2014 Fiscal Year Research-status Report
Mir-195 rescue B cell development independently of transcription factor in EBF KO hematopoietic progenitor cells
| Project/Area Number |
26860739
|
|---|
| Research Institution | Tokai University |
|---|
Principal Investigator |
チャンダ ビディシャ 東海大学, 医学部, 奨励研究員 (70725358)
|
|---|
| Project Period (FY) |
2014-04-01 – 2016-03-31
|
| Keywords | microRNA / B cell development / Leukemia / Lymphoma |
|---|
| Outline of Annual Research Achievements |
Over expression of miR-195 in EBF1 Konckout (KO) cells induces CD19 expression, completion of VDJ recombination, and upregulation of B cell related genes in EBF1 KO HPCs while neither PAX5 nor E2A are able to induce CD19 expression in the absence of EBF1. Furthermore, in vivo transplantation of mir-195 over expressed EBF1 KO cells into NOG mouse also expressed CD19 in bone marrow and splenocytes.I also investigated the effect of miR-195 on the maintenance of B cell fate in the absence of Pax5 and observed that miR-195 suppressed macrophage differentiation and maintained the expression of Ebf1, Tcfe2a (E2A), Rag1, andλ5 in cells lacking Pax5. However, Cd19 expression, which is directly regulated by Pax5 was not maintained by miR-195 and Cd79a, which is also a direct target of Pax5, was downregulated by miR-195 in Pax5 KO condition, suggesting that miR-195 transduced cells maintained B cell characteristics without upregulating the target genes of Pax5. In silico analysis we found 7 target genes of mir-195, among them TGF beta family genes such as Tgfβr3 and Acvr2a, were down regulated by luciferase assay and inhibition of Tgfβ by anti Tgfβ Ab increase the proliferation and B220 expression of EBF1 KO HPCs after 14 days coculture on TST4 cells.We hypothesize that by downregulating TGFβRIII, mir-195 might activate NFκB and rescues B cell development.
From our study we can conclude that miR-195 can rescue B cell developmental arrest and progressed toward pro B to pre B cell and acts more than a fine tuner in B cell lineage specification.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
1.About mouse model study: In Tokai University NOG mouse was available .So at the beginning of my study I did several NOG mouse experiments and derived in vivo result.
2.About identification of miR-195 role in developmental stages: I was able to maintain miR-195 over expressed fetal liver cells on Tst4-DLL4 cells and induced them to T cell differentiation without Bcl2 by our cell culture system.I also cultured miR-195 over expressed fetal liver cells on Tst4-DLL4 cells and induced them to Natural killer (NK) cells and found difference between miR-195 over expressed and control cells.
3.About molecular mechanism of rescue B cell lineage commitment and finding of target gene of miR-195: I had tried several ways to find out the target gene of miR-195. At the beginning we validated six target genes by luciferase assay and knock down one by one by shRNA in EBF1 KO cell line .But the poor infection efficiency of shRNA, we failed to derived any conclusion. Then I tried to knock down target genes by siRNA .I design two siRNA for each target genes and transfect them into EBF1 KO cell line. However, by this method I failed to derive any result in case of Tgfbr3 and Acvr2a. So I had tried pan-antibody against Tgfb and activin A and derived the result.
4.Investigation of function of miR-195 in Leukemia and Lymphoma: In vitro study of miR-195 in leukemia and lymphoma I did not get any favorable result in cell line study due to low infection efficiency of miR-195 over expressing retro viral vector. So I changed this study into in vivo mouse model and now I am preparing miR-195 KO mouse.
|
| Strategy for Future Research Activity |
1.Study the correlation of leukemia and miR-195 by mouse model:
I am preparing miR-195 KO homogenous miR-195 and miR-497 cluster deleted mice by using "CRISPER-Cas9" system. My next plan is to derive leukemia model, specially chronic lymphoid leukemia model by using this KO homo mice. Therefore, I will collect bone marrow (BM) cells from C57BL/6 Ly 5.1 mouse and sort lineage negative (lin-) cells by MACS sorter. Then pre stimulate those cells by SCF,Flt3l and TPO overnight and transducer with Bcr-Abl expressing green florescent protein (GFP) containing lenti viral vector and control GFP vector . It is reported that Bcr-Abl can cause CLL. So I will inject these transduced cells intravenously in miR-195 KO Ly 5.2 mouse and collect peripheral blood after every 2weeks of injection. I will check the PBMC morphology by FACS and May grunwald giemsa staining. After getting GFP+ cells in PBMC, I will sacrifice mice and check BM, spleen, lymph node (LN) and other abnormal phenotypes and compare it with control mice.
2.Mir-195 promoter assay: In present research I found that miR-195 rescues B lineage commitment in the absence of Ebf1 by suppressing Tgfbr3, Acvr2a,Pbx3 and Hgf. However, the miR-195 regulating factor still unknown. My hypothesis is that deregulation of these factors can cause change the expression of miR-195 and cause leukemia and lymphoma. So I have plan to analysis the promoter sequence upstream of miR-195 in chromosome 11 and find out the factors bind with this promoter sequence. Then knockdown these factors one by one by shRNA and study the phenomenon.
|
Research Products
(3 results)
-
[Presentation] A single micro-RNA can completely rescue B-cell differentiation arrest due to EBF1 deficiency-Can micro-RNA control cell fate as a potential alternative of transcriptional factor?2015
Author(s)
B Chanda, T Ikawa, K Okuyama, K Hozumi, K Ando, A Tojo, H Kawamoto,A Kotani
Organizer
Haematopoiesis (B6),Keystone Symposium,
Place of Presentation
Keystone, Colorado, USA
Year and Date
2015-02-22 – 2015-02-27
-
-
