Extracellular vesicles derived from cervical cancer cells carrying MCM3AP-AS1 promote angiogenesis and tumor growth in cervical cancer via the miR-93/p21 axis.

Tumor cells can promote angiogenesis by secreting extracellular vesicles (EVs). Meanwhile, tumor-derived EVs can carry long non-coding RNAs to activate pro-angiogenic signaling in endothelial cells. Here, we investigated the role of long non-coding RNA MCM3AP-AS1 carried by cervical cancer (CC) cell-derived EVs in the angiogenesis and the resultant tumor growth in CC, as well as the potential molecular mechanisms. LncRNAs significantly expressed in CC cell-derived EVs and CC were screened, followed by prediction of downstream target genes. EVs were isolated from HcerEpic and CaSki cell supernatants, followed by identification. The expression of MCM3AP-AS1 in CC was analyzed and its interaction with miR-93-p21 was confirmed. Following co-culture system, the role of MCM3AP-AS1 carried by EVs in HUVEC angiogenic ability, CC cell invasion and migration in vitro along with angiogenesis and tumorigenicity in vivo was assayed. MCM3AP-AS1 was overexpressed in CC cell-derived EVs as well as in CC tissues and cell lines. Cervical cancer cell-derived EVs could transfer MCM3AP-AS1 into HUVECs where MCM3AP-AS1 competitively bound to miR-93 and upregulate the expression of the miR-93 target p21 gene. Thus, MCM3AP-AS1 promoted angiogenesis of HUVECs. In the similar manner, MCM3AP-AS1 enhanced CC cell malignant properties. In nude mice, EVs-MCM3AP-AS1 induced angiogenesis and tumor growth. Overall, this study reveals that CC cell-derived EVs may transport MCM3AP-AS1 to promote angiogenesis and tumor growth in CC.

The zinc finger protein GATA4 induces mesenchymal-to-epithelial transition and cellular senescence through the nuclear factor-κB pathway in hepatocellular carcinoma.

BACKGROUND AND AIM: The high mortality and poor prognosis of hepatocellular carcinoma (HCC) have raised the public attention. Gene therapy is considered as a promising treatment option for cancer; thus, finding a new therapeutic target for HCC is urgently needed. GATA4 is a tumor suppressor gene in multiple cancers, but its role in HCC is unclear. In this study, we explored the function of GATA4 in HCC. METHODS: Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction were used to detect the mRNA expression of GATA4 in HCC cells and tissues. Cell viability, transwell, colony formation, and flow cytometry assays were applied to examine different aspects of biological effects of GATA4 in vitro. Xenografts, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assays were performed to evaluate the effect of GATA4 on tumorigenicity in vivo. Western blotting, immunofluorescence, and ß-galactosidase staining were used to investigate the mechanism underlying the function of GATA4. RESULTS: We found that GATA4 was silenced in 15/19 (79%) HCC tissues. Restoring the expression of GATA4 induced G0 /G1 phase arrest, promoted apoptosis, suppressed HCC proliferation in vitro, and inhibited HCC tumor growth in vivo. Our data further showed that the ectopic expression of GATA4 induced cellular senescence through regulating nuclear factor-κB and inducing mesenchymal-to-epithelial transition. CONCLUSIONS: Our data demonstrated that by inducing cellular senescence and mesenchymal-to-epithelial transition, GATA4 plays a crucial role as a tumor suppressor in HCC. It may thus be a potential cancer therapeutic target for HCC.

The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via ß-catenin/Cdc25c signaling and G2/M arrest.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear. METHODS: RT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs. RESULTS: We found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing ß-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin. CONCLUSION: Our results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing ß-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.

TET1 exerts its anti-tumor functions via demethylating DACT2 and SFRP2 to antagonize Wnt/ß-catenin signaling pathway in nasopharyngeal carcinoma cells.

