The Effect And Mechanism Of ROR2in Leukemia And The Application Of Transgenic Tool In Hematopoietic System

Objective:ROR2, an orphan cell surface receptor with strong homology to the tyrosine kinasedomain of growth factor receptors, is defined as one of the most important members in Wntnon-canonical signaling pathway. Loss-of-function mutation of this gene is associated with asevere skeletal dysplasia with generalized limb bone shortening, segmental defects of thespine, brachydactyly and a dysmorphic facial appearance.Recent studies have reported that ROR2plays diametrically opposite roles in differentkinds of tumors. ROR2acts as a tumor-suppressing factor in those cancers which are drivedby Wnt/β-catenin signaling pathway. On the other hand，ROR2may be an oncogene in somenon-canonical pathway tumorgenesis. Researches in our lab and abroad together revealed thatWnt5a binds to the Ror2receptor and subsequently inhibits leukemia development. Ourprevious work showed that wnt5a expression is downregulated by the alterations of itspromoter methylation in leukemia. Wnt5a can inhibit leukemic cell malignant proliferation byinhibiting the TCF/LEF function of canonical Wnt signaling and enhance the response ofCML cells to imatinib mesylate through JNK activation. In the study of mechanism, wecertificated that Wnt5a is involved in tumor inhibition through antagonizing Wnt/β-cateninsignaling by upregulating ROR2expression and binding to ROR2. However, the unique effectof ROR2in leukemia and the function of ROR2-induced bone marrow mesenchymal stemcell are not known and are investigated in this study.PiggyBac(PB) transposons system is improved and developed constantly in recent years.It has become one of the most popular gene transfer techniques in mammalian cells for itsremarkable advantage. With creative design，we constructed several gene expression vectorsand modified cells to support the following studies. Secondly, it is rare for gene knockdownmanipulation with PB transposons system. Considering the advantages of PB system, wedesigned and constructed a stable gene knockdown system at the base of PB system andproved its function in iMEFs cell. In addition, it was acknowledged that there is trouble to gain a high infection efficiency during the application of adenovirus system in leukemia cells.However，we improved the adenovirus infection system with a new component Polybrene,which helps to raise efficiency greatly in iMEFs and K562cells. In summary, we tried toillustrate the function and mechanism of ROR2in both leukemia cell K562and themesenchymal stem cell iMEFs with techniques such as Western blot, IHC,immunofluorescence, RT-PCR and siRNAs. Meanwhile, we constructed a couple of geneexpression plasmids, as well as improved the method of adenovirus infection in mesenchymalstem cell and leukemia cell to facilitate the transgenosis.We wish to clarify whether the ROR2could act as a tumor inhibitor and induceapoptosis of leukemia cells, and in which pathway that could ROR2mediate those funciton.In addition, we also want to investigate whether ROR2is involved in the regulation of theinteraction between the MSC and leukemia.This study discussed the tumor-suppressing roles and mechanism of ROR2through Wntnon-canonical pathway in chronic myelogenous leukemia and microenvironment. Theresearch could refine the theory of Wnt signaling and tumorgenesis, and provide a new viewof leukemia therapy and molecular compounds pharmacy.Methods：1. Study of the effect and mechanism of ROR2in leukemia1.1Westernblot and IHC were used to detect the expression of ROR2in clinical cases.1.2pMPB5-ROR2-luc, pMPB5-EV-luc and AdROR2were constructed by molecularcloning technique.1.3Testify the increase of infection efficiency of adenovirus system combinated withPolybrene with FACS examination, luciferase reporter assay, ALP staining and cell counting.1.4Proliferation of K562was detected by CCK-8.1.5K562cell cycle was detected by flow cytometry analysis.1.6Westernblot was used to detect the protein levels of JNK and MMP-9in K562cellsinfected with AdROR2.1.7AnnexinV-EGFP，PI and Hoechst33342combination test was used to determinateapoptosis of K562treated with ROR2or Wnt5a.1.8NOD-SCID mouse experiment was used to test the effect of ROR2on K562in vivo.2. The effect of ROR2on leukemia expressed in microenvironment 2.1Lipidosome transfection was used to construct the ROR2stable-expression iMEFscell line2.2Lipidosome transfection were used to construct the RFP stable-expression cell lineK562-RFP2.3Construction of non-contact and contact co-culture system2.4Fluorescence cell counting and morphology were used to observe the effect on K562of ROR22.5FACS was used to detect the proliferation of K562in co-culture system2.6RT-PCR was used to test the gene expression data of iMEFs treated with AdROR23. Construction and application of pMPBOS stable gene knockdown system in iMEFs3.1pMPBOS constructed by molecular cloning technique in PiggyBac-based vectors.3.2Lipidosome Transfection was used to construct the stable knockdown cellline”iMEFs-KDn” with pMPBOS.3.3Testify of the stability and long term effect of the pMPBOS in iMEFs-KDn withrelative activity of luciferase reporter assay, relative activity of ALP assay, RT-PCR andimmunofluorescence.Results1. Adenovirus-mediated gene transfer applied with cationic polymer Polybrene in C2C12and iMEFs exhibits a dependency between dose and effect.2. AdBMP9-mediated osteogenesis in mesenchymal stem cells can be significantlyenhanced by the Polybrene3. The efficiency of adenovirus-mediated gene transfer was significantly improved inK562with the help of Polybrene.4. The expression of ROR2is downregalated in acute or chronic myeloid leukemic casesand is upregalated in complete remission cases.5. K562with ROR2stable expression can not be proliferative.6. K562proliferation is inhibited by exogenous ROR2, while cell cycle is blocked in G2phase. Apoptosis of K562is upregulated.7. The p-JNK is activated by ROR2in K562.8. K562defomation and migration was observed in both two kinds of contact co-culturesystem. 9.ROR2reduced the defomation and migration of K562in contact co-culture system.10. Multi-gene expression change were detected in iMEFs treated with exogenousROR2.11. Establishment of gene one-step knockdown with the constuction of pMPBOS.12. Successful constuction of iMEFs-KD-β-catenin cell lines.13. Dependency between dose and effect of pMPBOS was testified in iMFEs cell.Conclusion1. ROR2could inhibit K562proliferation, increase its apoptosis, and block the cell at G2phase throgh activation of JNK pathway;2. ROR2could regulate multiple gene expression in iMEFs, such as c-Jun, CTGF,Pecam-1, L-selectin, E-Cadherin, LIF, Vcam-1and FOXO-1, and downregulate the migrationof K562into MSC niche，which may help to increase the contact between leukemia cells andthe blood to improve the pharmacy sensitivity.3. Adenovirus-mediated gene transfer in mesenchymal stem cells and leukemia cell canbe significantly enhanced by the Polybrene.4. A stable, long term gene knockdown cell line with dose dependent effect could be wellestablished with the application of the pMPBOS.