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. 2016 Oct 26;13:29.
doi: 10.1186/s12014-016-9128-7. eCollection 2016.

Proteomic Profiling of Retinoblastoma by High Resolution Mass Spectrometry

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Free PMC article

Proteomic Profiling of Retinoblastoma by High Resolution Mass Spectrometry

Ravikanth Danda et al. Clin Proteomics. .
Free PMC article

Abstract

Background: Retinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma.

Methods: We used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors.

Results: We identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies.

Conclusions: In the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma.

Figures

Fig. 1
Fig. 1
Schematic representation of work flow for the sample preparation and data analysis of total proteome
Fig. 2
Fig. 2
MS/MS spectra of the peptides with their reporter ions for the over expressed proteins in retinoblastoma. Relative intensities of reporter ions for a IGF2BP1 and b CHGA
Fig. 3
Fig. 3
MS/MS spectra of the peptides with their reporter ions for the over expressed proteins in retinoblastoma. Relative intensities of reporter ions for a AHSG and b MDK
Fig. 4
Fig. 4
a Sub-cellular classification of differentially regulated proteins in Retinoblastoma based on annotations from human proteome reference database. b Comparison of deregulated proteins involved in present study with previously reported transcriptome data of retinoblastoma. A total of 175 proteins showed positive correlation with the transcriptome
Fig. 5
Fig. 5
Immunohistochemistry of selected proteins IGF2BP1, RACGAP1, CHGA, MDK and ASHG in retinoblastoma primary tumors and non-neoplastic control retina
Fig. 6
Fig. 6
IGF2BP1 knockdown decreases Y79 RB cell proliferation and migration. a Comparison of mRNA expression of IGF2BP1 gene in siRNA untreated cells to treated cells. b Comparison of protein expression of IGF2BP1 in siRNA untreated to knockdown cells by western blot analysis. The top band shows IGF2BP1 expression and bottom band shows loading control, actin protein expression. c Comparison of percentage of cell viability in control and siRNA treated cells by MTT assay. d Comparison of migration of cells by wound healing assay in control (d1, d3) and siRNA treated cells (d2, d4). The top figure shows cell migration at 0 h and bottom shows figure cell migration at 48 h after the creation of the wound. *P < 0.05

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References

    1. De Falco G, Giordano A. pRb2/p130: a new candidate for retinoblastoma tumor formation. Oncogene. 2006;25:5333–5340. doi: 10.1038/sj.onc.1209614. - DOI - PubMed
    1. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323:643–646. doi: 10.1038/323643a0. - DOI - PubMed
    1. Comings DE. A general theory of carcinogenesis. Proc Natl Acad Sci USA. 1973;70:3324–3328. doi: 10.1073/pnas.70.12.3324. - DOI - PMC - PubMed
    1. Knudsen ES, Sexton CR, Mayhew CN. Role of the retinoblastoma tumor suppressor in the maintenance of genome integrity. Curr Mol Med. 2006;6:749–757. - PubMed
    1. Mallikarjuna K, Sundaram CS, Sharma Y, Deepa PR, Khetan V, Gopal L, et al. Comparative proteomic analysis of differentially expressed proteins in primary retinoblastoma tumors. Proteomics Clin Appl. 2010;4:449–463. doi: 10.1002/prca.200900069. - DOI - PubMed
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