BACKGROUND: TET1 is a tumor suppressor gene (TSG) that codes for ten-eleven translocation methyl cytosine dioxygenase1 (TET1) catalyzing the conversion of 5-methylcytosine to 5-hydroxy methyl cytosine as a first step of TSG demethylation. Its hypermethylation has been associated with cancer pathogenesis. However, whether TET1 plays any role in nasopharyngeal carcinoma (NPC) remains unclear. This study investigated the expression and methylation of TET1 in NPC and confirmed its role and mechanism as a TSG. RESULTS: TET1 expression was downregulated in NPC tissues compared with nasal septum deviation tissues. Demethylation of TET1 in HONE1 and HNE1 cells restored its expression with downregulated methylation, implying that TET1 was silenced by promoter hypermethylation. Ectopic expression of TET1 suppressed the growth of NPC cells, induced apoptosis, arrested cell division in G0/G1 phase, and inhibited cell migration and invasion, confirming TET1 TSG activity. TET1 decreased the expression of nuclear ß-catenin and downstream target genes. Furthermore, TET1 could cause Wnt antagonists (DACT2, SFRP2) promoter demethylation and restore its expression in NPC cells. CONCLUSIONS: Collectively, we conclude that TET1 exerts its anti-tumor functions in NPC cells by suppressing Wnt/ß-catenin signaling via demethylation of Wnt antagonists (DACT2 and SFRP2).

Facile Fabrication of Ordered Anodized Aluminum Oxide Membranes with Controlled Pore Size by Improved Hard Anodization.

We have fabricated highly ordered anodized aluminum oxide (AAO) membranes with different diameter through improved hard anodization (HA) at high temperature. This process can generate thick AAO membranes (30 µm) in a short anodizing time with high growth rate 20-60 µm h-1 which is much faster than that in traditional mild two-step anodization. We enlarged the AAO pore diameter by adjusting the voltage rise rate at the same time, which has a great influence on current density and temperature. The AAO pore diameter varies from 60-110 nm to 160-190 nm. The pore diameter (Dp) of the AAO prepared by this improved process is much larger than that prepared by HA (40-60 nm) when H2C2O4 as electrolyte. It can expand potential use of the AAO membranes such as for the template-based synthesis of nanowires or nanotubes with modulated diameters and also for practical separation technology. We also has used the AAO with different diameters prepared by this improved HA to fabricate Co nanowires and γ-Fe2O3 superparamagnetic nanorods.

Homeobox protein MSX1 inhibits the growth and metastasis of breast cancer cells and is frequently silenced by promoter methylation.

Deregulation of msh homeobox 1 (MSX1) has been identified to be associated with multiple human malignant neoplasms. However, the association of the expression and biological function of MSX1 with breast tumorigenesis, and the underlying mechanism remain largely unknown. Therefore, the present study examined the expression and promoter methylation of MSX1 in breast tumor cell lines, primary breast tumors and normal breast tissues using semi-quantitative, quantitative and methylation-specific reverse transcription­polymerase chain reaction. Colony formation assays, flow cytometric analysis, and wound healing and Transwell assays were used to assess various functions of MSX1. Western blot analyses were also conducted to explore the mechanism of MSX1. The results revealed that MSX1 was broadly expressed in normal human tissues, including breast tissues, but was frequently downregulated or silenced in breast cancer cell lines and primary tumors by promoter methylation. Methylation of the MSX1 promoter was observed in 7/9 (77.8%) breast cancer cell lines and 47/99 (47.5%) primary tumors, but not in normal breast tissues or surgical margin tissues, suggesting that tumor-specific methylation of MSX1 occurs in breast cancer. Pharmacological demethylation reduced MSX1 promoter methylation levels and restored the expression of MSX1. The ectopic expression of MSX1, induced by transfection with a lentiviral vector, significantly inhibited the clonogenicity, proliferation, migration and invasion of breast tumor cells by inducing G1/S cell cycle arrest and apoptosis. Ectopic MSX1 expression also inhibited the expression of active ß-catenin and its downstream targets c-Myc and cyclin D1, and also increased the cleavage of caspase-3 and poly (ADP-ribose) polymerase. In conclusion, MSX1 exerts tumor-suppressive functions by inducing G1/S cell cycle arrest and apoptosis in breast tumorigenesis. Its methylation may be used as an epigenetic biomarker for the early detection and diagnosis of breast cancer.

