mir-29 regulates Mcl-1 protein expression and apoptosis - PubMed (original) (raw)

mir-29 regulates Mcl-1 protein expression and apoptosis

J L Mott et al. Oncogene. 2007.

Abstract

Cellular expression of Mcl-1, an anti-apoptotic Bcl-2 family member, is tightly regulated. Recently, Bcl-2 expression was shown to be regulated by microRNAs, small endogenous RNA molecules that regulate protein expression through sequence-specific interaction with messenger RNA. By analogy, we reasoned that Mcl-1 expression may also be regulated by microRNAs. We chose human immortalized, but non-malignant, H69 cholangiocyte and malignant KMCH cholangiocarcinoma cell lines for these studies, because Mcl-1 is dysregulated in cells with the malignant phenotype. By in silico analysis, we identified a putative target site in the Mcl-1 mRNA for the mir-29 family, and found that mir-29b was highly expressed in cholangiocytes. Interestingly, mir-29b was downregulated in malignant cells, consistent with Mcl-1 protein upregulation. Enforced mir-29b expression reduced Mcl-1 protein expression in KMCH cells. This effect was direct, as mir-29b negatively regulated the expression of an Mcl-1 3' untranslated region (UTR)-based reporter construct. Enforced mir-29b expression reduced Mcl-1 cellular protein levels and sensitized the cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Transfection of non-malignant cells (that express high levels of mir-29) with a locked-nucleic acid antagonist of mir-29b increased Mcl-1 levels and reduced TRAIL-mediated apoptosis. Thus mir-29 is an endogenous regulator of Mcl-1 protein expression, and thereby, apoptosis.

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Figures

Figure 1

Figure 1. Putative microRNA binding sites on Mcl-1 messenger RNA

Panel a: Schematic of the Mcl-1 3’UTR (#NM_021960) nucleotides 1184−4020. Five microRNAs were predicted to bind and the position of binding to the Mcl-1 transcript is shown in parentheses. Panel b: Northern blot of the five putative Mcl-1 binding microRNAs. RNA enriched for small RNA (<200nt's) from H69 cells was separated and probed for the indicated microRNAs. Note the robust expression of mir-29. Size markers are from SybrGreen-stained gels prior to transfer; 5.8S and 5S are endogenous rRNAs, 26 nt and 18 nt refer to synthetic oligodeoxynucleotides run in an adjacent lane.

Figure 2

Figure 2. Reciprocal expression of mir-29b and Mcl-1

Panel a: Total cellular protein from immortalized non-malignant H69 cells and malignant KMCH cells was probed using a Mcl-1 antibody. Actin was used as a loading control. Panel b: Northern blot for mir-29b using RNA enriched for small RNAs from H69 and KMCH cells. Note that both precursor and mature mir-29b are decreased in KMCH cells. As a loading control, an RNA probe against the small housekeeping RNA U6 (106 nt) was used. Panel c: Quantitative RT-PCR for mir-29b using total RNA from H69 and KMCH cells. Results expressed as copies of mir-29b per copy of Z30 RNA (mean +/− SEM; p < 0.05).

Figure 3

Figure 3. Mcl-1 protein level is negatively regulated by mir-29b

Panel a: Fluorescently-labeled pre-mir-29b was generated by in vitro transcription (Supplemental Methods) and transfected into KMCH cells. After 24 hours, cells were counter-stained with the nuclear stain DAPI, and photographed. For comparison, untransfected cells are shown. Panel b: Northern blot using small RNA from KMCH cells transfected with 20 or 50 nM precursor mir-29b (not fluorescently-tagged) compared to mock-transfected cells. The mature, processed form (23 nt) is shown, demonstrating that the transfected RNA is processed. U6 was probed as a loading control. Panel c: Mcl-1 immunoreactivity in KMCH cells transfected with precursor mir-29b. A Texas red-conjugated secondary antibody was used, and cells were photographed under confocal microscopy. Panel d: Western blots of total cellular protein from KMCH cells transfected with a mir-29b expression vector (p29b). Control cells were transfected with 1 μg of pCDNA, experimentals were transfected with 0.5 or 1 μg p29b. Actin was used as a loading control. Panel e: Northern blot on total RNA isolated from KMCH cells stably transfected with p29b. Two lines were generated, K-mir-29−6 and K-mir-29−19. The housekeeping RNA U6 was probed as a loading control. Panel f: Western blot of total protein from KMCH, K-mir-29−6, and K-mir-29−19 (stably overexpress mir-29b). Actin was used as a loading control.

Figure 4

Figure 4. Effect of the putative mir-29 binding site derived from the Mcl-1 3’UTR on luciferase expression

Panel a: Alignment of mir-29b with the insert derived from the Mcl-1 3’UTR. Note the complementarity at the 5’ end of mir-29b, where the crucial seed region is located. A single-base mutant insert was also synthesized, as shown. Inserts were cloned into the 3’ UTR of the p-Mir-Report vector. Panel b: Luciferase activity in HeLa cells transiently transfected with the luciferase construct alone, or cotransfected with an expression plasmid for mir-29b (p29b). Luciferase vectors were parental (pLuc), luciferase with the Mcl-1-derived 3’UTR insert (pLuc-Mcl-1 3’UTR), or luciferase with the mutated insert (pLuc-Mutant 3’UTR). Mean ± SEM, *p<0.01.

Figure 5

Figure 5. Sensitivity to apoptosis is increased after transfection of mir-29b

Panel a: KMCH cells were transfected with pre-mir-29b RNA or control RNA of the same length (antisense) at 50 nM. After 22 hours, TRAIL was added where indicated at 2 ng/mL in fresh media and the cells were incubated for 4 hours. Cells were then stained with DAPI and cells with apoptotic morphology were counted. Mean ± SEM, **p<0.001. Panel b: In parallel, cells were transfected and treated with TRAIL as in panel a, but after 4 hours caspase 3/7-like (DEVDase) activity was measured. Mean ± SEM, *p<0.01.

Figure 6

Figure 6. Antagonism of mir-29b protects cells from apoptosis

Panel a: Western blot of total protein isolated from non-malignant H69 cells and malignant KMCH cells transfected with control RNA, pre-mir-29b, or the mir-29 antagonist oligonucleotide, LNA (50nM each). Panel b. Apoptotic morphology measured as in Figure 5 on H69 and KMCH cells transfected as above. Note that H69 cells are sensitive to TRAIL killing, as noted in previous studies, while KMCH cells are relatively resistant unless mir-29b is transfected. Mean ± SEM, **p<0.0001 compared to vehicle treated control; # p<0.01 compared to TRAIL-treated mir-29; ## p<0.0001 compared to TRAIL-treated mir-29. Panel c: Caspase 3/7-like activity (DEVDase) on cells transfected as above. Caspase activity parallels the apoptosis counts using morphology. Mean ± SEM, *p<0.01 compared to vehicle treated control; **p<0.0001 compared to vehicle treated control; ## p<0.0001 compared to TRAIL-treated mir-29.

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