Yangchao Chen

MiR-26a Inhibits Cell Growth and Tumorigenesis of Nasopharyngeal Carcinoma through Repression of EZH2

Several microRNAs (miRNA) have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer that is widely prevalent in southern China. In this study, we report that microRNA miR-26a is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. Ectopic expression of miR-26a dramatically suppressed cell proliferation and colony formation by inducing G(1)-phase cell-cycle arrest. We found that miR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. Similar to the restoring miR-26 expression, EZH2 downregulation inhibited cell growth and cell-cycle progression, whereas EZH2 overexpression rescued the suppressive effect of miR-26a. Mechanistic investigations revealed that miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclin-dependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner. Interestingly, cyclin D2 was regulated by miR-26a but not by EZH2, revealing cyclin D2 as another direct yet mechanistically distinct target of miR-26a. In clinical specimens, EZH2 was widely overexpressed and its mRNA levels were inversely correlated with miR-26a expression. Taken together, our results indicate that miR-26a functions as a growth-suppressive miRNA in NPC, and that its suppressive effects are mediated chiefly by repressing EZH2 expression.

microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, we identified miR-146b as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. We showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, we discovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, our findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

EZH2-Mediated Concordant Repression of Wnt Antagonists Promotes β-Catenin–Dependent Hepatocarcinogenesis

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that represses gene transcription through histone H3 lysine 27 trimethylation (H3K27me3). Although EZH2 is abundantly present in various cancers, the molecular consequences leading to oncogenesis remain unclear. Here, we show that EZH2 concordantly silences the Wnt pathway antagonists operating at several subcellular compartments, which in turn activate Wnt/β-catenin signaling in hepatocellular carcinomas (HCC). Chromatin immunoprecipitation promoter array and gene expression analyses in HCCs revealed EZH2 occupancy and reduced expression of Wnt antagonists, including the growth-suppressive AXIN2, NKD1, PPP2R2B, PRICKLE1, and SFRP5. Knockdown of EZH2 reduced the promoter occupancy of PRC2, histone deacetylase 1 (HDAC1), and H3K27me3, whereas the activating histone marks were increased, leading to the transcriptional upregulation of the Wnt antagonists. Combinatorial EZH2 and HDAC inhibition dramatically reduced the levels of nuclear β-catenin, T-cell factor-dependent transcriptional activity, and downstream pro-proliferative targets CCND1 and EGFR. Functional analysis revealed that downregulation of EZH2 reduced HCC cell growth, partially through the inhibition of β-catenin signaling. Conversely, ectopic overexpression of EZH2 in immortalized hepatocytes activated Wnt/β-catenin signaling to promote cellular proliferation. In human HCCs, concomitant overexpression of EZH2 and β-catenin was observed in one-third (61/179) of cases and significantly correlated with tumor progression. Our data indicate that EZH2-mediated epigenetic silencing contributes to constitutive activation of Wnt/β-catenin signaling and consequential proliferation of HCC cells, thus representing a novel therapeutic target for this highly malignant tumor.

Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin†

Enhancer of zeste homolog 2 (EZH2) has been shown to be overexpressed in hepatocellular (HCC). We investigated the potential role of EZH2 in HCC tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting EZH2 as a form of HCC treatment. Lentivirus‐mediated RNAi was employed to knock‐down EZH2 expression in human hepatoma cells to study the function of EZH2 in tumorigenesis and evaluate the treatment efficacy. Lentivirus‐mediated RNAi effectively reduced EZH2 expression. Suppression of EZH2 in HCC cells significantly reduced their growth rate in vitro and markedly diminished their tumorigenicity in vivo. Moreover, in a mice model of established large‐sized HCC, we showed that intratumor injection of lentiviral (Lenti)‐shRNA (short hairpin RNA) or siRNA (small interfering RNA) targeting EZH2 produced significant tumor regression. To understand its molecular mechanism of action, we employed proteomic profiling technique and found that stathmin 1 is the downstream target of EZH2, as Lenti‐shEZH2 treatment decreased stathmin protein expression, and ectopic overexpression of stathmin prevented Lenti‐shEZH2 mediated tumor growth inhibition. Conclusion: Results from our study suggested for the first time that EZH2 plays a key role in HCC tumorigenesis, and is a novel therapeutic target for HCC. (HEPATOLOGY 2007;46:200–208.)

MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells

MicroRNAs (miRNAs) are non-protein-coding RNAs that function as post-transcriptional gene regulators. Recent evidence has shown that miRNA plays a pivotal role in the development of many cancers including glioma, a lethal brain cancer. We have recently compared the miRNA expression profiles between normal brain and glioma tissues from Chinese patients by miRNA microarray and identified a panel of differentially expressed miRNAs. Here, we studied the function of one miRNA, miR-15b, in glioma carcinogenesis and elucidated its downstream targets. Over-expression of miR-15b resulted in cell cycle arrest at G0/G1 phase while suppression of miR-15b expression resulted in a decrease of cell populations in G0/G1 and a corresponding increase of cell populations in S phase. We further showed that CCNE1 (encoding cyclin E1) is one of the downstream targets of miR-15b. Taken together, our findings indicate that miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related molecules.

