Guiying Wang

Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation

Fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) by application of transcription factors octamer-binding protein 4 (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytomatosis oncogene (c-Myc) (OSKM), but the underlying mechanisms remain unclear. Here, we report that exogenous Oct4 and Sox2 can bind at the promoter regions of mir-141/200c and mir-200a/b/429 cluster, respectively, and induce the transcription activation of miR-200 family during the OSKM-induced reprogramming. Functional suppression of miR-200s with specific inhibitors significantly represses the OSKM-caused mesenchymal-to-epithelial transition (MET, an early event in reprogramming of fibroblasts to iPSCs) and iPSC generation, whereas overexpression of miR-200s promotes the MET and iPSC generation. Mechanistic studies showed that miR-200s significantly repress the expression of zinc finger E-box binding homeobox 2 (ZEB2) through directly targeting its 3′ UTR and direct inhibition of ZEB2 can mimic the effects of miR-200s on iPSC generation and MET process. Moreover, the effects of miR-200s during iPSC generation can be blocked by ZEB2 overexpression. Collectively, our findings not only reveal that members of the miR-200 family are unique mediators of the reprogramming factors Oct4/Sox2, but also demonstrate that the miR-200/ZEB2 pathway as one critical mechanism of Oct4/Sox2 to induce somatic cell reprogramming at the early stage.

microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming

Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM), but the mechanisms underlying this reprogramming remain poorly understood. Here, we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM- and OSK-mediated reprogramming. Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming. Moreover, the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression. Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events, such as mesenchymal-to-epithelial transition (MET) and Dlk1-Dio3 region transcription. Thus, our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a multistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.

MicroRNA-29a promotes colorectal cancer metastasis by regulating matrix metalloproteinase 2 and E-cadherin via KLF4.

Background:Growing evidence suggests that miR-29a has an important role in regulating tumourigenesis and development of various types of cancer. However, the role and the underlying mechanism of miR-29a in colorectal cancer (CRC) remain largely unknown.Methods:MiR-29a targeted gene was identified by the luciferase assay and western blot. MiR-29a function was analysed by invasion assays and the orthotopic transplantation mouse model. The miR-29a pathway was assayed by real-time PCR, western blot and chip analysis.Results:KLF4 was identified as a direct target gene of miR-29a. MiR-29a promoted CRC cell invasion, which was blocked by re-expression of KLF4. In addition, MMP2 was identified as a novel direct target of KLF4. Both miR-29a overexpression and KLF4 knockdown promoted MMP2 expression but inhibited E-cadherin expression. Furthermore, clinical data indicated that both miR-29a high expression and KLF4 mRNA low expression were associated with metastasis and poor prognosis in CRC patients, and KLF4 protein expression was inversely correlated with MMP2 but positively correlated with E-cad protein expression.Conclusion:Increased expression of miR-29a promoted CRC metastasis by regulating MMP2/E-cad through direct targeting KLF4, which highlights the potential of the miR-29a inhibitor as a novel agent against CRC metastasis.

MiR‐138 Promotes Induced Pluripotent Stem Cell Generation Through the Regulation of the p53 Signaling

Induced pluripotent stem (iPS) cells, especially those reprogrammed from patient somatic cells, have a great potential usage in regenerative medicine. The expression of p53 has been proven as a key barrier limiting iPS cell generation, but how p53 is regulated during cell reprogramming remains unclear. In this study, we found that the ectopic expression of miR‐138 significantly improved the efficiency of iPS cell generation via Oct4, Sox2, and Klf4, with or without c‐Myc (named as OSKM or OSK, respectively), without sacrificing the pluripotent characteristics of the generated iPS cells. Exploration of the mechanism showed that miR‐138 directly targeted the 3′ untranslated region (UTR) of p53, significantly decreasing the expression of p53 and its downstream genes. Furthermore, the ectopic expression of p53 having a mutant 3′‐UTR, which cannot be bound by miR‐138, seriously impaired the effect of miR‐138 on p53 signaling and OSKM‐initiated somatic cell reprogramming. Combined with the fact that miR‐138 is endogenously expressed in fibroblasts, iPS cells, and embryonic stem cells, our study demonstrated that regulation of the p53 signaling pathway and promotion of iPS cell generation represent an unrevealed important function of miR‐138. STEM Cells2012;30:1645–1654

Lysine Acetyltransferase GCN5 Potentiates the Growth of Non-small Cell Lung Cancer via Promotion of E2F1, Cyclin D1, and Cyclin E1 Expression

Background: The role of the lysine acetyltransferase GCN5 in cancer development remains largely unknown. Results: GCN5 expression correlates with lung cancer tumor size, directly enhances the expression of E2F1, cyclin E1, and cyclin D1, and potentiates lung cancer growth. Conclusion: GCN5 potentiates lung cancer growth in an E2F1-dependent manner. Significance: GCN5 is critical for lung cancer growth and represents a potential target for the treatment of lung cancer. The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment.

HDAC1 and Klf4 interplay critically regulates human myeloid leukemia cell proliferation.

