PRDM1 expression and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type / 中华血液学杂志

Objective@#To investigate the expression of PRDM1 and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type.@*Methods@#Immunocytochemistry and Western blot were used to detect the expression of PRDM1 and p-AKT in 10 EN-NK/T-NT tissue or 3 cell lines (PRDM1-positive YT cell line, PRDM1-negative NKL and NK92 cell lines). Nanostring gene expression profiling technique was used to detect the activation of the PI3K/AKT pathway in normal nasal mucosa, PRDM1-negative and positive EN-NK/T-NT tissue. MTS was used to detect cell proliferation, and flow cytometry was used to detect cell cycle and apoptosis.@*Results@#Nanostring gene expression profiling revealed that genes associated with PI3K/AKT signaling pathway (eg, IL-7, BRCA1, ITGA8, IL2RB, FASLG, CDK2, COL27A1, CSF3R, KITLG and IL-6) were highly expressed in EN-NK/T-NT cases (P<0.05). Also, we found that p-AKT was highly expressed in YT cell line, but lower or not expressed in NK92 and NKL cells. In addition, LY294002, a PI3K/AKT pathway inhibitor, increased PRDM1 and PTEN expression in a dose dependent manner in YT cells. More importantly, YT cell were treated with 20 μmol/L LY294002 48 h, the proliferation rate was significantly decreasing (58.18% vs 100.00%, t=12.770, P=0.006), and the proportion of cells in G1 phase was significantly increased (30.05% vs 76.93%, t=11.570, P<0.001). However, there was no significant difference in cell proliferation and cell cycle between NKL cells and control group (P>0.05).@*Conclusion@#The activation of PI3K/AKT pathway is positive associated with the expression of PRDM1 in EN-NK/T-NT, and inhibition of PI3K/AKT pathway may have significant therapeutic potential for PRDM1-positive EN-NK/T-NT.

PRDM1 expression and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type / 中华血液学杂志

Objective: To investigate the expression of PRDM1 and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type. Methods: Immunocytochemistry and Western blot were used to detect the expression of PRDM1 and p-AKT in 10 EN-NK/T-NT tissue or 3 cell lines (PRDM1-positive YT cell line, PRDM1-negative NKL and NK92 cell lines). Nanostring gene expression profiling technique was used to detect the activation of the PI3K/AKT pathway in normal nasal mucosa, PRDM1-negative and positive EN-NK/T-NT tissue. MTS was used to detect cell proliferation, and flow cytometry was used to detect cell cycle and apoptosis. Results: Nanostring gene expression profiling revealed that genes associated with PI3K/AKT signaling pathway (eg, IL-7, BRCA1, ITGA8, IL2RB, FASLG, CDK2, COL27A1, CSF3R, KITLG and IL-6) were highly expressed in EN-NK/T-NT cases (P<0.05). Also, we found that p-AKT was highly expressed in YT cell line, but lower or not expressed in NK92 and NKL cells. In addition, LY294002, a PI3K/AKT pathway inhibitor, increased PRDM1 and PTEN expression in a dose dependent manner in YT cells. More importantly, YT cell were treated with 20 μmol/L LY294002 48 h, the proliferation rate was significantly decreasing (58.18% vs 100.00%, t=12.770, P=0.006), and the proportion of cells in G(1) phase was significantly increased (30.05% vs 76.93%, t=11.570, P<0.001). However, there was no significant difference in cell proliferation and cell cycle between NKL cells and control group (P>0.05). Conclusion: The activation of PI3K/AKT pathway is positive associated with the expression of PRDM1 in EN-NK/T-NT, and inhibition of PI3K/AKT pathway may have significant therapeutic potential for PRDM1-positive EN-NK/T-NT.

