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. 2016 Mar 22;7(12):13742-53.
doi: 10.18632/oncotarget.7328.

BAP1 Suppresses Lung Cancer Progression and Is Inhibited by miR-31

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Free PMC article

BAP1 Suppresses Lung Cancer Progression and Is Inhibited by miR-31

Mengchao Yu et al. Oncotarget. .
Free PMC article

Abstract

BRCA1-associated protein-1 (BAP1) is an important nuclear-localized deubiquitinating enzyme that serves as a tumor suppressor in lung cancer; however, its function and its regulation are largely unknown. In this study, we found that BAP1 protein levels were dramatically diminished in lung cancer tissues while its mRNA levels did not differ significantly, suggesting that a post-transcriptional mechanism was involved in BAP1 regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that could potentially bind BAP1. We predicted and experimentally validated miR-31 as a direct regulator of BAP1. Moreover, we showed that miR-31 promoted proliferation and suppressed apoptosis in lung cancer cells and accelerated the development of tumor growth in xenograft mice by inhibiting BAP1. Taken together, this study highlights an important role for miR-31 in the suppression of BAP1 in lung cancer cells and may provide insights into the molecular mechanisms of lung carcinogenesis.

Keywords: BAP1; apoptosis; lung cancer; miR-31; proliferation.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Expression patterns of BAP1 in human lung cancer tissues
A. and B. Western blotting analysis of the expression levels of the BAP1 protein in 12 pairs of lung cancer tissue (LC) and lung noncancerous tissue (LN) samples. A. representative image; B. quantitative analysis (BAP1 protein vs. GAPDH protein). C. Quantitative RT-PCR analysis of the expression levels (BAP1 mRNA vs. GAPDH mRNA) of BAP1 mRNA in the same 12 pairs of LC and LN samples. (*** p < 0.005).
Figure 2
Figure 2. Prediction of the miR-31 binding site within the BAP1 3′-UTR
A. Schematic description of the hypothetical duplexes formed by the interactions between the binding site in the BAP1 3′-UTR (top) and miR-31 (bottom). The seed regions of miR-31 and the seed-recognition sites in the BAP1 3′-UTR are indicated in red. All nucleotides in the seed-recognition sites are completely conserved in several species. The predicted free energy values of each hybrid are indicated. B. Quantitative RT-PCR analysis of the expression levels (miR-31 vs. U6) of miR-31 in the same 12 pairs of LC and LN samples. (*** p < 0.005).
Figure 3
Figure 3. BAP1 is a direct target of miR-31
A. Quantitative RT-PCR analysis of the expression levels of miR-31 in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic (pre-miR-31), miR-31 inhibitor (anti-miR-31) or scrambled negative control RNA (pre-miR-control or anti-miR-control). B. and C. Western blotting analysis to detect BAP1 protein levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. B: representative image; C. quantitative analysis. D. Quantitative RT-PCR analysis of BAP1 mRNA levels in A549, H1975 and HCC827 cells transfected with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. E. Direct recognition of the BAP1 3′-UTR by miR-31. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-31 binding sites in the BAP1 3′-UTR were co-transfected into A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. Firefly luciferase values were normalized to β-galactosidase activity, and the results were calculated as the ratio of firefly luciferase activity in the miR-31-transfected cells normalized to the negative control RNA-transfected cells. The results are presented as the mean ± S.D. of three independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.005).
Figure 4
Figure 4. Effect of miR-31 and BAP1 on the proliferation and apoptosis of lung cancer cells
A. A cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 mimic or scrambled negative control RNA. B. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the miR-31 inhibitor or scrambled negative control RNA. C. The cell proliferation assay was performed 12, 24, 36 and 48 hours after the transfection of A549 cells with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. and E. The apoptosis assay was performed 24 hours after the transfection of A549 cells with equal doses of the miR-31 mimic, miR-31 inhibitor or scrambled negative control RNA or with equal doses of the pre-miR-control plus control plasmid, pre-miR-control plus BAP1 overexpression plasmid, miR-31 mimic plus control plasmid, or miR-31 mimic plus BAP1 overexpression plasmid. D. representative image; E. quantitative analysis. The results of cell proliferation are presented as the mean ± S.E. of three independent experience, and other results are presented as the mean ± S.D. of three independent experiments. (* p < 0.05; *** p < 0.005).
Figure 5
Figure 5. Effects of miR-31 and BAP1 on the growth of lung cancer cell xenografts in mice
A. Flow chart of the experimental plan. A549 cells were infected with a control lentivirus or a lentivirus to overexpress miR-31, transfected with a BAP1 overexpression plasmid, or co-transfected with a lentivirus to overexpress miR-31 and a BAP1 overexpression plasmid. A549 cells (2 × 106 cells per 0.1 mL) with different treatments were implanted subcutaneously into 6-week-old SCID mice (5 mice per group), and the tumor growth was evaluated on day 35 after cell implantation. B. Representative images of the tumors from the implanted mice. C. Quantitative analysis of the tumor weights. D. Quantitative RT-PCR analysis of miR-31 levels in the tumors from implanted mice. E. Quantitative RT-PCR analysis of BAP1 mRNA levels in the tumors from implanted mice. F. and G. Western blotting analysis of BAP1 protein levels in the tumors from implanted mice. F. representative image; G. quantitative analysis. H.-J. H&E-stained sections and immunohistochemical staining for Ki-67 and BAP1 in the tumors from implanted mice. H. representative image; I. and J. quantitative analysis. (* p < 0.05; ** p < 0.01; *** p < 0.005).

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References

    1. Mangia A, Partipilo G, Schirosi L, Saponaro C, Galetta D, Catino A, Scattone A, Simone G. Fine Needle Aspiration Cytology: A Tool to Study NHERF1 Expression as a Potential Marker of Aggressiveness in Lung Cancer. Molecular biotechnology. 2015;57:549–57. - PubMed
    1. National Lung Screening Trial Research T. Aberle DR, Berg CD, Black WC, Church TR, Fagerstrom RM, Galen B, Gareen IF, Gatsonis C, Goldin J, Gohagan JK, Hillman B, Jaffe C, Kramer BS, Lynch D, Marcus PM, et al. The National Lung Screening Trial: overview and study design. Radiology. 2011;258:243–253. - PMC - PubMed
    1. Jensen DE, Rauscher FJ., 3rd Defining biochemical functions for the BRCA1 tumor suppressor protein: analysis of the BRCA1 binding protein BAP1. Cancer letters. 1999;143(Suppl 1):S13–17. - PubMed
    1. Ventii KH, Devi NS, Friedrich KL, Chernova TA, Tighiouart M, Van Meir EG, Wilkinson KD. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer research. 2008;68:6953–6962. - PMC - PubMed
    1. Machida YJ, Machida Y, Vashisht AA, Wohlschlegel JA, Dutta A. The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1. The Journal of biological chemistry. 2009;284:34179–34188. - PMC - PubMed

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