Effect of viblastine on transcript expression of immune related genes in dendritic cell of chronic lymphocytic leukemia

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TITLE: EFFECT OF VIBLASTINE ON TRANSCRIPT EXPRESSION OF IMMUNE RELATED GENES IN DENDRITIC CELL OF CHRONIC LYMPHOCYTIC LEUKEMIA Student name : TRAN THI THUY DUNG Class : K63CNSHE Student code : 637407 Faculty : BIOTECHNOLOGY Supervisor : Assoc.Prof.NGUYEN THI XUAN MSc.NGUYEN THANH HUYEN HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - UNDERGRADUATE THESIS TITLE: EFFECT OF VIBLASTINE ON TRANSCRIPT EXPRESSION OF IMMUNE RELATED GENES IN DENDRITIC CELL OF CHRONIC LYMPHOCYTIC LEUKEMIA Student name : TRAN THI THUY DUNG Class : K63CNSHE Student code : 637407 Faculty : BIOTECHNOLOGY Supervisor : Assoc.Prof.NGUYEN THI XUAN MSc.NGUYEN THANH HUYEN HANOI – 2022 DECLARATION I hereby declare that the graduate thesis work is mine All research results have been results during the implementation of the topic The results, the data are completely true, never appeared in any scientific report I also guarantee that the references and useful information for the topic are clearly cited and all helps are appreciated Hanoi, December 5th, 2022 Student Tran Thi Thuy Dung ACKNOWLEDGEMENTS During the process of implementing my graduation project, I have received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Associate Professor Ph D Nguyen Thi Xuan and MSc Nguyen Thanh Huyen for giving me the opportunities to carry out this work, and their huge efforts, enthusiasm, and supports throughout the duration of the undergraduate thesis Secondly, I would like to thank the teachers in the Faculty of Biotechnology who helped and taught me during my training at the University Especially, the teachers of the Microbial Biotechnology department who gave me advice during carrying out Finally, I would like to sincerely thank my family members and friends who always trust, support and encourage me to complete this report Sincerely thank! Hanoi, December 5th, 2022 Student Tran Thi Thuy Dung ii CONTENTS DECLARATION i ACKNOWLEDGEMENTS .ii CONTENTS .iii LIST OF ABBREVIATIONS v LIST OF FIGURES vii LIST OF TABLES viii PART INTRODUCTION 1.1 Preface 1.2 Objectives and Requirements 1.2.1 Objectives 1.2.2 Requirements PART LITERATURE REVIEW 2.1 Leukemia 2.1.1 Acute Lymphocytic Leukemia (ALL) 2.1.2 Acute myeloid leukemia (AML) 2.1.3 Chronic myeloid leukemia (CML) 2.1.4 Chronic Lymphocytic Leukemia (CLL) 2.2 Leukemia in the world and in Vietnam 10 2.2.1 In the world 10 2.2.2 In Vietnam 11 2.3 Dendritic cell 12 2.4 The immune related genes 13 2.4.1 Deubiquitinase group 13 2.4.1.1 A20 Gene 14 2.4.1.2 OTUB1 Gene 15 2.4.1.3 OTUB2 Gene 16 2.4.1.4 OTUD7B (CEZANNE) Gene 17 2.4.1.5 CYLD Gene 17 2.4.2 Pro-apoptotic group 19 2.4.2.1 BAX gene 20 2.5 Vinblastine 21 PART MATERIALS AND METHODS 24 3.1 Materials 24 3.1.1 Subjects for research 24 3.1.2 Tools and Devices 24 iii 3.1.3 Chemicals 24 3.2 Methods 25 3.2.1 Collecting the samples 26 3.2.2 Culturing peripheral mononuclear blood cells (PBMCs) 26 3.2.2.1 PBMCs isolation 26 3.2.2.2 Cell culture 28 3.2.2.3 RNA extraction 28 3.2.2.4 Synthesis of cDNA from total RNA 29 3.2.2.5 Realtime-PCR 29 3.2.2.6 Results analysis 30 PART RESULTS AND DISCUSSION 32 4.1 PBMCs isolation 32 4.2 RNA extraction 32 4.3 Expression levels of A20, CYLD, OTUB1, OTUB2 and CEZANNE genes in patients with Chronic lymphocytic leukemia 34 4.3.1 Expression level of A20 gene in patients with Chronic lymphocytic leukemia 34 4.3.2 Expression level of CYLD gene in patients with Chronic Lymphocytic Leukemia 36 4.3.3 Expression level of OTUB1 gene in patients with Chronic lymphocytic leukemia 