scholarly journals MiR-28-5p mediates the anti-proliferative and pro-apoptotic effects of curcumin on human diffuse large B-cell lymphoma cells

2020 ◽  
Vol 48 (7) ◽  
pp. 030006052094379
Author(s):  
Tian Kang ◽  
Wei-Li Sun ◽  
Xiao-Fei Lu ◽  
Xin-Liang Wang ◽  
Lian Jiang
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Large B Cell Lymphoma ◽  
Apoptotic Effects ◽  
Large B Cell ◽  
Dual Luciferase Reporter Assay

Objective To investigate the anti-proliferative and pro-apoptotic effects of curcumin on diffuse large B-cell lymphoma (DLBCL) cells and explore the mechanism. Methods OCI-LY7 cells were treated with curcumin (2.5, 5, 10, 20, and 40 μM) for 24, 48, or 72 hours. Cell viability and apoptosis were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide assay and TdT-mediated dUTP nick-end labeling staining, respectively. MiR-28-5p expression was detected via qRT-PCR. The binding site of miR-28-5p was predicted using online databases and verified using the dual-luciferase reporter assay. MiR-28-5p overexpression and inhibition were achieved via transfection with an miR-28-5p mimic and inhibitor, respectively. Results Curcumin decreased the viability of OCI-LY7 cells in a concentration- and time-dependent manner, and these effects were attenuated by miR-28-5p inhibition. MiR-28-5p expression was upregulated by curcumin. Curcumin increased the numbers of apoptotic cells and upregulated cleaved caspase-3 expression, and these effects were attenuated by miR-28-5p inhibition. The dual-luciferase reporter assay confirmed that miR-28-5p directly targets the 3′-untranslated region of BECN1. Curcumin downregulated BECN1 and microtubule-associated protein 1 light chain 3 beta-II/I expression and upregulated p62 expression. Conclusions Our results described the curcumin exerted anti-proliferative and pro-apoptotic effects on OCI-LY7 cells through a mechanism potentially involving miR-28-5p.

scholarly journals hsa-MicroRNA-28-5p Inhibits Diffuse Large B-Cell Lymphoma Cell Proliferation by Downregulating 14-3-3ζ Expression

2022 ◽  
Vol 2022 ◽  
pp. 1-13
Author(s):  
Shufang Yan ◽  
Yang Chen ◽  
Meihong Yang ◽  
Qian Zhang ◽  
Jiajia Ma ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Dual Luciferase Reporter Assay

MicroRNAs (miRNAs) participate in the comprehensive biological process of several cancer types. In our former study, we found that hsa-microRNA- (miR-)28-5p was downregulated, but tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta (14-3-3ζ or YWHAZ) was upregulated in diffuse large B-cell lymphoma (DLBCL) tissues. We predicted that YWHAZ was a target gene for hsa-miR- 28-5p using bioinformatics analysis. Our goal was to reveal the role of hsa-miR-28-5p in DLBCL. YWHAZ was tested by immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) tissues of 137 DLBCL tissues, and the expression of hsa-miR-28-5p and YWHAZ was examined by quantitative real-time polymerase chain reaction (qRT-PCR) in 15 fresh and frozen DLBCL tissues. To study the functional roles of the downregulated hsa-miR-28-5p in DLBCL, a Cell Counting Kit-8 assay was conducted to estimate cell proliferation. Transient transfection of miRNA mimics was performed to overexpress hsa-miR-28-5p, and flow cytometry was performed to examine cell apoptosis and cell cycle progression. A dual-luciferase reporter assay was employed to explore the relationship between hsa-miR-28-5p and YWHAZ. Western blotting and qRT-PCR were used to investigate the function of hsa-miR-28-5p in YWHAZ expression. hsa-miR-28-5p was found to be significantly downregulated in DLBCL tissues and cell lines. Functional studies showed that hsa-miR-28-5p overexpression inhibited cell viability and proliferation, and YWHAZ was predicted to be a target of hsa-miR-28-5p. Dual-luciferase reporter assay, Western blotting, and qRT-PCR verified that hsa-miR-28-5p negatively regulated YWHAZ expression by directly targeting its 3′ untranslated regions in DLBCL cells. hsa-miR-28-5p may suppress the growth of DLBCL cells by inhibiting YWHAZ expression. These findings could provide novel targets for DLBCL diagnosis and therapy.

Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2963-2963
Author(s):  
Goldi A Kozloski ◽  
Xiaoyu Jiang ◽  
Shruti Bhatt ◽  
Rita Shaknovich ◽  
Ari M Melnick ◽  
...  
Keyword(s):  
B Cell ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Large B Cell Lymphoma ◽  
Animal Survival ◽  
Dlbcl Subtype ◽  
Large B Cell

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis. Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. Figure 1 Figure 1. Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals MiRNA-340-5p Mediates the Functional and Infiltrative Promotion of Tumor-Infiltrating CD8+ T Lymphocytes in Human Diffuse Large B Cell Lymphoma

2020 ◽  
Author(s):  
Yangyang Xu ◽  
Zhenchuan Liu ◽  
Lixin Lv ◽  
Ping Li ◽  
Aibin Liang
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: CD8+ tumor-infiltrating T lymphocytes (T-TILs) in the tumor microenvironment (TME) play an important role in tumor development, and miRNAs regulate tumor cell interactions with the microenvironment. T-TIL-based tumor immunotherapy provides a promising treatment strategy in diffuse large B-cell lymphoma (DLBCL). MiRNAs tend to be attractive targets for novel antitumor interventions.Methods: Weighted gene coexpression network analysis (WGCNA), CIBERSORT analysis and Cox regression analysis were used to identify CD8+ T-TIL-related miRNAs. RT-PCR, western blotting, immunohistochemistry (IHC), luciferase reporter assay, coimmunoprecipitation and ubiquitination analyses were used to detect miRNA, mRNA and protein expression and their combination. The viability and function of CD8+ T cells after stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), cytotoxicity assay, functional avidity assessment, flow cytometry and Cell Counting Kit-8 (CCK-8) assay. DLBCL cell lines, primary cells and a murine xenograft model established with A20 cell injection were used as in vitro and in vivo experimental models.Results: MiR-340-5p was positively correlated with CD8+ T-TILs in DLBCL patients, and KMT5A was a direct target gene of miR-340-5p. CD8+ T-cell function was significantly enhanced by miR-340-5p mimics both in vitro and in vivo, which was reversed by KMT5A overexpression. We demonstrated that COP1/CD73 was involved in the downstream mechanism of the miR-340-5p/KMT5A axis involving ubiquitination. In vivo, we validated an improved CD8+ T-TIL infiltration rate and tumor suppression with miR-340-5p treatment. Furthermore, miR-340-5p directly regulated the biological activity of DLBCL cells without CD8+ T-cell participation.Conclusions: MiR-340-5p promoted CD8+ T-TIL infiltration and antitumor function by regulating KMT5A and COP1 and further activating CD73 ubiquitination. MiR-340-5p is potentially a novel target for DLBCL immunotherapy.

scholarly journals GRK2 Is a Novel Modulator of the MALT1 Oncoprotein

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 775-775 ◽  
Author(s):  
Jing Cheng ◽  
Heejae Kang ◽  
Narayanan Parameswaran ◽  
Mathijs Baens ◽  
Ming Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Stimulation Of

