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. Author manuscript; available in PMC: 2014 Sep 19.
Published in final edited form as: Immunity. 2013 Sep 5;39(3):10.1016/j.immuni.2013.08.025. doi: 10.1016/j.immuni.2013.08.025

Figure 6. MicroRNA101 targets CtBP2 and controls cancer stemness.

Figure 6

(A) Target of microRNA101 in 3′UTR of CtBP2. The sequence of wild type (WT) and mutant 3′UTR of CtPB2 gene used in luciferase assay was shown.

(B) Effect of microRNA101 on WT-3′UTR-CtBP2 luciferase activity. 3′UTR-CtBP2 luciferase activity was measured in tumor cells transfected with WT-3′UTR and mutant. Results are expressed as the percentage of inhibition of 3′UTR-CtBP2 luciferase activity. 3 experiments with triplicates are shown. Results are expressed as the mean percentage of inhibition ± SEM. *, P<0.01 as compared to mutant-3′UTR- CtBP2.

(C) Effect of microRNA101 on CtBP2 protein expression. Primary ovarian cancer cells were transfected with lentiviral vector encoding microRNA101 and control. Cancer CtBP2 protein was detected by Western blotting. One of 3 experiments is shown.

(D) Effect of CtBP2 on stem cell core protein expression in primary ovarian cancer cells. Primary ovarian cancer cells were transfected with lentiviral vector encoding shCtBP2 and control. Expression of CtBP2, Nanog, Sox2 protein was detected by Western blotting. A, B are two shCtBP2. One of 3 experiments is shown.

(E) Effect of CtBP2 on ovarian cancer sphere formation. Primary ovarian cancer cells were transfected with lentiviral vector encoding shCtBP2. Sphere assay was performed in 3 experiments with triplicates. Results are expressed as the fold increase (mean + SEM). *, P<0.01 as compared to control.

(F) Effect of knock down of CtBP2 expression on tumor incidence. Primary ovarian cancer cells were transfected with lentiviral vector encoding shCtBP2. shCtBP2 cancer cells were injected into NSG mice. Tumor incidence was monitored. Results are expressed as the percentage of tumor development. n = 6 in Control group; n = 5 in sh-CtBP2 group. P < 0.05.

(G, H) Effects of microRNA101 on the binding of CtBP2 to core stem cell gene promoters. ChIP assay was performed in primary ovarian cancer cells expressing microRNA101 (G), shCtBP2 (H) or scramble. One of three experiments is shown.

(I) Effect of MDSCs on CtBP2 protein expression in primary ovarian cancer cells. Primary ovarian cancer cells were cultured with MDSCs. CtBP2 protein expression was detected with Western blot. One of three experiments is shown.

See also Figure S6