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Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.

Feng ZM, Qiu J, Chen XW, Liao RX, Liao XY, Zhang LP, Chen X, Li Y, Chen ZT, Sun JG - BMC Cancer (2015)

Bottom Line: The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05).A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs.Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 22346720@qq.com.

ABSTRACT

Background: Breast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs.

Methods: MUC1(-)ESA(+) cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments.

Results: In a 2-D clonogenic assay, MUC1(-)ESA(+) cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1(-)ESA(+) cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs.

Discussion: Accumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis.

Conclusions: PDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs.

No MeSH data available.


Related in: MedlinePlus

Microarray analysis of miRNA expression in MaSCs and qRT-PCR verification. a. Duplicate microarray analyses of miRNA expression in MaSCs. Triplicate dots on the microarray exhibit consistent signal intensity. b. Relative quantitative (RQ) expressions of 5 miRNAs in MCF-7/BCSCs/MaSCs shown as mean ± standard deviation (SD). (*, The expressions of miR-21, miR-296 and miR-200c are significantly higher in MCF-7 than in BCSCs. **, the expression of miR-373* is significantly higher in MaSCs than in BCSCs). c. Amplification curve of miR-200c in MCF-7/BCSCs/MaSCs. Comparison of miR-200c expressions tends to be MaSCs < BCSCs < MCF-7. d. Amplification curve of miR-21 in MCF-7/BCSCs/MaSCs. Comparison of miR-21 expressions tends to be MaSCs < BCSCs < MCF-7
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Fig2: Microarray analysis of miRNA expression in MaSCs and qRT-PCR verification. a. Duplicate microarray analyses of miRNA expression in MaSCs. Triplicate dots on the microarray exhibit consistent signal intensity. b. Relative quantitative (RQ) expressions of 5 miRNAs in MCF-7/BCSCs/MaSCs shown as mean ± standard deviation (SD). (*, The expressions of miR-21, miR-296 and miR-200c are significantly higher in MCF-7 than in BCSCs. **, the expression of miR-373* is significantly higher in MaSCs than in BCSCs). c. Amplification curve of miR-200c in MCF-7/BCSCs/MaSCs. Comparison of miR-200c expressions tends to be MaSCs < BCSCs < MCF-7. d. Amplification curve of miR-21 in MCF-7/BCSCs/MaSCs. Comparison of miR-21 expressions tends to be MaSCs < BCSCs < MCF-7

Mentions: The internal control U6 snRNA dots on all microarrays exhibited consistent signal intensity and the signal of all the detected dots in the replicate microarrays showed a high correlation efficiency (R = 0.9616 ± 0.0244), indicating the repetitiveness and reproducibility of the microarrays. Collectively, a profile of 72 mature miRNAs was detected (with signal value above 800) in MaSCs (Fig. 2a).Fig. 2


Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.

Feng ZM, Qiu J, Chen XW, Liao RX, Liao XY, Zhang LP, Chen X, Li Y, Chen ZT, Sun JG - BMC Cancer (2015)

Microarray analysis of miRNA expression in MaSCs and qRT-PCR verification. a. Duplicate microarray analyses of miRNA expression in MaSCs. Triplicate dots on the microarray exhibit consistent signal intensity. b. Relative quantitative (RQ) expressions of 5 miRNAs in MCF-7/BCSCs/MaSCs shown as mean ± standard deviation (SD). (*, The expressions of miR-21, miR-296 and miR-200c are significantly higher in MCF-7 than in BCSCs. **, the expression of miR-373* is significantly higher in MaSCs than in BCSCs). c. Amplification curve of miR-200c in MCF-7/BCSCs/MaSCs. Comparison of miR-200c expressions tends to be MaSCs < BCSCs < MCF-7. d. Amplification curve of miR-21 in MCF-7/BCSCs/MaSCs. Comparison of miR-21 expressions tends to be MaSCs < BCSCs < MCF-7
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581477&req=5

Fig2: Microarray analysis of miRNA expression in MaSCs and qRT-PCR verification. a. Duplicate microarray analyses of miRNA expression in MaSCs. Triplicate dots on the microarray exhibit consistent signal intensity. b. Relative quantitative (RQ) expressions of 5 miRNAs in MCF-7/BCSCs/MaSCs shown as mean ± standard deviation (SD). (*, The expressions of miR-21, miR-296 and miR-200c are significantly higher in MCF-7 than in BCSCs. **, the expression of miR-373* is significantly higher in MaSCs than in BCSCs). c. Amplification curve of miR-200c in MCF-7/BCSCs/MaSCs. Comparison of miR-200c expressions tends to be MaSCs < BCSCs < MCF-7. d. Amplification curve of miR-21 in MCF-7/BCSCs/MaSCs. Comparison of miR-21 expressions tends to be MaSCs < BCSCs < MCF-7
Mentions: The internal control U6 snRNA dots on all microarrays exhibited consistent signal intensity and the signal of all the detected dots in the replicate microarrays showed a high correlation efficiency (R = 0.9616 ± 0.0244), indicating the repetitiveness and reproducibility of the microarrays. Collectively, a profile of 72 mature miRNAs was detected (with signal value above 800) in MaSCs (Fig. 2a).Fig. 2

Bottom Line: The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05).A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs.Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 22346720@qq.com.

ABSTRACT

Background: Breast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs.

Methods: MUC1(-)ESA(+) cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments.

Results: In a 2-D clonogenic assay, MUC1(-)ESA(+) cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1(-)ESA(+) cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs.

Discussion: Accumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis.

Conclusions: PDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs.

No MeSH data available.


Related in: MedlinePlus