Abstract Background and Significance: Lymphocyte antigen receptor-dependent stimulation of the NF-κB transcription factor is a key event during the normal adaptive immune response. However, dysregulated activation of these signaling pathways can lead to lymphoma. Stimulation of the B-or T-cell receptor promotes assembly of the CARMA1-Bcl10-MALT1 (CBM) signaling complex, where MALT1 functions as the essential effector molecule by carrying out two important functions: First, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, acts as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of canonical NF-κB signaling. Activating mutations in either BCR or CARMA1 promote constitutive and exaggerated MALT1 activity and are seen as driver mutations in diffuse large B cell lymphoma (DLBCL). Aberrant MALT1 activity also underlies the pathogenesis of MALT lymphoma, another subtype of B-cell malignancy. As such, MALT1 has emerged as a potential new target for pharmaceutical inhibition in the treatment of lymphoma. Here, we describe our discovery of a novel MALT1-binding partner, G protein-coupled receptor kinase 2 (GRK2). We hypothesize that GRK2 plays a critical role in the pathogenesis of lymphoma by modulating MALT1 activity. Methods and Results: Using co-immunoprecipitation and liquid chromatography mass spectrometry we identified GRK2 as a novel MALT1-interacting protein. We validated our results by showing that endogenous GRK2 and MALT1 co-immunoprecipitate (CO-IP) in both B cells and T cells. We next utilized GST-tagged purified proteins to demonstrate a direct interaction between GRK2 and MALT1. We then performed a number of studies to investigate the functional consequences of GRK2 interaction with MALT1. A CO-IP assay demonstrated that GRK2 inhibits Bcl10-dependent binding to MALT1, and a luciferase reporter assay revealed that GRK2 inhibits Bcl10/MALT1-dependent NF-κB activity. In addition, we demonstrated that GRK2 also inhibits MALT1 proteolytic activity using Western blot analysis of two known MALT1 substrates, the deubiquitinase cylindromatosis (CYLD) and the NF-kB family member RelB. Moreover, in Jurkat T cells, overexpression of GRK2 impeded AgR-induced NF-κB activation while stable GRK2 knockdown resulted in enhanced AgR-induced phosphorylation of inhibitor of kappaB (IκB) and secretion of interleukin2 (IL-2). We next began to investigate the potential of GRK2 to function as a tumor suppressor by blocking MALT1 activity. Of note, utilizing a published data set to compare GRK2 mRNA expression in 44 DLBCL patient samples to that of 20 samples of healthy B-cell controls, we found that GRK2 mRNA levels are markedly lower in DLBCL cases. We also compared GRK2 mRNA levels among patients diagnosed with DLBCL grouped by vital status, using data from the Cancer Genome Atlas (TCGA). We found that tumors from patients who died of disease within three years expressed significantly lower levels of GRK2 as compared to tumors from patients alive after three years. We also wished to investigate the impact of GRK2 expression on activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) cells, which require MALT1 activity for growth and survival. We were successful in engineering GRK2 overexpression in control germinal center B-cell like (GCB) DLBCL cells, which do not depend on MALT1 activity for growth. In contrast, we found that in ABC-DLBCL cells, which do depend on MALT1 activity, GRK2 overexpression greatly impairs cell proliferation, making it difficult to grow these engineered cells. These data provide support for the concept that GRK2 may have a potential tumor suppressor role in MALT1-dependent lymphoma. Conclusions: Our study represents the first report of a negative modulator of MALT1 and provides critical new insight into mechanisms regulating its activity. We hope to harness this information in developing novel strategies for inhibiting MALT1-dependent lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals MiR-214 prevents the progression of diffuse large B-cell lymphoma by targeting PD-L1

2019 ◽  
Vol 24 (1) ◽  
Author(s):  
Jing-Ran Sun ◽  
Xiao Zhang ◽  
Ya Zhang
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Expression Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Objective We explored the role and mechanism of miR-214 involvement in the progression of diffuse large B-cell lymphoma (DLBCL). Methods The expression levels of miR-214 and PD-L1 in human DLBCL cell lines and in tissue samples from patients with DLBCL were determined using quantitative RT-PCR. The dual-luciferase reporter assay was employed to determine the correlation between the expressions of miR-214 and PD-L1. Cell viability, invasiveness and apoptosis were respectively examined in cells of the DLBCL line OCI-Ly3 using CCK-8, transwell and flow cytometry assays. The expression level of PD-L1 was determined via immunoblotting. Inflammatory cytokine secretion was determined via enzyme-linked immune sorbent assay (ELISA). Results miR-214 was downregulated and PD-L1 was upregulated in DLBCL tissues and cell lines in comparison to normal adjacent tissues or normal B-cell. This indicates a negative correlation in the expression levels. Overexpression of miR-214 inhibited cell viability and invasion and induced apoptosis of OCI-Ly3 cells. Moreover, miR-214 was shown to target PD-L1 mRNA by binding to its 3′-untranslated region (UTR). Knockdown of PD-L1 attenuated the malignant phenotype of OCI-Ly3 cells. Overexpression of miR-214 inhibited tumor growth by targeting PD-L1 in vivo. Conclusion By targeting PD-L1, miR-214 regulates the progression of DLBCL in vitro and in vivo.

