| Background: Breast cancer is one of most common malignant tumors occurringin women. There are over1.38million new breast cancer patients around the world,accounting for about30%of the new female malignant tumors. It has beendemonstrated recently that the incidence rates of breast cancer increased in women agedless than40years, thereby threatening the lives of women. Breast cancer is a systemicdisease. The mainstay of breast cancer treatment is surgery when the tumor is localized,and chemotherapy and radiotherapy or other adjuvant hormonal therapy are theimportant methods for breast invasion tumors. The significant obstacle in breast cancertreatment is chemotherapy or radio-therapy resistance, whiledistant metastasis and tumor recurrence are the main reasons for the failure inmalignant tumor treatments. It has been thought that cancer stem cells are the sources ofmalignant tumor recurrence andmetastasis, and microenvironment plays an importantrole in regulating stem cell self-renewal, mμlti-directional differentiation, drugresistance etc. To further study the mechanism of its regulation could provide atheoretical basis for developing new therapeutic strategies targeting cancer stem cellsand reducing tumor recurrence. Recently, interleukin8(IL-8), a CXC chemokine,attracted our attention on tumorigenesis and development. Objective: To understand the expression of IL8and its receptor CXCR1in breastcancer stem cells (CD44+/CD24-); and further to investigate the effects ofoverexpression of exogenous IL8on breast cancer stem cells and its mechanism.Methods: First of all, sorting breast cancer CD44+/CD24-cells from MCF-7by usingmagnetic beads, and then detecting the expressions of IL-8and its receptor CXCR1byqRT-PCR, ELISA and Western Blot assays. Then constructing recombinant plasmidpcDNA3.1/myc-HisC-IL-8. After transfecting into MCF-7cells, the expressios of IL8and CXCR1were detected by qRT-PCR, ELISA and Western Blot. The effect of IL-8onproliferation, migration and invasion of MCF-7cells was evaluated by using CCK-8assay and transwell assay. The effect of IL-8on mammosphere formation capacity,percentage of CD44+/CD24-in MCF-7, the expression of ALDH1and BMI1weremeasured through mammosphere formation assay, FCM and Western Blot. Afterinocμlating MCF-7cells with overexpression IL8into nude mice, tumor growth wasobserved. And21days after inocμlating, blood and tumor tissues were obtained. BloodIL8was measured by ELISA; while ALDH1was detected by IHC and CXCR1, ALDH1and BMI1were demonstrated by Western Blot.Results:1. The expressions of IL-8mRNA are increased in both CD44+/CD24-cells andnon-CD44+/CD24-cells; while the level of IL-8protein in CD44+/CD24-cells is higherthan that in non-CD44+/CD24-cells. The expression of CXCR1is increased in bothmRNAand protein levels in CD44+/CD24-cells.2. We successfully constructed the recombinant plasmid pcDNA3.1/myc-HisC-IL-8by using molecμlar cloning techniques; after transfecting into MCF-7cells, theexpressions of IL8and CXCR1were increased by qRT-PCR, ELISA and Western Blotassays.3. In migration assay, the number of MCF-7cells that migrated to the lower surface ofthe filter membrane was255.3±22.72in IL-8transfection group, higher than that in non-IL8transfection and non-transfection groups105.3±7.1and126.7±11.6; Ininvasion experiment, the number of MCF-7cells that migrated through matrigel to thelower surface of the filter membrane was247.3±17.6in IL8transfection group, higherthan that in non-IL8transfection and non-transfection groups76.7±3.1and86.3±7.3.However, there was no obvious difference in the proliferation between MCF-7cellswith or without IL8overexpression.4. The size and the number of mammosphere were more increased in MCF7cells withIL8transfection than that with non-IL8transfection and non-transfection. Thepercentage of CD44+CD24-in MCF7cells were16.39%±0.56%, significantly higherin MCF7cells with IL8transfection than that5.28%±1.1%and6.39%±0.59%withnon-IL8transfection and non-transfection respectively. And the expressions of ALDH1and BMI1protein were up-regulated in MCF7cells with IL8transfection than withnon-IL8transfection and non-transfection. The tumor growth was faster intumor-bearing nude mice with IL8transfection than in that with non-IL8transfection.Nude mice were killed in21days after tumor cell inoculation. The tumor weight in IL-8transfected group was2.07times than that non-IL8transfected group. The IL8level washigher in the peripheral blood of nude mice with IL8transfection than that in mice withnon-IL8transfection. Immunohistochemistry showed that the expression of ALDH1was higher in tumor tissue with IL8transfection than that with non-IL8transfection.The expressions of CXCR1, ALDH1and BMI1protein were more in tumor tissue withIL8transfection than that with non-IL8transfection.Conclusion:1. Comparing with non-CD44+/CD24-cells, breast cancer CD44+/CD24-cells coμldexpress more IL8and CXCR1.2. The expression of CXCR1was increased in MCF-7cells after overexpression ofexogenous IL8, accompanied by enhancing the capacity of cellular migration andinvasion. The ability of mammosphere formation, the percentage of CD44+/CD24-cells,and the expression of ALDH1and BMI1proteins were all increased after overexpression of exogenous IL8. What ‘s more, overexpression of exogenous IL8coμld significantly promote the growth of tumor-bearing mude mice. |
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