The Expression And Significance Of Transcription Factor Nanog In Triple-negative Breast Cancer Stem Cells

Triple negative breast cancer,which refers to the immunohistochemical ER negative,PR negative and HER-2 negative special pathological subtype,occurs in premenopausal young women.This cancer has high recurrence risk,prone to visceral metastasis,poor response to drug treatment and prognosis dangerous etc.The expression of transcription factor Nanog is found in tumor tissue and this factor is related to tumor recurrence,metastasis and drug resistance.Therefore,in triple negative breast cancer stem cells,investigation of expression and regulation of Nanog will provide a theoretical and experimental basis for the triple negative breast cancer treatment.In this study,breast cancer stem cells were isolated and enriched from ER-,PR-,HER2-triple negative breast cancer cell line-MDA-MB-231 by serum-free suspension culture.The characteristics of the breast cancer stem cells were measured by flow cytometry.Meanwhile,the stem cell characteristics of the cells were evaluated by paclitaxel inhibition test and subcutaneous tumorigenesis experiment in NOD/SCID mouse.The expression level of Nanog is down regulated in MDA-MB-231 breast cancer stem cells and MDA-MB-231 stem cells after infected with lentivirus encoding sequences of Nanog siRNA.The regulation of Nanog expression at molecular and protein level was confirmed by Real-time PCR,RT-PCR and Western blot.The effect of MDA-MB-231 breast cancer stem cell proliferation,invasion and resistance to chemotherapeutic agents after knock down of Nanog expression was studied in NOD/SCID mice.The above results indicated that the small population of MDA-MB-231 breast cancer stem cells could be grown in serum-free cell culture medium and was existed in the form of suspension micro-balls.The proportion of CD44 +/CD24-/low cell subsets(76.6%)in MDA-MB-231 breast cancer stem cellswas much lower than that of MDA-MB-231 breast cancer cells(92.7%)(P<0.05).100%The paclitaxel could inhibit proliferation of MDA-MB-231 in a concentration dependent manner.The median inhibitory concentration(IC50)of paclitaxel to MDA-MB-231 cells was(4.25± 0.22)ug/ml,which was much lower than that to MDA-MB-231 cells(8.13 ±0.34)ug/ml(t = 9.549,P = 0.0004<0.05).All mice(10/10)inoculated with 0.2×106 breast cancer stem cells formed tumors.However,when the mice inoculated the same number of cells with MDA-MB-231 breast cancer cells only 60%(6/10)of the mice formed tumors.The mean volume(1040 ± 52.65)mm3 of tumors in the MDA-MB-231 stem cell group was much larger than that in the MDA-MB-231 cell group(558.4 ± 32.72)mm3(t=7.763,P<0.05)after 8 weeks.The real-time PCR,RT-PCR and Western Blot results showed that the expression level of Nanog in MDA-MB-231 stem cells was higher than that in MDA-MB-231 cells(P<0.05).The expression of Nanog in MDA-MB-231 stem cells could be down-regulated by small RNA interference fragment.After inhibition of Nanog expression,the rate of tumor formation was 80%in Nanog siRNA group.Its mean tumor weight(0.73 ± 0.04g)and volume were lower than that in control and negative siRNA groups.Nanog expression was lower than that of Control group and Negative siRNA group(P<0.05).In nanog siRNA group paclitaxel IC50(5.27 ± 0.22)ug/ml was less than that in control and negative siRNA groups.After treated by 1ml of paclitaxel(16?g/ml),the tumor volume and tumor weight in the Nanog siRNA group were(732.6 ± 19.23)mm3 and(0.752 ± 0.053)g,respectively.They were smaller than those in the control group and the Negative siRNA(P<0.05).The tumor inhibition rate of Nanog siRNA group was 58%.Compared to control group and Negative siRNA group,the tumor necrosis rate of Nanog siRNA group was high,but no lung metastasis and abdominal metastasis were observed.HE staining of tumors showed that less tumors formed in Nanog siRNA group.Compared to control group and Negative siRNA group,the histone expression level of Nanog siRNA was significantly decreased(P<0.05).MDA-MB-231 breast cancer stem cells were successfully obtained by serum-free suspension culture.Nanog was associated with tumorigenicity and chemotherapeutic antagonism of MDA-MB-231 breast cancer stem cells.Nanog could be a gene therapy target for breast cancer.The down-regulation of Nanog can inhibit the growth of MDA-MB-231 breast cancer stem cells in nude mice and reduce the resistance to chemotherapy drug paclitaxel,which could provide a new approach for breast cancer therapy.