A novel alternative splicing isoform of NF2 identified in human Schwann cells

Su,Fang; Zhou,Zhengguang; Su,Wen; Wang,Zishu; Wu,Qiong

doi:10.3892/ol.2016.4685

A novel alternative splicing isoform of NF2 identified in human Schwann cells

Authors:
- Fang Su
- Zhengguang Zhou
- Wen Su
- Zishu Wang
- Qiong Wu
View Affiliations

Affiliations: Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui 233004, P.R. China
Published online on: June 8, 2016 https://doi.org/10.3892/ol.2016.4685
Pages: 977-982

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Vestibular schwannoma (VS) is a benign, slow‑growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full‑length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full‑length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI‑193 cell line and cell proliferation assays were performed using Cell Counting Kit‑8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3'‑end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild‑type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein merlin, they are not merlin‑null, since they express the AS isoform. Although the AS merlin isoform has no suppressive effect on cell growth, certain mechanisms may exist that underlie this phenomenon, and this may be associated with the genesis and development of VS.

View Figures

View References

1	Mahanthappa NK, Anton ES and Matthew WD: Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth. J Neurosci. 16:4673–4683. 1996.PubMed/NCBI
2	Shepard TH, Tucci DL, Grant GA and Kaylie DM: Management of hearing in pediatric NF2. Otol Neurotol. 33:1066–1070. 2012.PubMed/NCBI
3	Diensthuber M, Lenarz T and Stöver T: Neurotrophic factor expression in vestibular schwannoma. An overview. Laryngorhinootologie. 85:731–737. 2006.(In German). View Article : Google Scholar : PubMed/NCBI
4	Hexter A, Jones A, Joe H, Heap L, Smith MJ, Wallace AJ, Halliday D, Parry A, Taylor A, Raymond L, et al: Clinical and molecular predictors of mortality in neurofibromatosis 2: A UK national analysis of 1192 patients. J Med Genet. 52:699–705. 2015. View Article : Google Scholar : PubMed/NCBI
5	Drouet A, Le Moigne F, Salamé D, Quesnel L, Motolese C, des Portes V, Guilloton L and Pinson S: Type 2 neurofibromatosis: Intergenerational differences in genetic and clinical expression. Arch Pediatr. 21:1233–1240. 2014.(In French). View Article : Google Scholar : PubMed/NCBI
6	Chen H, Zhang X, Zhang Z, Yang T, Wang Z and Wu H: The role of NF2 gene mutations and pathogenesis-related proteins in sporadic vestibular schwannomas in young individuals. Mol Cell Biochem. 392:145–152. 2014. View Article : Google Scholar : PubMed/NCBI
7	Kuga Y, Ohnishi H, Kodama Y, Takakura S, Hayashi M, Yagi R, Fukutome K, Matsushima K, Okamoto K, Taomoto K and Takahashi H: Cerebral and spinal cord tanycytic ependymomas in a young adult with a mutation in the NF2 gene. Neuropathology. 34:406–413. 2014.PubMed/NCBI
8	Harvey KF, Zhang X and Thomas DM: The Hippo pathway and human cancer. Nat Rev Cancer. 13:246–257. 2013. View Article : Google Scholar : PubMed/NCBI
9	Petrilli A, Copik A, Posadas M, Chang LS, Welling DB, Giovannini M and Fernández-Valle C: LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2. Oncogene. 33:3571–3582. 2014. View Article : Google Scholar : PubMed/NCBI
10	Li W, Cooper J, Karajannis MA and Giancotti FG: Merlin: A tumour suppressor with functions at the cell cortex and in the nucleus. EMBO Rep. 13:204–215. 2012. View Article : Google Scholar : PubMed/NCBI
11	Schmucker B, Tang Y and Kressel M: Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules. Hum Mol Genet. 8:1561–1570. 1999. View Article : Google Scholar : PubMed/NCBI
12	Rustgi AK, Xu L, Pinney D, Sterner C, Beauchamp R, Schmidt S, Gusella JF and Ramesh V: Neurofibromatosis 2 gene in human colorectal cancer. Cancer Genet Cytogenet. 84:24–26. 1995. View Article : Google Scholar : PubMed/NCBI
13	Lepont P, Stickney JT, Foster LA, Meng JJ, Hennigan RF and Ip W: Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity. Mutat Res. 637:142–1451. 2008. View Article : Google Scholar : PubMed/NCBI
14	Chang LS, Akhmametyeva EM, Wu Y, Zhu L and Welling DB: Multiple transcription initiation sites, alternative splicing and differential polyadenylation contribute to the complexity of human neurofibromatosis 2 transcripts. Genomics. 79:63–76. 2002. View Article : Google Scholar : PubMed/NCBI
15	Barker CL, Baillie BK, Hammond-Kosack KE, Jones JD and Jones DA: Dominant-negative interference with defence signalling by truncation mutations of the tomato Cf-9 disease resistance gene. Plant J. 46:385–399. 2006. View Article : Google Scholar : PubMed/NCBI
16	Doherty JK, Ongkeko W, Crawley B, Andalibi A and Ryan AF: ErbB and Nrg: Potential molecular targets for vestibular schwannoma pharmacotherapy. Otol Neurotol. 29:50–57. 2008. View Article : Google Scholar : PubMed/NCBI
17	Angelo LS, Maxwell DS, Wu JY, Sun D, Hawke DH, McCutcheon IE, Slopis JM, Peng Z, Bornmann WG and Kurzrock R: Binding partners for curcumin in human schwannoma cells: Biologic implications. Bioorg Med Chem. 21:932–939. 2013. View Article : Google Scholar : PubMed/NCBI
18	Prabhakar S, Taherian M, Gianni D, Conlon TJ, Fulci G, Brockmann J, Stemmer-Rachamimov A, Sena-Esteves M, Breakefield XO and Brenner GJ: Regression of schwannomas induced by adeno-associated virus-mediated delivery of caspase-1. Hum Gene Ther. 24:152–162. 2013. View Article : Google Scholar : PubMed/NCBI
19	Cockburn K, Biechele S, Garner J and Rossant J: The Hippo pathway member Nf2 is required for inner cell mass specification. Curr Biol. 23:1195–1201. 2013. View Article : Google Scholar : PubMed/NCBI
20	Cačev T, Aralica G, Lončar B and Kapitanović S: Loss of NF2/Merlin expression in advanced sporadic colorectal cancer. Cell Oncol (Dordr). 37:69–77. 2014. View Article : Google Scholar : PubMed/NCBI
21	Thurneysen C, Opitz I, Kurtz S, Weder W, Stahel RA and Felley-Bosco E: Functional inactivation of NF2/merlin in human mesothelioma. Lung Cancer. 64:140–147. 2009. View Article : Google Scholar : PubMed/NCBI
22	Kros J, de Greve K, van Tilborg A, Hop W, Pieterman H, Avezaat C, Lekanne Dit Deprez R and Zwarthoff E: NF2 status of meningiomas is associated with tumour localization and histology. J Pathol. 194:367–372. 2001. View Article : Google Scholar : PubMed/NCBI
23	Dilwali S, Patel PB, Roberts DS, Basinsky GM, Harris GJ, Emerick KS and Stankovic KM: Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol. Hear Res. 315:25–33. 2014. View Article : Google Scholar : PubMed/NCBI
24	Sainio M, Jääskeläinen J, Pihlaja H and Carpén O: Mild familial neurofibromatosis 2 associates with expression of merlin with altered COOH-terminus. Neurology. 54:1132–1138. 2000. View Article : Google Scholar : PubMed/NCBI