Study On The Effect And Mechnism Of Ror2 In The Proliferation Of Chronic Myeloid Leukemia Cell Line K562

Objective Leukemia is a clonal disease of hematopoietic stem cell abnormality.It is mainly treated by chemotherapy,but chemotherapy has a lot of disadvantages of toxic and side effects.Currently,biological therapy has become a new research hotspot.Wnt signaling pathway is closely related to the occurrence of a variety of tumors,the classic Wnt signal(Wnt/ beta-catenin)is a cancer promoting pathway,the non classical Wnt pathway is considered to be a tumor suppressor pathway.Our previous studies indicate that Wnt5 a,as a member of the Wnt family,has priority to combine Ror2 receptor and activate the non classical pathway,thereby inhibiting chronic myeloid leukemia cell line K562 proliferation.In the pre-study,cell apoptosis was found when Ror2 was overexpressed in the K562 cells,howerver,no cell apotosis occured when Wnt5 a was overexpressed.The purpose of this study is to further clarify whether Ror2 can induce apoptosis of K562 cells and to explore its possible mechanism.The orphan receptor Ror2 expressed the highest in the early stage of embryonic development,playing the important role in the tissue and organ infomation.The Ror2 family have been reported to increase in recent years,but mainly Ror1,such as Ror1,which is highly expressed in human lymphocytic leukemia,is a kind of carcinogenic factor.Even though,Ror2's report is still very little.We examined bone marrow samples from 26 patients with non hematologic malignancies and 9 samples with acute/chronic leukemia,and found that Ror2 expression decreased in leukemia cases.The expression of non malignant hematologic disease and leukemia remission cases was increased.There was apotosis in K562 cells when Ror2 was overexpression,suggesting that Ror2 in leukemia may be a tumor suppressor.Ror2 induced apoptosis is one of suppression of proliferation of myeloid leukemia cells.The mechanism may be related to the upregulation of the pro apoptotic protein caspase-3,bax,and downregulation of the antiapoptotic proteins survivin and bcl-2.In order to establish the K562 cell line of the overexpression of Ror2 receptor,Polybrene was used to promote the transfection efficience of recombinant adenovirus,Ror2 was overexpressed efficently,which effectively solved the difficult problem of blood cell transfection efficience,laying a good foundation for the following experiments.Method1The study of polybrene promoting the recombinant adenovirus infection efficience of K562 cells1.1 The recombinant adenovirus Ror2(Ad Ror2)with different dosage was used to infect K562 cells,and the optimal dosage of recombinant adenovirus was obtained1.2 The different concentrations of Polybrene and the best dosage of AdRor2 were used to infect K562 cells,the optimal Polybrene titer was obtained.1.3 The optimal concentration polybrene and optimal dosage of AdRor2 infected K562 cells at 1day,2day,3day,4day,5day,the best Polybrene infection time was obtained.1.4 K562 cells were infected with the best Polybrene concentration and the best dosage of AdRor2.The expression of Ror2 in mRNA and protein levels were detected by RT-PCR and Western blot.2 Ror2 inhibits the proliferation and induces apoptosis of K562 cells2.1 The cell proliferation was detected by CCK8 after AdRor2 was overexpressed in K562 cells2.2 The cell cycle and apoptosis were detected by Flow Cytometry after Ad Ror2 infected K562 Cells at 30 h,48h.3 A preliminary study on the mechanism of K562 cell apoptosis that induced by Ror2.3.1After AdRor2 infected the K562 cells,the expression of pro-apoptotic protein caspase-3 and bax was detected by Western blot.3.2 After AdRor2 infected the K562 cells,the expression of anti-apototic protein survivinand bcl-2 was detected by Western blot.Res?lt1.The optimal dosage of AdRor2 infecting K562 cells was 8 ?l/105 cells;the optimal concentration that polybrene enhanced AdRor2 infection efficiency was 3?g/ml,and the best time was 2d.2.Compared with the control group,Ror2 in m RNA and protein expression levels were higher than the control group after the best concentration of polybrene and the best dosage of Ad Ror2 infecting K562 cells with two days.3.Compared with the control group,the cell proliferation was decreased when AdRor2 infected K562 cells at 1day,2day,3day,4day,5day.4.Cell cycle was blocked in G2/M phase after Ad Ror2 infected K562 cells at 30 h and 48 h.compared with the cells(34.96 + 0.16)% of control group,the cells(59.88 + 0.25)% increased significantly in the G2/M phase.The percentage of apoptosis(13.52 + 0.48)% was higher than the control group(10.71 + 0.50)%.5.Compared with the control group,the expression of Pro apoptotic protein caspase-3 and bax were significantly higher when AdRor2 infected K562 cells,while the expression of anti apoptotic protein survivin and bcl-2 were lower after Ad Ror2 infected K562 cells with 2 days.ConlusionIn chronic myeloid leukemia cell line K562,polybrene promotes the infecting efficiency of recombinant adenovirus Ror2 in K562 cells,which could solve the difficult problem of blood cells with import genes.Overexpression of Ror2 could significantly inhibit the proliferation of K562 cells,prevent cells in G2/M phase of the cell cycle and induce cell apoptosis.The mechanism may be related to the upregulation of the expression of Pro apoptotic protein caspase3 and bax and downregulation the expression of anti apoptotic protein survivin and bcl-2 by Ror2 participation.