Dickkopf-related protein 2 induces G0/G1 arrest and apoptosis through suppressing Wnt/ß-catenin signaling and is frequently methylated in breast cancer.

Dickkopf-related protein 2 (DKK2) is one of the antagonists of Wnt/ß-catenin signaling, with its downregulation reported in multiple cancers. However, how DKK2 contributes to breast tumorigenesis remains unclear. We examined its expression and promoter methylation in 10 breast tumor cell lines, 98 primary tumors, and 21 normal breast tissues. Compared with normal tissues, DKK2 was frequently silenced in breast cell lines (7/8). DKK2 promoter methylation was detected in 77.8% of cell lines and 86.7% of breast tumors; while rarely detected in normal breast tissues (19%), indicating common DKK2 methylation in breast cancer. Ectopic expression of DKK2 changed breast tumor cell morphology, inhibited cell proliferation and colony formation by inducing G0/G1 cell cycle arrest and apoptosis, and suppressed tumor cell migration by reversing epithelial-mesenchymal transition (EMT) and downregulating stem cell markers. Moreover, restored expression of DKK2 in MCF7 cells disrupted the microtube formation of human umbilical vein endothelial cells on Matrigel®. In vivo, the growth of MDA-MB-231 cells in nude mice was markedly decreased after stable expression of DKK2. DKK2 suppressed canonical Wnt/ß-catenin signaling by inhibiting ß-catenin activity with decreased active ß-catenin protein. Thus, our findings demonstrate that DKK2 functions as a tumor suppressor through inhibiting cell proliferation and inducing apoptosis via regulating Wnt signaling during breast tumorigenesis.

A Way of Tailoring Magnetic Anisotropy of Co Nanowire Arrays: Magnetic Field Annealing.

Hexagonal close-packed Co nanowire arrays in anodic aluminum oxide template with the diameter of 50 nm have been fabricated using an ac electrodeposition method. The effect of magnetic field annealing on the thermal stability and magnetic properties of these nanwire arrays was studied. XRD measurements indicate the increase of diffraction intensity with the increase of heat-treatment temperature without magnetic field. Furthermore, the intensity of diffraction peak decreases rapidly if the sample undergoes the magnetic field annealing. Influence of different annealing process on the magnetic properties of Co nanowire arrays has also been studied. It is found that the magnetocrystalline anisotropy of hcp Co becomes weaker after magnetic field annealing, which lead to increase of the total anisotropy of Co nanowire arrays.

Electrostatic complexation of polyelectrolyte and magnetic nanoparticles: from wild clustering to controllable magnetic wires.

We present the electrostatic complexation between polyelectrolytes and charged nanoparticles. The nanoparticles in solution are γ-Fe2O3 (maghemite) spheres with 8.3 nm diameter and anionic surface charges. The complexation was monitored using three different formulation pathways such as direct mixing, dilution, and dialysis. In the first process, the hybrids were obtained by mixing stock solutions of polymers and nanoparticles. A 'destabilization state' with sharp and intense maximum aggregation was found at charges stoichiometry (isoelectric point). While on the two sides of the isoelectric point, 'long-lived stable clusters state' (arrested states) were observed. Dilution and dialysis processes were based on controlled desalting kinetics according to methods developed in molecular biology. Under an external magnetic field (B = 0.3 T), from dialysis at isoelectric point and at arrested states, cationic polyelectrolytes can 'paste' these magnetic nanoparticles (NPs) together to yield irregular aggregates (size of 100 µm) and regular rod-like aggregates, respectively. These straight magnetic wires were fabricated with diameters around 200 nm and lengths comprised between 1 µm and 0.5 mm. The wires can have either positive or negative charges on their surface. After analyzing their orientational behavior under an external rotating field, we also showed that the wires made from different polyelectrolytes have the same magnetic property. The recipe used a wide range of polyelectrolytes thereby enhancing the versatility and applied potentialities of the method. This simple and general approach presents significant perspective for the fabrication of hybrid functional materials.