial cells undergoing transforming growth factor beta-induced apoptosis.Mol Cell Biol. 2006; 26: 4474-4488Crossref PubMed Scopus (141) Google Scholar Similarly, we found that RUNX3 inhibits cell invasion and migration, and induces apoptosis in human breast cancer stem cells. Acetylating of RUNX3 enhanced binding of the RUNX3/p300 complex to the miR-125a-5p promoter and silencing of RUNX3 has the same biological effect as decreasing miR-125a-5p in human breast cancer cells. Therefore, we believe that RUNX3 is an important role for transcription regulations and biological effect of miR-125a-5p. Our data further indicated that HDAC5 silencing in breast cancer cells is due to increased miR-125a-5p and its inhibitory effect on HDAC5. Thus, RUNX3 may mediate apoptosis through a regulatory loop involving miR-125a-5p and HDAC5. In summary, our study identified a critical regulatory RUNX3/p300/HDAC5/miR-125a-5p loop network that modulates HDAC5 levels when HDACi are used to treat breast cancer cells. Furthermore, miR-125a-5p and RUNX3 appear to play similar biological roles in mediating apoptosis and tumorigenesis in human breast cancer stem cells. Thus, the present study provides valuable insight that will be of great use in clinical applica" @default.
• W1998780829 created "2016-06-24" @default.
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• W1998780829 date "2015-04-01" @default.
• W1998780829 modified "2023-09-26" @default.
• W1998780829 title "HDAC Inhibitors Target HDAC5, Upregulate MicroRNA-125a-5p, and Induce Apoptosis in Breast Cancer Cells" @default.
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