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MiR-27a modulates radiosensitivity of triple-negative breast cancer (TNBC) cells by targeting CDC27.

Ren YQ, Fu F, Han J - Med. Sci. Monit. (2015)

Bottom Line: CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.The miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells.Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, The Central Hospital of Yishui, Linyi, Shandong, China (mainland).

ABSTRACT

Background: MiR-27a is significantly overexpressed in triple-negative breast cancer (TNBC). However, the exact biological function of MiR-27a in TNBC is not fully understood. In this study, we verified miR-27a expression in TNBC cells and explored how its overexpression modulates radiosensitivity of the cells.

Material/methods: qRT-PCR analysis was performed to study miR-27a expression in TNBC lines MDA-MB-435 and MDA-MB-231 and in normal human breast epithelial cell line MCF10A. Dual luciferase assay was performed to verify a putative downstream target of miR-27a, CDC27. CCK-8 assay was used to assess the influence of miR-27a-CDC27 axis on cell proliferation under irradiation (IR) treatment.

Results: We confirmed significantly higher miR-27a expression in 2 TNBC cell lines--MDA-MB-435 and MDA-MB-231--than in human breast epithelial cell line MCF10A. miR-27a could modulate proliferation and radiosensitivity of TNBC cells. CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.

Conclusions: The miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells. Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.

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MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. (A) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. (B, C) The relative firefly luciferase activity in HEK-293T (B) and MDA-MB-435 (C) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. (D, E) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 (D) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles (E). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
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f3-medscimonit-21-1297: MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. (A) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. (B, C) The relative firefly luciferase activity in HEK-293T (B) and MDA-MB-435 (C) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. (D, E) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 (D) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles (E). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

Mentions: Although the regulative role of miR-27a in TNBC cells has been verified, its downstream targets are still not clear. Through searching and comparison in online bioinformatics databases, we found CDC27 has a highly conserved 3′UTR sequence with miR-27a in mammals (Figure 3A). Then, we verified this binding using dual-luciferase assay in both HEK-293T and MDA-MB-435 cells. In both of these 2 cell lines, miR-27a mimics could only inhibit luciferase activity of reporters carrying the wide-type binding sites, but had no effect on the mutant reporter (Figure 3B, 3C). MDA-MB-435 cells transfected with miR-27a mimics had significantly reduced CDC27 expression at protein level, while the cells transfected with antagomiR-27a had significantly enhanced CDC27 expression (Figure 3D, 3E). These results suggest that miR-27a can directly target CDC27 and regulate its expression in TNBC cells.


MiR-27a modulates radiosensitivity of triple-negative breast cancer (TNBC) cells by targeting CDC27.

Ren YQ, Fu F, Han J - Med. Sci. Monit. (2015)

MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. (A) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. (B, C) The relative firefly luciferase activity in HEK-293T (B) and MDA-MB-435 (C) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. (D, E) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 (D) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles (E). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4548742&req=5

f3-medscimonit-21-1297: MiR-27a directly targets CDC27 and regulates its expression in TNBC cells. (A) The putative binding site between miR-27a and CDC27 and the designed mutant sequence without the pairing. (B, C) The relative firefly luciferase activity in HEK-293T (B) and MDA-MB-435 (C) cells co-transfected with 150 ng reporter plasmids and 50 nM miR-27a mimics. Both firefly and Renilla luciferase activities were measured 24 h after transfection and the firefly luciferase activity was normalized to the Renilla luciferase activity. (D, E) Western blot analysis of CDC27 protein expression in MDA-MB-435 transfected with miR-27a mimics or siCDC-27 (D) or transfected with antagomiR-27a or infected with CDC-27 lentiviral particles (E). Data are shown as mean ±S.D. by 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
Mentions: Although the regulative role of miR-27a in TNBC cells has been verified, its downstream targets are still not clear. Through searching and comparison in online bioinformatics databases, we found CDC27 has a highly conserved 3′UTR sequence with miR-27a in mammals (Figure 3A). Then, we verified this binding using dual-luciferase assay in both HEK-293T and MDA-MB-435 cells. In both of these 2 cell lines, miR-27a mimics could only inhibit luciferase activity of reporters carrying the wide-type binding sites, but had no effect on the mutant reporter (Figure 3B, 3C). MDA-MB-435 cells transfected with miR-27a mimics had significantly reduced CDC27 expression at protein level, while the cells transfected with antagomiR-27a had significantly enhanced CDC27 expression (Figure 3D, 3E). These results suggest that miR-27a can directly target CDC27 and regulate its expression in TNBC cells.

Bottom Line: CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.The miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells.Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, The Central Hospital of Yishui, Linyi, Shandong, China (mainland).

ABSTRACT

Background: MiR-27a is significantly overexpressed in triple-negative breast cancer (TNBC). However, the exact biological function of MiR-27a in TNBC is not fully understood. In this study, we verified miR-27a expression in TNBC cells and explored how its overexpression modulates radiosensitivity of the cells.

Material/methods: qRT-PCR analysis was performed to study miR-27a expression in TNBC lines MDA-MB-435 and MDA-MB-231 and in normal human breast epithelial cell line MCF10A. Dual luciferase assay was performed to verify a putative downstream target of miR-27a, CDC27. CCK-8 assay was used to assess the influence of miR-27a-CDC27 axis on cell proliferation under irradiation (IR) treatment.

Results: We confirmed significantly higher miR-27a expression in 2 TNBC cell lines--MDA-MB-435 and MDA-MB-231--than in human breast epithelial cell line MCF10A. miR-27a could modulate proliferation and radiosensitivity of TNBC cells. CDC-27 is a direct target of miR-27a and its downregulation conferred increased radioresistance of the cells.

Conclusions: The miR-27a-CDC27 axis might play an important role in modulating response to radiotherapy in TNBC cells. Testing miR-27a expression might be a useful way to identify a subgroup of patients who will benefit from an IR-based therapeutic approach.

Show MeSH
Related in: MedlinePlus