Establishment of drug screening model targeting PRDM1 gene promoter / 重庆医学

Objective To construct a double luciferase reporter gene vector with PRDM1 gene promoter as target,and establish drug screening cell model in vitro,hope to find small molecule compounds in Chinese herbal medicine library by this model.Methods Total DNA was extracted from 293T cells and it was used for amplifying the fragment contained PRDM1 gene promoter (267 bp/1 257 bp) by PCR.The amplified product was inserted into pGL3-Basic vector.The PCR product and pGL3-PRDM1 vector were verified by sequencing and alignment.The pGL3-PRDM1 and pRL-TK vector were co-transfected into engineer cells.The activity of PRDM1 gene promoter could be assayed by measuring luciferase.The method was optimized by changing ratio of two vectors.Results The highest transfection efficiency and luciferase activity were found in ratio of n(pGL-PRDM1) ∶ n(pRL-TK)=2 ∶1,and with the recombinant luciferase report gene vector contained the length of 1 257 bp amplified fragment transfecting into U266 cells.Moreover,the inductor (5-Aza-CdR) of PRDM1 gene was used for authenticating the method(P＜0.01),the fluoresscence in tensity and promoter activity of PRDM1 gene were enhanced in a dose dependent manner with 5-Aza-CdR.The activity of the promoter of PRDM1 gene was significantly decreased from the concentration of 5 μmol/L of Artemisinid(P＜0.05).The total glucosides of paeoniflorin promoted the promoter activity of PRDM1 gene at a low concentration,and inhibited the promoter activity of PRDM1 gene at a high concentration.Artesunate has no effect on cell proliferation.The effect of total glucosides of paeony on cell proliferation was more complicated.Conclusion A drug selection model targeting PRDM1 gene promoter has been successfully established,and artesunate has been screened to inhibit the promoter activity of PRDM1 gene.

Generation of a Genetically Engineered Aggressive Nk-Cell Leukemia Cell Line with Stable IL2 Expression.

Introduction: Aggressive NK-cell leukemia (ANKL) is a highly aggressive disease with extremely poor prognosis. A few malignant NK cell lines reflecting ANKL biology have been generated; however, these NK cell lines require the inclusion of exogeneous IL2 in the culture medium continuously, which increases the cost of cell culture significantly. Methods: IL2 coding sequenced was cloned into MSCV-IRES-YFP (PMIY) vector by directional PCR-cloning. IL2 was ectopically expressed in KHYG1 cell line through retroviral transduction. The transduced cells were cultured in limiting IL2 concentrations during which they were quantified with flow cytometry in regular time intervals to track the transduced population. IL2 transduced KHYG1 cells were then sorted to generate IL2-KHYG1 cells. PRDM1 was ectopically expressed in IL2-KHYG1 cells through retroviral transduction. Trypan blue count was performed to test proliferation of IL2-KYHG1 cells in the absence of IL2. Results: IL2-KHYG1 cells were enriched (from ~7% to ~16% YFP+ cells) during cell culture in limiting (6.25IU) IL2 concentrations. Complete removal of IL2 further enriched the transduced portion to 27% YFP-positivity, which did not increase with additional culturing. IL2 expressing KHYG1 cells were further enriched by sorting transduced IL2-KHYG1 cells with high level IL2 expression. IL2-KHYG1 cells survived and continued to grow without IL2. IL2-KHYG1 cells were transduced successfully using a PRDM1 construct having GFP as a marker under cell culture conditions having no IL2. Conclusion: IL2-KHYG1 cell line may decrease the cost associated with culturing ANKL cell lines, and it may facilitate in vitro investigation of the molecular basis of this malignancy.

Expression of miR-9 in H/RS cells and its regulation on target PRDM1 / 白血病·淋巴瘤

Objective To explore the expression of miR-9 in H/RS cells and its regulation on target PRDM1.Methods miR-9 expression in normal CD19+ B-cell subsets and eight lymphoma cell lines was detected by fluorescence quantitative RT-PCR and in situ hybridization (ISH),for quantification and location,respectively.Chemically synthesizcd antisense oligonucleotide of miR-9 was transiently transfected into L428 for its silence,and the PRDM1 expression was tested.Results Fluorescence quantitative RT-PCR showed that the expression of miR-9 in L428 cells was marked higher than that of normal CD19+ B-cell subsets and other lymphoma cell lines (the expression of miR-9 in L428 cells was 47-fold of OCI-Ly1,50-fold of Raji cells,7-fold of EBV+ immortalized B cell line,and 6-fold of ALCL cell line).ISH indicated that miR-9 located in cytoplasm,it was a diffuse and strong positive in L428,scattered and weak in DLBCL and Burkitt' s lymphoma cell lines,while negative in KARPAS-299 or Jurkat cell lines.Transient down-regulation of miR-9 in L428 leded to the increase of PRDMI protein.Conclusion miR-9 plays the role of cancer gene in cHL,and may exert a potential function in regulating terminal B cell differentiation through a post transcription regulation of PRDM1 gene.