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Fig. 5 | Breast Cancer Research

Fig. 5

From: LncRNA IPW inhibits growth of ductal carcinoma in situ by downregulating ID2 through miR-29c

Fig. 5

miR-29c suppresses self-renewal ability of DCIS by targeting ID2. A Figure showing the potential binding site of miR-29c on ID2 mRNA 3’UTR. The 12 base miR-29c binding region (colored in red) was deleted from the ID2 3’UTR. B DCIS.com cells were co-transfected with wild-type ID2 3’UTR luciferase reporter and miR-29c-expressing vector or empty vector, and luciferase activity was measured using luminometer. Similarly, DCIS.com cells were co-transfected with mutated ID2 3’UTR luciferase reporter (region of mutation are red colored in Figure A) and miR-29c-expressing vector or empty vector, and luciferase activity was quantified using luminometer (n = 4/group). Statistical inference between the groups was determined by ANOVA with Tukey multiple comparison post hoc test. C DCIS.com cells transduced with either empty or miR-29c-expressing vector and cell lysates were prepared. ID2 protein expression was examined in the lysates by western blot and imaged by Amersham imager. D ID2 expression was evaluated in 10A(control) and 10A shIPW cells by qRT-PCR (lower panel) and western blot (upper panel). For qRT-PCR (n = 5/group), statistical inference was calculated using unpaired two-tailed Student’s t test and represented as mean + SEM. E Effect of IPW on ID2 mRNA expression was examined by qRT-PCR in DCIS.com cells transduced with pSIN-IPW or empty vector followed by RNA isolation. Actin was used as internal control (n = 5/group, statistical inference was made using unpaired two-tailed Student’s t-test and represented as mean + SEM). F–H SOX2 (F), OCT4 (G) and NANOG (H) mRNA expression was examined in DCIS.com (control), DCIS-IPW and DCIS-IPW cells ectopically expressed with ID2 (n = 3/group) by qRT-PCR actin used as internal control. One-way ANOVA with Tukey multiple comparison post hoc test was performed for statistical comparison. I DCIS (control), DCIS-IPW and DCIS-IPW were transfected with miR-29c-locked nucleic acid (LNA). ID2 expression was evaluated by qRT-PCR (n = 4/group; lower panel) and western blot (upper panel). One-way ANOVA with Tukey multiple comparison post hoc test was performed for statistical comparison of qRT-PCR results. J DCIS.com (control), DCIS-IPW and DCIS-IPW cells ectopically expressed with ID2 were seeded on ultra-low attachment 96-well plate (1000 cells/well, n = 5/group), and number of spheres formed by these cells were counted at day 5. K Tumor-initiating stem cell population (CD24lowCD44highESAhigh) was examined in DCIS.com(control), DCIS-IPW and DCIS-IPW cells transduced with ID2-expressing vector by flow cytometry (n = 3/group). One-way ANOVA with Tukey multiple comparison post hoc test was performed for statistical comparison and data are represented as mean + S.E.M. (*p < 0.05, **p < 0.01, ***p < 0.001)

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