scholarly journals PiRNA MW557525 Promotes the Vitality and Pluripotency of Piwil2-iCSCs by Regulating NOP56.

2021 ◽  
Author(s):  
Zhaoxia Zhang ◽  
Zhang Wang ◽  
Xiaojun Tan ◽  
Liming Jin ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Target Gene ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Tumor Development ◽  
High Mobility ◽  
P Element ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Go Analysis ◽  
Protein Levels

Abstract Objective Cancer stem cells (CSCs) play an important role in tumor development. Some studies have demonstrated that P-element–induced wimpy testis (Piwi)–interacting ribonucleic acids (piRNAs) participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. The aim of the present study was to investigate the effect of the uknown upregulated piRNA MW557525 and its predicted target gene nucleolar protein 56 (NOP56) inPiwi-like protein 2 (Piwil2)–induced CSCs (Piwil2-iCSCs).Methods We screened differential piRNAs of Piwil2-iCSCs using high-throughput sequencing (HTS). Target genes were predicted by the miRanda algorithm and subjected to Gene Ontology (GO) analysis. One of the differential piRNAs, MW557525, and its target gene NOP56 were transfected and silenced in Piwil2-iCSCs, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect expression levels of piRNA MW557525 and NOP56 in Piwil2-iCSCs after transfection. We measured protein levels of NOP56 in different groups via Western blot (WB), verified interactions using a dual luciferase reporter assay (LRA) and investigated the effect of piRNA MW557525 and NOP56 on Piwil2-iCSC proliferation using a Cell Counting Kit-8 (CCK-8). In addition, we evaluated cell migratory and invasive abilities via transwell assay and detected cell apoptotic ability via flow cytometry (FCM) assay. Protein levels of Cluster of Differentiation 24 (CD24), CD133, Krüppel-like factor 4 (KLF4) and sex-determining region Y–related high-mobility group (HMG) box 12 (SOX2) were measured to evaluate the change in Piwil2-iCSC pluripotency after transfection.Results Via HTS, we screened out 204 differential piRNAs, and miRanda predicted 77 target genes. GO analysis showed that the biological processes (BPs) of these target genes were mainly involved in regulating the calcium concentration of cells and their molecular functions (MFs) were mainly involved in ATPase activity.The expression of piRNA MW557525 and NOP56 were significantly upregulated,and piRNA MW557525 was negatively associated with NOP56 in Piwil2-iCSCs. PiRNA MW557525 promoted proliferation, migration, invasion and pluripotency and inhibited apoptosis, while NOP56 suppressed proliferation, migration, invasion and pluripotency and induced apoptosis, in Piwil2-iCSCs.Conclusion Taken together, these findings suggested that piRNA MW557525 promoted and maintained the vitality and pluripotency of Piwil2-iCSCs, while NOP56 inhibited these characteristics. Therefore, piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

scholarly journals PiRNA MW557525 Promotes the Vitality and Pluripotency of Piwil2-iCSCs by Regulating NOP56.

2022 ◽  
Author(s):  
Liming Jin ◽  
Zhaoxia Zhang ◽  
Zhang Wang ◽  
Xiaojun Tan ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Target Gene ◽  
Opposite Effect ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Tumor Development ◽  
Luciferase Reporter ◽  
Transwell Assay ◽  
Kegg Analysis ◽  
Detailed Function ◽  
Dual Luciferase Reporter Assay

Abstract Background: CSCs play an important role in tumor development. Some studies have demonstrated that piRNAs participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. This study aimed to investigate the significance of some piRNAs in Piwil2-iCSCs. Methods and Results: Differentially expressed piRNAs in Piwil2-iCSCs were screened by high-throughput sequencing. Target genes were predicted by the miRanda algorithm and subjected to GO and KEGG analysis. One of the differential piRNAs, novel piRNA MW557525, was transfected and its target gene NOP56 was silenced in Piwil2-iCSCs, respectively. RT-qPCR, western blot and dual luciferase reporter assay were used to investigate the interaction of piRNA MW557525 and NOP56. We identified the effect of piRNA MW557525 and NOP56 knockdown on cell proliferation, migration, invasion, and apoptosis via CCK-8, transwell assay, and flow cytometry. The expressions of CD24, CD133, KLF4, and SOX2 were detected via WB. The results showed that piRNA MW557525 was negatively correlated with NOP56, and it promoted the proliferation, migration, invasion, and inhibited apoptosis in Piwil2-iCSCs, and it also promoted the expressions of CD24, CD133, KLF4, and SOX2, while NOP56 showed the opposite effect. Conclusions: These findings suggested that novel piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

scholarly journals Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2

2021 ◽  
Vol 41 (3) ◽  
Author(s):  
Fuyuan Xie ◽  
Longgen Li ◽  
Yuting Luo ◽  
Rensheng Chen ◽  
Jinhong Mei
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cell ◽  
Target Gene ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Thyroid Cancer Cell ◽  
Gene Assay