38 4.3.4 Expression level of OTUB2 gene in patients with Chronic lymphocytic leukemia 39 4.3.5 Expression level of CEZANNE gene in patients with Chronic lymphocytic leukemia 41 4.3.6 Expression level of BAX gene in patients with Chronic lymphocytic leukemia 42 PART CONCLUSION AND SUGGESTION 45 5.1 Conclusion 45 5.2 Suggestion 45 REFERENCES 46 iv LIST OF ABBREVIATIONS CLL Chronic lymphocytic leukemia ALL Acute lymphocytic leukemia AML Acute myeloid leukemia CML Chronic myeloid leukemia MOM Mitochondria outer membrane PTP Permeability transition pore cDCs conventional dendritic cells DUB Deubiqitinase NK-κB Nuclear factor kappa-light-chain-enhancer of activated B CCR7 C-C chemokine receptor HLA Human Leucocyte Antigen GM-CSF Granulocyte-macrophage colony- stimulating factor PBMC Peripheral blood mononuclear cell DC Dendritic cell VinB Vinblastine ABL Abelson murine leukemia BCR Breakpoint TKI Tyrosine kinase inhibitor FLT3 FM’s Related Receptor Tyrosine Kinase PCR Polymerase-Chain-Reaction Ag Antigen OTU Ovarian tumor domain (OTU) HCC Hepatocellular carcinoma v RNA Ribonucleic acid DNA Deoxyribonuclec acid vi LIST OF FIGURES Figure 2.1: Acute Lymphocytic Leukemia (ALL) Figure 2.2: Acute myeloid leukemia (AML) Figure 2.3: Chronic myeloid leukemia (CML) Figure 2.4: Chronic Lymphocytic Leukemia (CLL) and normal blood cell Figure 2.5: Comparison between normal blood and Leukemia 11 Figure 2.6: The structure of A20 15 Figure 2.7: The structure of OTUB1 16 Figure 2.8: The structure of CYLD 18 Figure 2.9: Chemical structure of Vinblastine 22 Figure 3.1: Phase cleavage after centrifugation to isolate PBMCs 27 Figure 4.1: DCs are differentiated from PBMCs: A DCs at 40X microscopy, B PBMCs at 20X microscopy 32 Figure 4.2 Electrophoresis image after RNA extraction 33 Figure 4.3: The graph of transcript expression of A20 34 Figure 4.4: The graph of transcript expression of CYLD 36 Figure 4.5: The graph of transcript expression of OTUB1 38 Figure 4.6: The graph of transcript expression of OTUB2 39 Figure 4.7: The graph of transcript expression of OTUD7 41 Figure 4.8: The graph of transcript expression of BAX 43 vii LIST OF TABLES Table 3.1: Sequence of immune related genes 29 Table 4.1: List of RNA extraction from the cultured cell sample 33 viii a significant different from the control sample when these p-value is higher than 0.01 Cezanne is an OTU deubiquinase with sequence homology to the A20 gene, which has been shown to inhibit the NF-κB pathway (Evans, Smith et al 2003, Enesa, Zakkar et al 2008).In my study, the expression of CEZANNE have been shown higher than the control sample Effect of VinB make a significant change the transcript expression of CEZANNE gene in dendritic cells of CLL compared to the control sample leading to inhibit the NF-κB and active the apoptosis process This results could lead to increase inhibition of NF-kB pathway Cezanne is highly expressed in both squamous cell lung cancer adenocarcinoma lung cancer and hepatocellular carcinoma (Luong le, Fragiadaki et al 2013, Wang, Zhong et al 2017) Therefore, VinB have a significant impact on the transcript of CEZANNE in dendritic cell of CLL However, this project need futher study to evaluate whether the overpression of CEZANNE have negative or positive impact on dendritic cell of CLL as well as to find out the optimum concentration of VinB to prevent the overexpression of CEZANNE 4.3.6 Expression level of BAX gene in patients with Chronic lymphocytic leukemia 42 Figure 4.8: The transcript expression of BAX at different concentration of VinB The expression of BAX gene in CLL patients at different concentration of Vinblastine is shown by the diagram with the