OBJECTIVETo investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937.

METHODSFour different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry.

RESULTSLentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells [(43.9±0.7)%] increased in the interference group (P<0.05). Wright's staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group [(27.1±1.4)%] was significantly higher than in the negative [(19.4±1.9)%] and untreated groups [(5.5±1.3)%] (P<0.05).

CONCLUSIONSLentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.

Apoptosis ; Cell Proliferation ; Gene Silencing ; Homeodomain Proteins ; antagonists & inhibitors ; genetics ; Humans ; Lentivirus ; genetics ; RNA Interference ; Sequence Analysis, DNA ; U937 Cells

Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.
Animals ; Animals, Genetically Modified ; metabolism ; Binding Sites ; Cell Differentiation ; Cell Proliferation ; DNA-Binding Proteins ; metabolism ; Drosophila ; metabolism ; Drosophila Proteins ; antagonists & inhibitors ; metabolism ; Enhancer Elements, Genetic ; Eye Proteins ; antagonists & inhibitors ; metabolism ; Homeodomain Proteins ; antagonists & inhibitors ; metabolism ; PAX6 Transcription Factor ; Paired Box Transcription Factors ; antagonists & inhibitors ; metabolism ; Protein Structure, Tertiary ; Repressor Proteins ; antagonists & inhibitors ; metabolism ; Retina ; cytology ; metabolism ; Wings, Animal ; growth & development

The aim of the present study was to investigate the regulatory effects of histone methylation modifications on the expression of miR-200c, as well as invasion and migration of gastric carcinoma cells. Gastric carcinoma cell line, MGC-803, were treated by 2.5 μmol/L histone methyltransferase inhibitor, DZNep. The expression of miR-200c was detected by real-time quantitative PCR (qRT-PCR). The epithelial-mesenchymal transition (EMT) indicators (ZEB1/2 and E/N-cadherin), EZH2, EED, SUZ12 and H3K27me3 expressions were detected by Western blot. Cell migration and invasion abilities were detected by Transwell and scratch tests. The result showed that, compared with DMSO (control) group, DZNep significantly increased the expression of miR-200c to about 2.1 times, inhibited ZEB1, ZEB2, and N-cadherin expressions, and activated E-cadherin expression; Also, DZNep decreased the protein expressions of EZH2, EED, SUZ12 and H3K27me3; Moreover, DZNep could inhibit MGC-803 cell invasive and migrative abilities, as well as MMP9 expression. These results suggest DZNep raises miR-200c expression to delay the invasion and migration of gastric carcinoma cells, and the underlying mechanisms involve the regulations of EMT-related proteins and polycomb repressive complex 2.
Adenosine ; analogs & derivatives ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Homeodomain Proteins ; metabolism ; Humans ; MicroRNAs ; metabolism ; Protein Methyltransferases ; antagonists & inhibitors ; Repressor Proteins ; metabolism ; Transcription Factors ; metabolism ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

AIMTo investigate the effect of DAPT (gamma-secretase inhibitor) on the growth of human tongue carcinoma cells and to determine the molecular mechanism to enable the potential application of DAPT to the treatment of tongue carcinoma.

METHODOLOGYHuman tongue carcinoma Tca8113 cells were cultured with DAPT. Cell growth was determined using Indigotic Reduction method. The cell cycle and apoptosis were analyzed by flow cytometry. Real-time PCR and Immuno-Fluorescence (IF) were employed to determine the intracellular expression levels.

RESULTSDAPT inhibited the growth of human tongue carcinoma Tca8113 cells by inducing G0-G1 cell cycle arrest and apoptosis. The mRNA levels of Hairy/Enhancer of Split-1 (Hes-1), a target of Notch activation, were reduced by DAPT in a dose-dependent manner. Coincident with this observation, DAPT induced a dose-dependent promotion of constitutive Caspase-3 in Tca8113 cells.

CONCLUSIONDAPT may have a therapeutic value for human tongue carcinoma. Moreover, the effects of DAPT in tumor inhibition may arise partly via the modulation of Notch-1 and Caspase-3.

Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Basic Helix-Loop-Helix Transcription Factors ; drug effects ; Carcinoma ; pathology ; Caspase 3 ; drug effects ; Cell Line, Tumor ; Cell Membrane ; drug effects ; Cell Nucleus ; drug effects ; Cyclin D1 ; drug effects ; Dipeptides ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; G1 Phase ; drug effects ; Homeodomain Proteins ; drug effects ; Humans ; Receptor, Notch1 ; drug effects ; Repressor Proteins ; drug effects ; Resting Phase, Cell Cycle ; drug effects ; Tongue Neoplasms ; pathology ; Transcription Factor HES-1

TNF-alpha, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-alpha signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-alpha-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-alpha down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-alpha induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-alpha. TNF-alpha activated the NF-kappaB and the JNK pathways. Inhibition of NF-kappaB or JNK activation reduced the inhibitory effect of TNF-alpha on ALP expression, whereas TNF-alpha-induced Msx2 expression was only suppressed by the inhibition of the NF-kappaB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-alpha on BMP2-regulated osteoblast differentiation and that the TNF-alpha-activated NF-kappaB pathway is responsible for Msx2 induction.
Alkaline Phosphatase/genetics/metabolism ; Animals ; Animals, Newborn ; Bone Morphogenetic Protein 2/pharmacology ; Cell Culture Techniques ; Cell Differentiation/*drug effects/genetics ; Cell Proliferation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/genetics ; Down-Regulation/drug effects ; Gene Expression Regulation/drug effects ; Homeodomain Proteins/antagonists & inhibitors/genetics/*physiology ; Mice ; Mice, Inbred ICR ; Mice, Transgenic ; Osteoblasts/*drug effects/metabolism/physiology ; RNA, Small Interfering/pharmacology ; Tumor Necrosis Factor-alpha/*pharmacology