| Background and objective:The relationship between the hematopoietic microenvironment and leukemia has become a hot topic in recent years.The leukemic hematopoietic microenvironment contributes to leukemia onset and progression.At the same time,leukemia cells can also remodel the hematopoietic microenvironment so that it is conducive to leukemia cell survival.Targeting the abnormal hematopoietic microenvironment,blocking the vicious circle between hematopoietic microenvironment and leukemia cells may be able to improve the treatment of leukemia.Bone marrow stromal cells(BMSCs),as an important functional component of hematopoietic microenvironment,have been found to have cytogenetic and functional abnormalities in leukemia bone marrow.Our previous study found that connexin43(Cx43)expression and gap junction intercellular communication(GJIC)were decreased in leukemic BMSCs.Decreased expression of Cx43 in BMSCs can protect leukemia cells from spontaneous and drug-induced apoptosis.Recent studies have shown that hypoxia is present in CML hematopoietic microenvironment,and the degree of hypoxia is further increased with the progression of the disease.In addition,a study shows that hypoxia can induce down-regulation of Cx43 in Rat H9C2 cardiomyocytes.Therefore,we hypothesized that hypoxia in CML hematopoietic microenvironment may lead to a decrease of Cx43 expression in CML microenvironment and further protection of CML cells.In this study,we investigated hypoxia induced downregulation of Cx43 in BMSCs and the mechanisms of its reduction of imatinib(IM)induced K562 cells apoptosis by establishing an in vitro model to provide a potential target for leukemia treatment.Methods:1.The expression of Cx43 and hypoxia maker HIF-1α in CML and normal control bone marrow biopsy specimens was detected by immunohistochemical staining.2.BMSCs from healthy donors were isolated according to our labs methods.The hypoxia model of BMSCs was established by CoCl2,and BMSCs were divided into hypoxia group and control group.Hypoxia group treated with CoCl2(100μmol/L)for 72 h,the control group treated with normal saline.The expression of Cx43 in hypoxia group and control group was detected by RT-qPCR and Western blot.3.The expression of hypoxia inducible factor-1α(HIF-1α)in BMSCs was downregulated by adenovirus Ad-shHIF-1α-GFP,and BMSCs were divided into Ad-shHIF-1α-GFP group,Ad-GFP group and blank control group.BMSCs of Ad-shHIF-1α-GFP group and Ad-GFP group were treated with CoCl2(100μmol/L)for 72 h,the blank control group treated with normal saline.The expression of HIF-1α and Cx43 in BMSCs was detected by Western blot.4.The expression of Cx43 in BMSCs was down-regulated by adenovirus Ad-shCx43-GFP(BMSCs-shCx43),and Ad-GFP was used as a control(BMSCs-NC).The expression of Cx43 in BMSCs was detected by Western blot.5.A co-culture model of K562 cells was established in vitro,cells were divided into K562 group,K562+IM group,K562+BMSCs-NC+IM group and K562+BMSCs-shCx43+IM group.The apoptosis and cell cycle of co-cultured K562 cells were detected by flow cytometry.6.A co-culture model of K562 cells was established in vitro,cells were divided into K562 group,K562+IM group,K562+BMSCs-NC+IM group and K562+BMSCs-shCx43+IM group.The expression of Akt,Erk1/2,Stat5,P-Crkl and β-catenin in co-cultured K562 cells was detected by Western blot.7.Co-cultured K562 cells were treated with Akt inhibitor MK-2206,and the apoptosis of co-cultured K562 cells was detected by flow cytometry.Results:1.The expression of Cx43 in some CML bone marrow biopsy specimens was lower than that in normal controls,and the expression of HIF-1α in Cx43 decreased CML bone marrow biopsy specimens was higher than that in normal controls.2.In the hypoxia model of BMSCs,the mRNA of Cx43 in hypoxia group was significantly lower than that in control group(p<0.01),which was about 1/2 of that in control group.The expression of HIF-1α protein in hypoxia group was significantly higher than that in control group,while the expression of Cx43 protein in hypoxia group was also significantly reduced.3.In the hypoxia model of BMSCs,the expression of HIF-1α protein in Ad-shHIF1α-GFP group was significantly lower than that in Ad-GFP group,while the expression of Cx43 protein in Ad-shHIF1α-GFP group was significantly increased close to that in blank control group.4.The expression of Cx43 protein in Ad-shCx43-GFP group was significantly lower than that in Ad-GFP group.5.The apoptosis ratio of K562 cells in K562 group,K562+IM group,K562+BMSCsNC+IM group and K562+BMSCs-shCx43+IM group was 8.20%±1.39%,36.71%±1.61%,24.43%±2.88% and 18.19%±3.49%,respectively.The apoptosis of K562 cells in K562+ BMSCs-NC+IM group was significantly lower than that in K562+IM group(p<0.01),and the apoptosis of K562 cells in K562+BMSCs-shCx43+IM group was significantly lower than that in K562+BMSCs-NC+IM group(p<0.05).6.K562+BMSCs-NC+IM group increased the percentage of cells in G0 / G1 phase from 51% to 61%(p<0.01)and decreased the percentage of cells in S phase from 42% to 26%(p<0.01)compared with K562 + IM culture group.K562+BMSCs-shCx43+IM group increased the percentage of cells in G0 / G1 phase from 61% to 72%(p<0.01)compared with K562+BMSCs-NC+IM group,and the percentage of cells in S phase was not statistically significant.7.High levels of P-Akt,P-Erk1/2,P-Stat5 and P-Crkl was detected in K562 cells.After imatinib treatment for 48 h,the expression of P-Erk1/2,P-Stat5 and P-Crkl in K562 cells was decreased in the presence and absence of BMSCs.However,the expression of P-Akt in K562+BMSCs-NC+IM group was significantly higher than that in K562+IM group,and the expression of P-Akt in K562+BMSCs-shCx43+IM group was further increased compared with K562+BMSCs-NC+IM group.The expression of β-catenin in K562+BMSCs-shCx43+ IM group was higher than that in K562+BMSCs-NC+IM group.8.The Akt inhibitor MK-2206 can increase imatinib-induced apoptosis of K562 cells cocultured with BMSCs or BMSCs-shCx43.Conclusions:1.Cx43 was negatively correlated with hypoxia marker HIF-1α in CML patients.In the hypoxia model,hypoxia can induce down-regulation of Cx43 in BMSCs from healthy donors through HIF-1α pathway.2.Down-regulation of Cx43 in BMSCs can reduce imatinib-induced apoptosis in co-cultured K562 cells and increase the proportion of co-cultured K562 cells in G0 / G1 phase.3.Down-regulation of Cx43 in BMSCs can reduce the apoptosis of imatinib-treated K562 cells by activation of BCR-ABL independent Akt signaling pathway in K562 cells to mediate chemoresistance. |
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