Abstract Objective: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis. Methods: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2′-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer. Results: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer. Conclusion: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.

scholarly journals circITGA7 Acts as a miR-370-3p Sponge to Suppress the Proliferation of Prostate Cancer

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Gang Luo ◽  
Guohao Li ◽  
Zhihua Wan ◽  
Yuanjie Zhang ◽  
Dong Liu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Profiles ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Loss Of Function

Prostate cancer (PCa) refers to one of the most common tumors in male’s genitourinary system. Emerging research has confirmed that circRNAs play an important role in the occurrence and development of tumors. However, the correlation between circular RNA circITGA7 and PCa still remains unclear. Here, the role of circITGA7 in PCa was explored and the underlying mechanism was investigated as well. The circRNA expression profiles in PCa and the paracancerous tissues were established by high-throughput sequencing. The expression levels of circITGA7 in PCa tissues and cells were detected by qRT-PCR. Cell Counting Kit-8, colony formation, EdU, and flow cytometry assays were used to detect the effects of circITGA7 on PCa cell proliferation. To further explore the underlying mechanisms, bioinformatics analysis on downstream target genes was carried out. RNA immunoprecipitation and dual-luciferase reporter assays were used to verify the direct relationship between miR-370-3p and circITGA7 or P21CIP1. The present results demonstrated that circITGA7 was downregulated in PCa tissues and cells. Gain- or loss-of-function assays showed that circITGA7 inhibited the proliferation of PCa cells in vivo and in vitro. Mechanically, circITGA7 served as a sponge for miR-370-3p, and miR-370-3p could target P21CIP1 in PCa cells. The inhibition of cell proliferation induced by circITGA7 could be reversed by transfecting miR-370-3p mimic. Collectively, our data indicated that circITGA7 played an important role in inhibiting tumor proliferation in PCa and might be a potential therapeutic target.

scholarly journals circITGA7 Acts As miR-370-3p Sponge to Suppress The Proliferation of Prostate Cancer

2021 ◽  
Author(s):  
Yonglian Guo ◽  
Guohao Li ◽  
Zhihua Wan ◽  
Yuanjie Zhang ◽  
Dong Liu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Profiles ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Loss Of Function

Abstract Objective: Prostate cancer (PCa) refers to one of the most common tumors in male’s genitourinary system. Emerging research has confirmed that circRNAs play an important role in the occurrence and development of tumors. However, the correlation between circular RNA circITGA7 and PCa still remains unclear. Here, the role of circITGA7 in PCa was explored and the underlying mechanism was investigated as well.Methods: The circRNA expression profiles in PCa and the paracancerous tissues were established by high-throughput sequencing. The expression levels of circITGA7 in PCa tissues and cells were detected by qRT-PCR. Cell Counting Kit-8, colony formation, EdU and flow cytometry assays were used to detect the effects of circITGA7 on PCa cell proliferation. To further explore the underlying mechanisms, bioinformatics analysis on downstream target genes was carried out. RNA immunoprecipitation and dual-luciferase reporter assays were used to verify the direct relationship between miR-370-3p and circITGA7 or P21CIP1.Results: circITGA7 was downregulated in PCa tissues and cells. Gain- or loss-of-function assays showed that circITGA7 inhibited the proliferation of PCa cells in vivo and in vitro. Mechanically, circITGA7 served as a sponge for miR-370-3p and miR-370-3p could target P21CIP1 in PCa cells. The inhibition of cell proliferation induced by circITGA7 could be reversed by transfecting miR-370-3p mimic. Conclusion: Our data indicated that circITGA7 played an important role in inhibiting tumor proliferation in PCa and might be a potential therapeutic target.

scholarly journals MicroRNA-29a Inhibits Growth, Migration and Invasion of Melanoma A375 Cells in Vitro by Directly Targeting BMI1

2018 ◽  
Vol 50 (1) ◽  
pp. 385-397 ◽  
Author(s):  
Ying Xiong ◽  
Liqian Liu ◽  
Ying Qiu ◽  
Lili  Liu
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Target Gene ◽  
Target Genes ◽  
Malignant Tumors ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Bmi1 Expression ◽  
A375 Cells

