[Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment].

OBJECTIVE: To explore the effects of silencing hypoxia inducible factor-2α (HIF-2α) by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment. METHODS: The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell line. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT-4 and KLF-4 protein in xenograft tumor tissues were detected by Western blot. RESULTS: The expression vector of HIF-2α-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group (P < 0.05). The expression of HIF-2α protein of interference group (0.42 ± 0.01) was much lower than that of the empty vector group (0.89 ± 0.03, P < 0.05), the expression of CD44 protein of interference group (0.21 ± 0.01) was much lower than the empty vector group (0.78 ± 0.03, P < 0.05), the expression of OCT-4 protein of interference group (0.42 ± 0.01)was much lower than the empty vector group (0.68 ± 0.03, P < 0.05) and the expression of KLF-4 protein of interference group (0.34 ± 0.01) was much lower than the empty vector group (0.72 ± 0.03, P < 0.05). CONCLUSION: Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stem cells. HIF-2α is expected to become a new target for gene therapy of breast cancer.

[Expression of breast cancer resistance protein and p-glycoprotein in residual breast cancer tissue after chemotherapy and its correlation with cancer stem cells].

OBJECTIVE: To compare the expression differences of breast cancer resistance protein(BCRP/ABCG2) and P-glycoprotein(P-gp) in breast cancer tissue before chemotherapy and in residual breast cancer tissue, and to explore its correlation with breast cancer stem cells. METHODS: Immunohistochemistry was used to detect the expression of ABCG2, P-gp, and breast cancer stem cells(BCSCs) markers(CD44 and CD24) in breast cancer tissue before chemotherapy and residual breast cancer tissue after chemotherapy. Immunofluorescence was applied for determination of the CD44 and CD24 protein expressions of BCSCs microspheres cells. The monoclone-forming ability of BCSCs microspheres cells was detected by limited dilution assay. The expressions of ABCG2, P-gp, CD44, and CD24 proteins were detected by Western blot. RESULTS: Compared with those in breast cancer tissue before chemotherapy, the expression levels of ABCG2 and P-gp were positively correlated with the expression level of CD44 protein(Χ(2)=41.34, r=0.83;Χ(2)=22.81, r=0.61) in residual breast cancer tissue after chemotherapy;meanwhile, they were negatively correlated with the expression of CD24 protein(Χ(2)=-21.25, r=0.72;Χ(2)=-17.26, r=0.65) (all P<0.05) .The diameter of BCSCs microspheres were increased significantly after chemotherapy.The content of BCSCs increased by about 2.5 times after chemotherapy.The expressions of ABCG2, P-gp and CD44 proteins significantly increased and that of CD24 protein significantly declined(P<0.05) . CONCLUSION: Chemotherapy endows residual breast cancer tissue with cancer stem cells-like features, leading to multidrug resistance of breast cancer.