The Role Of MXRA7 In The Development Of Acute Promyelocytic Leukemia

Objective:Matrix-remodeling associated 7(MXRA7)belongs to MXRA family which was co-expressed with a group of matrix remodeling related genes.Previous studies have demonstrated MXRA7 was involved in some critical physiological and pathological processes.MXRA7 deficiency alleviated CC;4-indcue acute liver injury in mice,MXRA7 promoted differentiation of bone marrow mesenchymal stem cells toward osteoblasts,and MXRA7 might function as a negative modulator in psoriasis development.Public databases showed that MXRA7 was expressed higher in the bone marrow cells of acute promyelocytic leukemia than in normal bone marrow,suggesting that MXRA7 may be involved in mediating the development of acute promyelocytic leukemia(APL).Therefore,we investigated the function and mechanism of MXRA7 in acute promyelocytic leukemia using the NB4 cell line.Methods:(1)Analyze the expression of MXRA7 in acute myelocytic leukemia using BloodSpot and Leukemia Gene Atlas databases.(2)Collect bone marrow samples of APL(AML-M3)patients,acute B lymphocytic leukemia(B-ALL)patients,acute T lymphocytic leukemia(T-ALL)patients,normal healthy donors and leukemia cell lines and detect the MXRA7 expression using real-time quantitative PCR(RT-qPCR)and western blot.(3)Construct MXRA7 overexpressing and knockdown NB4 cell lines using lentivirus system.The expression of MXRA7 in these cell lines was detected by RT-qPCR and western blot.(4)The cell proliferation was tested by CCK8 method and agarose colony formation assay.The cell cycle distribution was detected by flow cytometry with PI staining.The transwell cell invasion assay in vitro was performed and the expression of matrix metalloproteinases was detected by RT-qPCR.(5)The cell apoptosis of transfected NB4 cell lines induced by chemotherapy drugs or not was analyzed with flow cytometry.The expression of apoptosis-related protein was detected by western blot.(6)Flow cytometry was used to detect the expression of CD11b,a surface marker molecule for differentiation of promyelocytic cells toward granulocyte cells.Analyze the effect of MXRA7 on the differentiation of NB4 cells by Wright-Giemsa staining and NBT assay.(7)Western blot was used to detect the effect of MXRA7 on the expression of fusion genes PML-RARa,PML and RARa during the differentiation of NB4 cells induced by all-trans retinoic acid(ATRA).RT-qPCR was used to detect the expression of the transcription factor CEBP family,which plays an important role in the differentiation of myeloid cells.(8)The mouse model of acute promyelocytic leukemia was constructed using NOD-SCID immunodeficient mice.NB4-NC or NB4 MXRA7iow cells were injected into the tail vein of mice to observe the survival time and body weight change of mice.The percentage and number of leukemia cells in bone marrow cells of femur were analyzed.Results:(1)The analysis of Bloodspot and Leukemia Gene Atlas databases revealed MXRA7 mRNA level in AML-t(15;17)(APL)was highest among all leukemia categories.(2)RT-qPCR and western blot results showed that the mRNA and protein expression levels of MXRA7 in the bone marrow of patients with AML-M3,B-ALL and T-ALL were higher than that of normal donors.MXRA7 was also expressed highly in the cell lines of AML,B-ALL and T-ALL.(3)The YFP positive cells were sorted with flow cytometry to establish the NB4-V and NB4-MXRA7high cell lines.Stably transfected NB4 cell lines were selected using puromycin to establish the NB4-NC and NB4-MXRA7iow cell lines.The stable transfected cell lines were constructed successfully which was verified by RT-qPCR and western blot.(4)The cell viability assay showed that MXRA7 did not significantly change the viability of NB4 cells.MXRA7 also did not affect the cycle distribution of NB4 cells.Colony formation assay showed that the ability of clone-forming was enhanced after MXRA7overexpression.Moreover,MXRA7overexpression also enhanced the invasion potency of NB4 cells and up-regulated the expression of MMP-2 and MMP-9.However,knockdown of MXRA7 did not significantly affect the invasion potency of NB4 cells.(5)Knockdown of MXRA7 increased apoptosis when treated with two chemical cytotoxins,MTX and Ara-C.Correspondingly,cleavage of caspases 3,8 and 9 in MTX or Ara-C treated NB4-MXRA7-low cells were increased compared with control cells.However,compared with control NB4 cells,NB4-MXRA7high cells did not manifested significant alteration in their response to apoptosis-induction.(6)Knockdown of MXRA7 increased that the expression of granulocyte cells surface marker CD11b and increased the NBT redox ability.Morphologically,upon ATRA treatment,knockdown of MXRA7 promoted the differentiation of NB4 cells detected by Wright-Giemsa staining.Overexpression of MXRA7 decreased the expression of CD11b and weakened the NBT redox ability,but did not change the differentiation in morphologically.(7)NB4 cells with increased MXRA7 levels(NB4-MXRA7h,gh)showed elevated PML-RARα proteins level and decreased PML and RARa proteins level after ATRA treatment.Knockdown of MXRA7 in NB4 cells decreased PML-RARa level and increased PML and RARa levels.Moreover,the expression of CEBPB,CEBPD and CEBPG were decreased in NB4-MXRA7high cells,and CEBPD and CEBPE transcription were slightly increased in NB4-MXRA7low cells.(8)NB4-MXRA7low or control NB4-NC cells were inoculated into NOD-SCID mice as an artificial APL model.It was found that MXRA 7low cells caused less weight loss than control NB4-NC cells did.And the mice inoculated with MXRA7low cells contained less leukemic cells than the control mice.These results indicated that knock-down of MXRA7 in NB4 cells decreased the malignancy of these cells in vivo and improved the survival state of mice.Conclusion:In this study,we found that MXRA7 behaved like a blockade of cell differentiation through mediating the expression of PML-RARa in acute promyelocytie leukemia cells.Therefore,MXRA7 can affect the development of acute promyelocytic leukemia.Moreover,it is noteworthy that MXRA7 expression was elevated in several other types of leukemia as well,such as T-ALL and B-ALL.The novel findings about the role of MXRA7 in leukemia not only shed lights over the biology of MXRA7 but also proposed a new target for manipulating leukemia,and more studies are deserved to dissect the mechanisms of MXRA7 modulation in leukemia.