Background/Aims: Melanoma is one of the most aggressive malignant tumors, with increasing incidence, poor prognosis, and lack of any effective targeted therapies. Abnormal expression of miR-29a has been found in several types of cancers, including melanoma. In this study, experiments were performed to investigate the role of miR-29a in melanoma, and the molecular mechanism by which miR-29a represses melanoma. Methods: miR-29 and Bmi1 expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability, apoptosis, migration and invasion were respectively determined by Cell Counting Kit-8 assay, Propidium iodide (PI) fluorescein isothiocynate (FITC)-Annexin V staining assay, wound healing assay and transwell assay. Luciferase reporter assay was performed to determine a target gene of miR-29a. Western blot was used to analyze protein expression of apoptosis-related proteins, Bmi1, Wnt/β-catenin and Nuclear factor-κB (NF-κB) pathway target genes. Results: miR-29a was down-regulated in all tested melanoma cell lines. Up-regulation of miR-29a effectively inhibited cell viability, migration, and invasion, but promoted apoptosis in A375 cells. Bmi1 was a direct target gene of miR-29a. Transfection with miR-29a mimic decreased cell migration and invasion and Bmi1 expression in Malme-3M cells, SK-MEL-2, SK-MEL-5, and M14 cell lines. Moreover, miR-29a might suppress growth, migration and invasion of A375 cells by negatively regulating Bmi1. In addition, our results demonstrated that transfection with miR-29a mimic effectively blocked Wnt/β-catenin and NF-κB pathways via down-regulating Bmi1. Conclusion: miR-29a could be functioned as a potential tumor suppressor through direct regulation of Bmi1 in melanoma cells.

scholarly journals Circ_0116061 regulated the proliferation, apoptosis, and inflammation of osteoarthritis chondrocytes through regulating the miR-200b-3p/SMURF2 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei Zheng ◽  
Guanhua Hou ◽  
Yong Li
Keyword(s):  
Circular Rna ◽  
Cell Counting ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Biological Functions ◽  
Protein Levels ◽  
Osteoarthritis Chondrocytes ◽  
Oa Chondrocytes ◽  
Labeled Rna ◽  
Apoptosis And Inflammation

Abstract Background Circular RNA (circRNA) has been shown to be associated with osteoarthritis (OA) progression. Circ_0116061 has been found to be highly expressed in OA cartilage tissues, but its role and mechanism in OA progression remain unclear. Methods Expression levels of circ_0116061, microRNA (miR)-200b-5p, and Smad ubiquitin regulatory factor 2 (SMURF2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay, colony formation assay, and flow cytometry. Furthermore, the protein levels of proliferation-related marker, apoptosis-related markers, inflammatory factors, and SMURF2 were tested using western blot (WB) analysis. In addition, the interaction between miR-200b-3p and circ_0116061 or SMURF2 was examined using dual-luciferase reporter assay and biotin-labeled RNA pull-down assay. Results Circ_0116061 and SMURF2 were highly expressed, and miR-200b-3p was lowly expressed in OA cartilage tissues. Knockdown of circ_0116061 could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. MiR-200b-3p could be sponged by circ_0116061, and its inhibitor could reverse the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes. SMURF2 was a target of miR-200b-3p, and its expression was positively regulated by circ_0116061. Silencing of SMURF2 also could enhance the proliferation and suppress the apoptosis and inflammation of OA chondrocytes. Furthermore, the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes also could be reversed by SMURF2 overexpression. Conclusion Our data showed that circ_0116061 might regulate the miR-200b-3p/SMURF2 axis to promote the progression of OA.

scholarly journals HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

2015 ◽  
Vol 35 (8) ◽  
pp. 1390-1400 ◽  
Author(s):  
Nancy Yu ◽  
Michael Kakunda ◽  
Victoria Pham ◽  
Jennie R. Lill ◽  
Pan Du ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Target Gene ◽  
Target Genes ◽  
Glycogen Synthase ◽  
Protein Phosphatase 2A ◽  
Breast Cancer Patients ◽  
Poor Overall Survival ◽  
Protein Levels ◽  
Phosphatase 2A ◽  
Unidentified Component

The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study.

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

2019 ◽  
Vol 86 (4) ◽  
pp. 425-431 ◽  
Author(s):  
Zhi Chen ◽  
Jingpeng Zhou ◽  
Xiaolong Wang ◽  
Yang Zhang ◽  
Xubin Lu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Differential Expression ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Interleukin 1 ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Expression Of Genes ◽  
Chinese Holstein Cows ◽  
Reporter Assays

AbstractWe established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA–mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

scholarly journals The Expression of microRNA in Adult Rat Heart with Isoproterenol-Induced Cardiac Hypertrophy

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1173 ◽  
Author(s):  
Mailin Gan ◽  
Shunhua Zhang ◽  
Yuan Fan ◽  
Ya Tan ◽  
Zhixian Guo ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Signaling Pathway ◽  
Natriuretic Peptide ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Pathological Condition ◽  
Wnt Signaling Pathway ◽  
Differentially Expressed ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.

