scholarly journals MicroRNA-130a–mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors

Blood ◽  
2011 ◽  
Vol 118 (25) ◽  
pp. 6649-6659 ◽  
Author(s):  
Mattias Häger ◽  
Corinna Cavan Pedersen ◽  
Maria Torp Larsen ◽  
Mette Klarskov Andersen ◽  
Christoffer Hother ◽  
...  
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Mrna Level ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Smad4 Protein ◽  
Tgf Β1

Abstract Smad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3′-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1–induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia–derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1–mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.

2′,5′-Oligoadenylate-Antisense Chimeras Cause RNase L to Selectively Degrade bcr/abl mRNA in Chronic Myelogenous Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4336-4343 ◽  
Author(s):  
Avudaiappan Maran ◽  
Cornelius F. Waller ◽  
Jayashree M. Paranjape ◽  
Guiying Li ◽  
Wei Xiao ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Chronic Myelogenous Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Rnase L ◽  
Hematopoietic Stem

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2′,5′-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210bcr/abl kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of β-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.

2′,5′-Oligoadenylate-Antisense Chimeras Cause RNase L to Selectively Degrade bcr/abl mRNA in Chronic Myelogenous Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4336-4343 ◽  
Author(s):  
Avudaiappan Maran ◽  
Cornelius F. Waller ◽  
Jayashree M. Paranjape ◽  
Guiying Li ◽  
Wei Xiao ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Chronic Myelogenous Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Rnase L ◽  
Hematopoietic Stem

Abstract We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2′,5′-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210bcr/abl kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of β-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.

HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

2018 ◽  
Vol 18 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Maristella Bergamo dos Reis ◽  
Vanessa Silva Silveira ◽  
Regia Caroline Peixoto Lira ◽  
Carlos Gilberto Carlotti Jr ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Relevant Information ◽  
Protein Stabilization ◽  
Mrna Levels ◽  
Medulloblastoma Cell Line ◽  
Pediatric Medulloblastoma ◽  
Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

scholarly journals Regulation of β-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2

1996 ◽  
Vol 317 (3) ◽  
pp. 925-931 ◽  
Author(s):  
Volker DANGEL ◽  
Jeanette GIRAY ◽  
Dieter RATGE ◽  
Hermann WISSER
Keyword(s):  
Cell Line ◽  
Half Life ◽  
Rat Heart ◽  
Radioligand Binding ◽  
Mrna Level ◽  
Fold Increase ◽  
Heart Cell ◽  
H9c2 Cell ◽  
Heart Cells ◽  
Mrna Levels

The regulation of the expression of β-adrenoceptors (β-ARs) is not thoroughly understood. We demonstrate that the rat heart cell-line H9c2 expresses both β1- and β2-ARs. In radioligand-binding experiments, the maximal binding capacity of (-)-[125I]-iodocyanopindolol was determined as 18±0.6 fmol/mg of protein with a KD of 35.4±4.1 pM. Competitive radioligand-binding experiments with subtype-specific β-antagonists reveal a subtype ratio of β1- to β2-ARs of 29%:71%. With competitive reverse-transcriptase PCR we found β2-mRNA to be up to 1600 times more frequent than β1-mRNA. Treatment of the H9c2 cell-line with the β-adrenergic agonist (-)-isoproterenol (10-6 M), the antagonist (-)-propranolol (10-6 M) and the glucocorticoid dexamethasone (500 nM) induces regulatory effects on both the β-AR protein and mRNA level. Isoproterenol treatment leads to down-regulation of the total receptor number by 56±4%, due to a decrease in β2-ARs, while maintaining the β1-AR number constant. On the transcription level, both β1-and β2-mRNAs are decreased by 30% and 42% respectively. mRNA stability measurements reveal a reduced half-life of β2-mRNA from 9.3 h to 6.5 h after isoproterenol treatment. Incubation of cells with (-)-propranolol does not affect the amounts of β-ARs and their mRNAs. Dexamethasone induces a 1.8±0.2-fold increase in β-AR number over the basal level as well as a 1.9±0.2-fold increase in the amount of β2-mRNA. Because the half-life of β2-mRNA was unaffected by dexamethasone, the increased β2-mRNA level must be due to an enhanced transcription rate. The β1-mRNA levels are unchanged during dexamethasone-incubation of the cells. Our data clearly demonstrate that treatment of H9c2 rat heart cells with isoproterenol and dexamethasone induces alterations in the level of RNA stability as well as gene transcription, leading to altered receptor numbers.

scholarly journals Mediation of the inhibitory effect of thyroid hormone on proliferation of hepatoma cells by transforming growth factor-beta

2006 ◽  
Vol 36 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Chun-Che Yen ◽  
Ya-Hui Huang ◽  
Chu-Yu Liao ◽  
Cheng-Jung Liao ◽  
Wan-Li Cheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Thyroid Hormone ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Transforming Growth Factor ◽  
Cyclin E ◽  
Hepatoma Cell ◽  
Stable Cell Line ◽  
Mrna Levels

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)α1 or TRβ1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-β neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-β was studied to delineate the repression mechanism. TGF-β was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-β repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-β neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.

Assessment of mRNA Levels for Matrix Molecules and TGF-β1 in Rabbit Flexor and Peroneus Tendons Reveals Regional Differences in Steady-State Expression

2004 ◽  
Vol 29 (2) ◽  
pp. 165-169 ◽  
Author(s):  
M. BERGLUND ◽  
M. WIIG ◽  
M. TORSTENSSON ◽  
C. RENO ◽  
D. A. HART
Keyword(s):  
Regional Differences ◽  
Flexor Tendon ◽  
Mrna Level ◽  
Rna Extraction ◽  
Rabbit Model ◽  
Tendon Sheath ◽  
Mrna Levels ◽  
Collagen Iii ◽  
Polymerase Chain ◽  
Tgf Β1

This study analysed the differences on a molecular level between two segments of the deep flexor tendon, and compared the intrasynovial flexor tendon with the tendon sheath and the extrasynovial peroneus tendon in a rabbit model. The TRIspin method of RNA extraction was combined with the reverse transcription polymerase chain reaction to assess mRNA levels in the tissue segments. Significant differences were detected for all genes studied. mRNA levels for aggrecan, biglycan and collagen III were significantly higher in the fibrocartilaginous proximal segment of the flexor tendon. Collagen I was higher in the flexor tendon than the sheath and the peroneus tendon, and TGF-β1 was significantly lower in the peroneus tendon. This study demonstrates differences at the mRNA level between different segments of tendon, indicating that the tendon tissue may be adapted to its environment.

Effects of rmhTRAIL Combined with Daunorubicin in Inducing Apoptosis of Leukemia Cell Lines and Its Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mrna Level ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Expression Level ◽  
U937 Cells ◽  
Leukemia Cell Lines ◽  
Before And After

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.

Sign in / Sign up

Export Citation Format

Share Document