scholarly journals MiRNA-340-5p mediates the functional and infiltrative promotion of tumor-infiltrating CD8+ T lymphocytes in human diffuse large B cell lymphoma

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Yangyang Xu ◽  
Zhenchuan Liu ◽  
Lixin Lv ◽  
Ping Li ◽  
Bing Xiu ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background CD8+ tumor-infiltrating T lymphocytes (T-TILs) in the tumor microenvironment (TME) play an important role in tumor development, and miRNAs regulate tumor cell interactions with the microenvironment. T-TIL-based tumor immunotherapy provides a promising treatment strategy in diffuse large B-cell lymphoma (DLBCL). MiRNAs tend to be attractive targets for novel antitumor interventions. Methods Weighted gene coexpression network analysis (WGCNA), CIBERSORT analysis and Cox regression analysis were used to identify CD8+ T-TIL-related miRNAs. RT-PCR, western blotting, immunohistochemistry (IHC), in situ hybridization (ISH), luciferase reporter assay, coimmunoprecipitation and ubiquitination analyses were used to detect miRNA, mRNA and protein expression and their combination. The viability and function of CD8+ T cells after stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), cytotoxicity assay, functional avidity assessment, flow cytometry and Cell Counting Kit-8 (CCK-8) assay. DLBCL cell lines, primary cells and a murine xenograft model established with A20 cell injection were used as in vitro and in vivo experimental models. Results MiR-340-5p was positively correlated with CD8+ T-TILs in DLBCL patients, and KMT5A was a direct target gene of miR-340-5p. CD8+ T-cell function was significantly enhanced by miR-340-5p mimics both in vitro and in vivo, which was reversed by KMT5A overexpression. We demonstrated that COP1/CD73 was involved in the downstream mechanism of the miR-340-5p/KMT5A axis involving ubiquitination. In vivo, we validated an improved CD8+ T-TIL infiltration rate and tumor suppression with miR-340-5p treatment. Furthermore, miR-340-5p directly regulated the biological activity of DLBCL cells without CD8+ T-cell participation. Conclusions MiR-340-5p promoted CD8+ T-TIL infiltration and antitumor function by regulating KMT5A and COP1 and further activating CD73 ubiquitination. MiR-340-5p is potentially a novel target for DLBCL immunotherapy.

Lymphome

Praxis ◽  
2016 ◽  
Vol 105 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Andreas Lohri
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Staging System ◽  
Maligne Lymphome ◽  
Interim Pet ◽  
Large B Cell Lymphoma ◽  
Ann Arbor ◽  
Large B Cell

Zusammenfassung. Maligne Lymphome unterteilen sich zwar in über 60 Entitäten, das grosszellige B-Zell-Lymphom, das follikuläre Lymphom, der Hodgkin und das Mantelzell-Lymphom machen aber mehr als die Hälfte aller Lymphome aus. Im revidierten Ann Arbor staging system gelten die Suffixe «A» und «B» nur noch für den Hodgkin. «E» erscheint nur noch bei Stadien I und II. Eine Knochenmarksuntersuchung wird beim Hodgkin nicht mehr verlangt, beim DLBCL (Diffuse large B cell lymphoma) nur, falls das PET keinen Knochenmark-Befall zeigt. Der PET-Untersuchung, speziell dem Interim-PET, kommt eine entscheidende Bedeutung zu. PET-gesteuerte Therapien führen zu weniger Toxizität. Gezielt wirkende Medikamente mit eindrücklicher Wirksamkeit wurden neu zugelassen. Deren Kosten sind hoch. Eine strahlen- und chemotherapiefreie Behandlung maligner Lymphome wird in Zukunft möglich sein.

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