Over-expression of Mir-29a inhibits growth of MDA-MB-453 cells To further study whether Mir-29a negatively regulates cancer cell growth, Mir-29a was over-expressed in MDA-MB-453 cells. As shown in Figure 3A, Mir-29a expression level was 5.6-fold higher GS-1101 cell line in cells transduced with Mir-29a over-expression construct than vector control. MDA-MB-453 cells over-expressed with Mir-29a displayed significantly slower growth rate than control cells (Figure 3B). To further determine if slower cell growth rate was due to perturbation of cell cycles progression, cell cycle profile was investigated by monitoring cell numbers at different stages (Figure 3C-E). Interestingly, compared to vector control, over-expression

of Mir-29a caused 15% (P < 0.01) more cells

to stay at G0/G1 phase (Figure 3E). This data suggested that over-expression of Mir-29 resulted in the arrest of cell cycle in G0/G1 phase LY333531 supplier and prevention of cells from entering into the S phase. Figure 3 Over-expression of miR-29a in MDA-MB-453 cells inhibits growth of cells. A, relative levels of mir-29a in cells with or without mir-29a over-expression, n = 5, Mean ± SD. B, the growth curve of above cells, n = 5, Mean ± SD. C and D, representative figures of cell cycle analysis using Guava assay. E, quantitative analysis of the results of cell cycle examination, n = 5, Mean ± SD. Mir-29a knockdown facilitates growth of MCF-10A cells To confirm the inhibitory role of Mir-29a, cell growth and cell cycle profile were investigated in MCF-10A cells with Mir-29a knockdown. Suppression Sodium butyrate of Mir-29a resulted in a higher cell growth rate than empty vector control (Figure 4A and 4B). In MCF-10A cells with knockdown of Mir-29a, the percentage of cells at G0/G1 phase was 12% (P < 0.01) lower than that in control cells (Figure 4C-E).

This data suggested that knockdown of Mir-29a in normal cells caused more cells entering to S phase and thus promote cell growth. These results, together with data of over-expression of Mir29a in breast cancer cells, strongly suggested Mir-29a participates in arresting cells at G0/G1 phase and thus inhibiting tumor cell growth. Figure 4 Knockdown of miR-29a in MCF-10A cells increases growth of cells. A, relative levels of mir-29a in cells with or without mir-29a knockdown, n = 5, Mean ± SD. B, the growth curve of above cells, n = 5, Mean ± SD. C and D, representative figures of cell cycle analysis using Guava assay. E, quantitative analysis of the results of cell cycle examination, n = 5, Mean ± SD. Mir-29a negatively regulates cell growth through its depression on B-Myb expression The next question is how Mir-29a inhibits growth of cells. To further investigate this question, we searched the literature and found Mir-29a might inhibit growth of cells by down-regulating the transcription factor, B-Myb [22]. To evaluate the direct effect of mir-29a on B-Myb expression, we used pMIR-REPORT System.