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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

miR-200c inhibits the self-renewal of BCSCs and MaSCs. a. In qRT-PCR assay, miR-200c agomir significantly upregulates miR-200c expression in both BCSCs and MaSCs (*, P < 0.01); miR-200c antagomir significantly downregulates miR-200c expression in both BCSCs and MaSCs (**, P < 0.01). b. In MaSCs, miR-200c agomir significantly decreases the colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases the colonies (P < 0.01, n = 5). c. In BCSCs, miR-200c agomir significantly decreases colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases colonies (P < 0.01, n = 5). d. No tumor was observed in the test group (miR-200c agomir) in 2 months after inoculation of 10 K cells. In the miR-control group and parental BCSC group, average tumor volumes are 137.4 ± 13.7 mm3 and 124.1 ± 18.6 mm3, respectively. e. A limiting dilution assay for tumorigenesis in vivo and TIC Frequency calculation. f. Surface markers (ESA+CD44+CD24-/low) of BCSCs were detected on day 10 after transfecting miR-agomir or miR-control
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Fig3: miR-200c inhibits the self-renewal of BCSCs and MaSCs. a. In qRT-PCR assay, miR-200c agomir significantly upregulates miR-200c expression in both BCSCs and MaSCs (*, P < 0.01); miR-200c antagomir significantly downregulates miR-200c expression in both BCSCs and MaSCs (**, P < 0.01). b. In MaSCs, miR-200c agomir significantly decreases the colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases the colonies (P < 0.01, n = 5). c. In BCSCs, miR-200c agomir significantly decreases colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases colonies (P < 0.01, n = 5). d. No tumor was observed in the test group (miR-200c agomir) in 2 months after inoculation of 10 K cells. In the miR-control group and parental BCSC group, average tumor volumes are 137.4 ± 13.7 mm3 and 124.1 ± 18.6 mm3, respectively. e. A limiting dilution assay for tumorigenesis in vivo and TIC Frequency calculation. f. Surface markers (ESA+CD44+CD24-/low) of BCSCs were detected on day 10 after transfecting miR-agomir or miR-control

Mentions: To test biological function of miR-200c, we introduced miR-200c agomir and miR-200c antagomir into both BCSCs and MaSCs, respectively. In qRT-PCR assay, miR-200c was expressed much higher after miR-200c agomir transfected into BCSCs and MaSCs than the control (P < 0.01) analyzed by Tamhane’s test or non-parametric statistics analysis (SPSS 18.0). miR-200c expression reached 281 and 408 times higher in BCSCs and MaSCs, respectively. And miR-200c antagomir significantly downregulated miR-200c expression in BCSCs and MaSCs than the control (P < 0.01). The fold changes were 0.40 and 0.52, respectively (Fig. 3a).Fig. 3


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)

miR-200c inhibits the self-renewal of BCSCs and MaSCs. a. In qRT-PCR assay, miR-200c agomir significantly upregulates miR-200c expression in both BCSCs and MaSCs (*, P < 0.01); miR-200c antagomir significantly downregulates miR-200c expression in both BCSCs and MaSCs (**, P < 0.01). b. In MaSCs, miR-200c agomir significantly decreases the colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases the colonies (P < 0.01, n = 5). c. In BCSCs, miR-200c agomir significantly decreases colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases colonies (P < 0.01, n = 5). d. No tumor was observed in the test group (miR-200c agomir) in 2 months after inoculation of 10 K cells. In the miR-control group and parental BCSC group, average tumor volumes are 137.4 ± 13.7 mm3 and 124.1 ± 18.6 mm3, respectively. e. A limiting dilution assay for tumorigenesis in vivo and TIC Frequency calculation. f. Surface markers (ESA+CD44+CD24-/low) of BCSCs were detected on day 10 after transfecting miR-agomir or miR-control
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Fig3: miR-200c inhibits the self-renewal of BCSCs and MaSCs. a. In qRT-PCR assay, miR-200c agomir significantly upregulates miR-200c expression in both BCSCs and MaSCs (*, P < 0.01); miR-200c antagomir significantly downregulates miR-200c expression in both BCSCs and MaSCs (**, P < 0.01). b. In MaSCs, miR-200c agomir significantly decreases the colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases the colonies (P < 0.01, n = 5). c. In BCSCs, miR-200c agomir significantly decreases colonies (P < 0.01, n = 5) while miR-200c antagomir significantly increases colonies (P < 0.01, n = 5). d. No tumor was observed in the test group (miR-200c agomir) in 2 months after inoculation of 10 K cells. In the miR-control group and parental BCSC group, average tumor volumes are 137.4 ± 13.7 mm3 and 124.1 ± 18.6 mm3, respectively. e. A limiting dilution assay for tumorigenesis in vivo and TIC Frequency calculation. f. Surface markers (ESA+CD44+CD24-/low) of BCSCs were detected on day 10 after transfecting miR-agomir or miR-control
Mentions: To test biological function of miR-200c, we introduced miR-200c agomir and miR-200c antagomir into both BCSCs and MaSCs, respectively. In qRT-PCR assay, miR-200c was expressed much higher after miR-200c agomir transfected into BCSCs and MaSCs than the control (P < 0.01) analyzed by Tamhane’s test or non-parametric statistics analysis (SPSS 18.0). miR-200c expression reached 281 and 408 times higher in BCSCs and MaSCs, respectively. And miR-200c antagomir significantly downregulated miR-200c expression in BCSCs and MaSCs than the control (P < 0.01). The fold changes were 0.40 and 0.52, respectively (Fig. 3a).Fig. 3

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