Overexpression of GATA1 Confers Chemotherapy Resistance in Pediatric Acute Megakaryocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2039-2039
Author(s):  
Holly Edwards ◽  
Chengzhi Xie ◽  
Alan Dombkowski ◽  
Maggie Keller ◽  
Mark Stout ◽  
...  
Keyword(s):  
Cell Line ◽  
Histone Deacetylase Inhibitors ◽  
Target Genes ◽  
Negative Control ◽  
Pi3 Kinase ◽  
Luciferase Reporter ◽  
Toxic Dose ◽  
Protein Levels ◽  
Acute Megakaryocytic Leukemia ◽  
Shrna Knockdown

Abstract Abstract 2039 Poster Board II-16 Acute megakaryocytic leukemia (AMkL; M7) is a biologically heterogeneous form of AML, representing ∼10% of pediatric and 1-2% of adult AML cases. AMkL is the most common AML subtype of children with Down syndrome (DS). DS children with AMkL have an excellent prognosis with EFS rates of 80-100% when treated with ara-C/anthracycline-based protocols, in contrast to the <30% EFS rates of non-DS children with AMkL. This also contrasts to the ∼50% EFS rates of non-DS children with AML overall, indicating that AMkL is an extremely poor risk group amongst non-DS children with AML despite the use of intensive chemotherapy-based protocols. These clinical data make a compelling argument that new therapies are essential to improve the treatment outcome of this aggressive disease. Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected uniformly in nearly all DS AMkL cases, but not in non-DS AML and non-AMkL DS leukemia cases. The net effect of GATA1 mutations is an introduction of early stop codons and synthesis of a shorter GATA1 protein (designated GATA1s) that has altered transactivation activity, potentially contributing to the uncontrolled proliferation of immature megakaryocytes. It is conceivable that the altered GATA1 function between DS and non-DS AMkL may account for differential expression of GATA1 target genes in these two groups of patients. On the other hand, overexpression of GATA1 in megakaryoblasts from non-DS children with AMkL compared to myeloblasts from non-DS children with other subtypes of AML may contribute to differences in chemotherapy sensitivity via regulation of GATA1 target genes. We previously reported that GATA1 mutations in DS AMkL are associated with decreased expression of cytidine deaminase (encodes an enzyme which can convert ara-C to ara-U, the inactive form of the drug), thus contributing to the enhanced ara-C sensitivity of DS AMkL blasts. Further, when GATA1 was ectopically expressed in a DS AMkL cell line, CMK, it caused significantly increased resistance to ara-C. In the present study, we confirmed overexpression of GATA1 in non-DS AMkL blasts compared to non-DS AML blasts by real-time RT-PCR quantitation of GATA1 transcripts in our cohort of patient samples. shRNA knockdown of GATA1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivities to ara-C and daunorubicin, the two main drugs used for AML treatment, and significantly increased basal level apoptosis. This was accompanied by significantly decreased Bcl-xL transcript and protein levels in the GATA1 shRNA knockdown clones compared to a shRNA negative control. Binding of GATA1 to the two GATA elements in Bcl-x promoter and transactivation of Bcl-x promoter activity by GATA1 was demonstrated by ChIP assays and luciferase reporter assays, respectively, in Meg-01 cells. In our cohort of non-DS AMkL and AML patient samples, significant overexpression of Bcl-xL in non-DS AMkL compared to non-DS AML cases and a significant correlation between Bcl-xL and GATA1 transcripts were detected. Besides Bcl-xL, additional GATA1 targets (e.g. TNF) related to apoptosis were also identified by gene expression and ChIP-on-ChIP microarray analyses. Interestingly, our microarray data also suggest that GATA1 may have an impact on PI3-kinase/Akt pathway through modulating directly or indirectly a group of genes within the pathway. Western blotting revealed increased phosphorylation of Akt in the GATA1 knockdown clones compared to the negative control cells. Previous studies reported that histone deacetylase inhibitors (HDACIs) treatment causes hyperacetylation and subsequent degradation of GATA1, suggesting that these agents may be effective in targeting GATA1 in AMkL. Treatment of Meg-01 cells with an HDACI, valproic acid (VPA), resulted in decreased protein levels for GATA1 and Bcl-xL and increased phosphorylation of Akt. Co-treatment of Meg-01 cells with VPA and ara-C resulted in synergistic induction of apoptosis and activation of caspase-3. This drug synergy was amplified when a non-toxic dose of the PI3-kinase inhibitor LY294002 was added. Our results demonstrate that GATA1 causes resistance to chemotherapy in non-DS AMkL by promoting AMkL blast survival through regulating its target genes. Treatment of AMkL may be improved by integrating HDACI and PI3-kinase or Akt inhibitors into the chemotherapy of this disease. Disclosures: No relevant conflicts of interest to declare.

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