Revisit complexation between DNA and polyethylenimine — Effect of uncomplexed chains free in the solution mixture on gene transfection

Our revisit of the complexation between anionic DNA and cationic polyethylenimine (PEI) in both water and phosphate buffered saline (PBS) by using a combination of laser light scattering (LLS) and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~3, but the PEI/DNA polyplexes have a high in-vitro gene transfection efficiency only when N:P≥10. Putting these two facts together, we not only conclude that this extra 7 portions of PEI chains are free in the solution mixture, but also confirmed that it is these free PEI chains that substantially promote the gene transfection no matter whether they are applied hours before or after the administration of the much less effective PEI/DNA polyplexes (N:P=3). The uptake kinetics measured by flow cytometry shows that the addition of free PEI leads to a faster and more efficient cellular internalization of polyplexes, but these free PEI chains mainly contribute to the subsequent intracellular trafficking. In contrast, the bound PEI chains mainly play a role in the DNA condensation and protection, leading to a different thinking in the development of non-viral vectors.

Revisit the complexation of PEI and DNA — How to make low cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure?

The commercially available branched polyethyleneimine (PEI) with a molar mass of 25 kD (PEI-25K) is an effective in vitro vector to transfer genes, but its cytotoxicity limits its applications in bio-related research. To solve such an efficiency-versus-cytotoxicity catch-22 problem, the disulfide bond has been previously used to link less toxic short PEI chains (2 kD), but previous literature results are controversial. Recently, we found that it is vitally important to remove both carbon dioxide and water in the linking reaction as well as to control the structure of the resultant chains linked by dithiobis(succinimidyl propionate) (DSP). Under a programmable mixing of PEI and DSP, we can use laser light scattering (LLS) to in-situ monitor the linking reaction kinetics in DMSO in terms of the change of the average molar mass (M(w)). Therefore, we were able to withdraw a series of linked PEI chains with different molar masses from one reaction mixture. Two such linked PEI samples (M(w) approximately 7 kD, PEI-7K-L and approximately 400 kD, PEI-400K-L) were used to illustrate the effect of the sample preparation and the chain structure on the in vitro gene transfection and cytotoxicity. Our results reveal that PEI-7K-L is less cytotoxic and more effective in the gene transfection than both PEI-25K and Lipofectamine 2000 in the in vitro gene transfection. However, PEI-400K-L has no gene transfection efficiency even though it is non-toxic.

Bone Morphogenic Protein-4 Impairs Endothelial Function Through Oxidative Stress–Dependent Cyclooxygenase-2 Upregulation. Implications on Hypertension

Rationale: Bone morphogenic protein (BMP)4 can stimulate superoxide production and exert proinflammatory effects on the endothelium. The underlying mechanisms of how BMP4 mediates endothelial dysfunction and hypertension remain elusive. Objective: To elucidate the cellular pathways by which BMP4-induced endothelial dysfunction is mediated through oxidative stress–dependent upregulation of cyclooxygenase (COX)-2. Methods and Results: Impaired endothelium-dependent relaxations, exaggerated endothelium-dependent contractions, and reactive oxygen species (ROS) production were observed in BMP4-treated mouse aortae, which were prevented by the BMP4 antagonist noggin. Pharmacological inhibition with thromboxane prostanoid receptor antagonist or COX-2 but not COX-1 inhibitor prevented BMP4-induced endothelial dysfunction, which was further confirmed with the use of COX-1−/− or COX-2−/− mice. Noggin and knockdown of BMP receptor 1A abolished endothelium-dependent contractions and COX-2 upregulation in BMP4-treated aortae. Apocynin and tempol treatment were effective in restoring endothelium-dependent relaxations, preventing endothelium-dependent contractions and eliminating ROS overproduction and COX-2 overexpression in BMP4-treated aortae. BMP4 increased p38 mitogen-activated protein kinase (MAPK) activity through a ROS-sensitive mechanism and p38 MAPK inhibitor prevented BMP4-induced endothelial dysfunction. COX-2 inhibition blocked the effect of BMP4 without affecting BMP4-induced ROS overproduction and COX-2 upregulation. Importantly, renal arteries from hypertensive rats and humans showed higher levels of COX-2 and BMP4 accompanied by endothelial dysfunction. Conclusions: We show for the first time that ROS serve as a pathological link between BMP4 stimulation and the downstream COX-2 upregulation in endothelial cells, leading to endothelial dysfunction through ROS-dependent p38 MAPK activation. This BMP4/ROS/COX-2 cascade is important in the maintenance of endothelial dysfunction in hypertension.

Targeting cadherin‐17 inactivates Wnt signaling and inhibits tumor growth in liver carcinoma

Hepatocellular carcinoma (HCC) is a lethal malignancy for which there are no effective therapies. To develop rational therapeutic approaches for treating this disease, we are performing proof‐of‐principle studies targeting molecules crucial for the development of HCC. Here, we show that cadherin‐17 (CDH17) adhesion molecule is up‐regulated in human liver cancers and can transform premalignant liver progenitor cells to produce liver carcinomas in mice. RNA interference–mediated knockdown of CDH17 inhibited proliferation of both primary and highly metastatic HCC cell lines in vitro and in vivo. The antitumor mechanisms underlying CDH17 inhibition involve inactivation of Wnt signaling, because growth inhibition and cell death were accompanied by relocalization of β‐catenin to the cytoplasm and a concomitant reduction in cyclin D1 and an increase in retinoblastoma. Conclusion: Our results identify CDH17 as a novel oncogene in HCC and suggest that CDH17 is a biomarker and attractive therapeutic target for this aggressive malignancy. (HEPATOLOGY 2009.)

The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat.

Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had been proposed as a powerful alternative method of cancer therapy. The present study was aimed to evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was evaluated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts using the combined treatment. These results demonstrated that the combined treatment generated additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising therapeutic strategy in treating glioma.