Acute myeloid leukemia (AML) is recognized as a complex disease of hematopoietic stem cell disorders, but its pathogenesis mechanisms, diagnosis, and treatment remain unclear. General histone deacetylase (HDAC) inhibitors have been used in blood cancers including AML, but the lack of gene specificity greatly limits their anti-cancer effects and clinical applications. Here, we found that HDAC1 expression was negatively correlated with that of Krüppel-like factor 4 (Klf4) and that AML patients with lower HDAC1 level had better prognosis. Further, knockdown of HDAC1 in leukemia cells K562, HL-60, and U937 significantly increased Klf4 expression and inhibited cell cycle progression and cell proliferation, similar results were found for HDAC inhibitors (VPA and mocetinostat). Moreover, overexpression or knockdown of Klf4 could markedly block the effects of HDAC1 overexpression or knockdown on leukemia cells in vitro and in vivo, respectively. Mechanistic analyses demonstrated that HDAC1 and Klf4 competitively bound to the promoter region of Klf4 and oppositely regulated Klf4 expression in myeloid leukemia. We identified HDAC1 as a potential specific target for repressing cell proliferation and inducing cell cycle arrest through interplay and modulation of Klf4 expression, suggests that HDAC1 and Klf4 are potential new molecular markers and targets for clinical diagnosis, prognosis, and treatment of myeloid leukemia.

Euphol arrests breast cancer cells at the G1 phase through the modulation of cyclin D1, p21 and p27 expression

Euphorbia tirucalli is a long‑established treatment for a wide variety of cancers. However, the mechanism of its anticancer effect is yet to be elucidated. In the present study, we examined the anticancer effect of euphol, a tetracyclic triterpene alcohol isolated from the sap of Euphorbia tirucalli, in T47D human breast cancer cells. Following the treatment of cells with different doses of euphol for 24, 48 and 72 h, the cell proliferation, cell cycle, and mRNA and protein levels of cell cycle regulatory molecules were analyzed, respectively. Treatment of the cells with euphol resulted in decreased cell viability, which was accompanied by an accumulation of cells in the G1 phase. Further studies demonstrated that euphol treatment downregulated cyclin D1 expression and the hypophosphorylation of Rb. Furthermore, this effect was correlated with the downregulation of cyclin‑dependent kinase 2 (CDK2) expression and the upregulation of the CDK inhibitors p21 and p27. Reduced expression levels of cyclin A and B1 were also observed, corresponding to the decreased distribution of cells in the S and G2/M phases, respectively. These findings indicated that euphol is an active agent in Euphorbia tirucalli that exerts anticancer activity by arresting the cell cycle of cancer cells.

A miR-590/Acvr2a/Rad51b Axis Regulates DNA Damage Repair during mESC Proliferation

Summary Embryonic stem cells (ESCs) enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF), which maintains the self-renewal capability of mouse ESCs (mESCs), significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB) and double-strand break (DSB) damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a) to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR) gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

Synergetic Cooperation of microRNAs with Transcription Factors in iPS Cell Generation

Induced pluripotent stem (iPS) cells were first generated by forced expression of transcription factors (TFs) in fibroblasts. Recently, iPS cells have been generated more rapidly and efficiently using miRNAs with or without other transcription factors. However, the specific and collaborative roles of miRNAs and transcription factors in pluripotency acquisition and maintenance remain to be further investigated. Here, based on the miRNA profiling in mouse embryonic fibroblasts (MEFs), MEFs infected with Oct3/4, Sox2, Klf4 and c-Myc (OSKM) for 1, 2, 4, or 8 day, two iPS cell lines and ES cells, representing iPS activation and maintenance steps, we found that two unique miRNA sets are responsible for different steps of iPS generation, and the miRNA expression profiles of iPS cells are very similar to that of ES cells. Furthermore, we searched for transcription factors binding sites at the promoter regions of up-regulated miRNAs, and found that up-regulated miRNAs such as the miR-429-200 and miR-17 clusters are directly activated by exogenous TFs. The GO and pathway enrichment for candidate target gene sets of miRNAs or OSKM provided a clear picture of division and collaboration between miRNAs and OSKM during completion of the iPS process. Compared with the pathways regulated by OSKM, we found that miRNAs play critical roles in regulating iPS-specific pathways, such as the adherens junction and Wnt signaling pathways. Furthermore, we blocked miRNA expression using Dicer knockdown, and found that the level of miRNAs was decreased following this treatment, and the efficiency of iPS generation was significantly repressed. By combining high-throughput analysis, biostatistical analysis and functional experiments, this study provides new ideas for investigating the important roles of miRNAs, the mechanisms of miRNAs and related signaling pathways, and the potential for many more applications of miRNAs in somatic cell reprogramming.

Curcumin p38-dependently enhances the anticancer activity of valproic acid in human leukemia cells

Valproic acid (VPA) is a broad-spectrum inhibitor of histone deacetylase, which has been used in cancer therapy. Recently, the combination of VPA with other anticancer agents has been considered as a useful and necessary strategy to specifically induce anticancer gene expression. Curcumin (Cur) is a promising natural anticancer agent that can specifically regulate the expression of NF-kappaB, bcl-2, and bax in leukemia cells. However, no literature is available on the anticancer effects of the combination of VPA and Cur. Here we show that this combination significantly increases Sp1 binding, histone H3 and H4 acetylation in the promoter region of bax, but not in that of bcl-2. This specifically up-regulates bax expression and leads to HL-60 cell proliferation arrest, sub-G1 DNA accumulation and cell death. Further studies reveal that Cur specifically activates p38 MAPK, an essential factor for Sp1 binding at the bax promoter. Moreover, both inhibition of p38 MAPK and knock-down of bax expression significantly prevent VPA and Cur-induced proliferation arrest and death in HL-60 cells. These results suggest that Cur could p38-dependently promote bax expression and hence enhance the anticancer activity of VPA in human leukemia cells.