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. 2013 May 22;2(7):675–685. doi: 10.1242/bio.20134507

Fig. 4. hsa-miR29b regulates MDM2 expression in NSCLC cells.

Fig. 4.

(A) In silico identification of complimentary sites for hsa-miR29b on the 3′-UTR of MDM2, PTEN and CDK2. A549 or H157 cells were transfected either with empty vector or phsa-miR29b plasmid. After 24 h, total RNA was extracted, reverse transcribed, and real-time PCR analysis was carried out using hsa-miR29b specific primers (B) or MDM2 specific primers (forward: 5′-TTGACCTGTCTATAAGAGAATTATATATTTC-3′, reverse: 5′-GTCTTACGGGTAAATGGTGGCT-3′) (C). RNU6B and GAPDH were used as internal controls for normalization. Data represent mean ± SEM of three separate experiments performed in duplicates. **P<0.01; versus empty vector control, ##P<0.01; versus empty vector control. A549 or H157 cells were transfected either with empty vector or phsa-miR29b plasmid for 24 h. The lysates were later immunoblotted for MDM2 (D), PTEN (E) and Cdk2 (F) expression. Upper panel represents mean ± SEM of the densitometry values of the immunoreactive bands from three independent highly reproducible experiments, while representative blots are displayed in the lower panels. ##P<0.01; versus empty vector control. (G) A549 cells were transfected either with empty vector or phsa-miR29b plasmid. After 24 h, the lysates were assayed for p53 expression by using anti-p53 antibodies. Upper panel represents mean ± SEM of the densitometry values of the p53 immunoreactive band from three independent highly reproducible experiments, while representative blots were displayed in the lower panels. **P<0.01; versus empty vector control. (H) A549 cells were co-transfected either with empty vector or phsa-miRNA29b plasmid with p53-luciferase (Stratagene) reporter construct, with luciferase under the control of 14 repeats of p53-binding sequence (TGCCTGGACTTGCCTGG). After 24 h, the lysates were assayed for p53-dependent luciferase activities as described in Materials and Methods. Data represent mean ± SEM of normalized luciferase activities obtained from three independent experiments. **P<0.01; versus empty vector control. (I) Beas2B cells were transfected either with negative control or miR29b precursors together with p53-luciferase reporter plasmid. After 24 h, the cells were treated either with or without PPARγ inhibitor (T0070907, 10 µM) as described in Materials and Methods. The cell lysates were later assayed for luciferase reporter expression as described in Materials and Methods. Data represent mean ± SEM of normalized luciferase activities obtained from three independent and highly reproducible experiments. #P<0.05; ##P<0.01; versus empty vector control.