control sample which is not treated by Vinblastine Generally, the expression level of BAX gene at different concentration of VinB is higher than the control sample With the relative expression of the control sample is while the expression level of BAX gene at 20nM, 60nM and 120nM VinB is 55.71, 6.1 and 76.52 relatively The transcript expression of BAX gene also rises significantly after treating dendritic cells of CLL patients with different concentrations of VinB Nevertheless, the expression of BAX gene in CLL patients at different concentration of VinB does not indicate a significant different from the control sample when these p-value is higher than 0.01 BAX gene causes irreparable damage to mitochondria, while precipitating downstream events that finish off a dying ganglion cell BAX gene goes through a series of ordered events that result in pore formation in the MOM, allowing signaling molecules like cytochrome c to be released This final event is often referred to as the irreversible step in the apoptotic pathway, or the "switch" from life to death Extrinsic apoptosis is distinguished by the activity of the tumor 43 necrosis factor (TNF) family of ligands, such as TNFα, FasL, and the TNF-related apoptosis-inducing ligand (TRAIL) These ligands can bind to specialized receptors on cells containing “death domains” (Brady and Gil-Gomez 1998) DCs have a short life span because they are subject to apoptotic cell death mediated by T cells, limiting their ability to prime antigen-specific T cells in the long term Antiapoptotic BAX proteins can be used to allow transfected DCs to resist T cell killing in vivo (Peng, Kim et al 2005) Overexpression of BAX in T-cells increases their sensitivity to apoptotic stimuli such as DNA damage or glucocorticoids but not to anti-Fas antibodies, and these effects can be blocked by Bcl-2 (Brady, Salomons et al 1996) The expression of BAX gene has been shown that high significantly in Peripheral Blood Lymphocytes of Patients with differentiated Thyroid Cancer who are treated with Iodine-131 that is used in the treatment of thyroid cancer with dosage of 100 mCi (Hamivand, Haddadi et al 2018) This finding is consistent with the obtained results of my research; when dendritic cells of CLL are treated with different of concentration of VinB gave similar results when increasing the expression of BAX gene compared with the control sample Therefore, VinB may have an effect by increasing the expression of BAX gene in dendritic cells of CLL 44 PART CONCLUSION AND SUGGESTION 5.1 Conclusion The experimental result showed that PBMCs are cultivated and differentiated into DCs after 72h to ensure sufficient density, quality as well as the level of differentiation into DCs to proceed to the next step of processing with Vinb This study provides the effect of VinB on the expression levels of DUB and pro-apoptotic genes as well as the role of the expression of immune-related genes in the signaling pathways on dendritic cells of CLL In my study, the results showed that VinB make a significant decrease in the transcript expression of A20 and an increase in the transcript expression of OTUB1, OTUB2, CEZANNE, CYLD and BAX Therefore, this result also leads to the effect of the signaling pathway and apoptosis process in dendritic cell of CLL depending on the transcript function of immune-related genes 5.2 Suggestion Due to the limited time in the research process, the report has some limitations Because of this, we would like to give some recommendations: Basing on this results to test other concentrations of VinB in dendritic cell of CLL to evaluate the 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