NRP‑1 silencing suppresses hepatocellular carcinoma cell growth in vitro and in vivo

Xu,Jing; Xia,Jinglin

doi:10.3892/etm.2012.803

NRP‑1 silencing suppresses hepatocellular carcinoma cell growth in vitro and in vivo

Authors:
- Jing Xu
- Jinglin Xia
View Affiliations

Affiliations: The Fifth People's Hospital, Fudan University, Shanghai 200032, P.R. China, Liver Cancer Institute, Fudan University, Shanghai 200032, P.R. China
Published online on: November 6, 2012 https://doi.org/10.3892/etm.2012.803
Pages: 150-154

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

Neuropilin-1 (NRP‑1) is a novel receptor of vascular endothelial growth factor 165 that promotes angiogenesis, tumor growth, tumor invasion and metastasis. However, its role in tumorigenesis and progression of human hepatocellular carcinoma (HCC) is unknown. In this study, lentivirus‑mediated short hairpin RNA (shRNA) was used to silence NRP‑1 in the HCCLM6 cell line to explore its role in regulating the growth of HCC. Recombinant NRP‑1 shRNA lentivirus was prepared and transfected into HCCLM6 cells. Transfection efficiencies of the lentivirus were observed by flow cytometry. Protein and mRNA expression of NRP‑1 were examined by western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT‑PCR), and the effect of the lentivirus on cell growth was determined using MTT assay. Different cell groups were inoculated into nude mice to establish cancer xenografts, and tumor growth was monitored. Protein expression of NRP‑1 in tumor tissues was detected by western blot assay. Microvessel density (MVD) in tumor tissues was assessed by immunohistochemistry (IHC). Lentivirus‑mediated shRNA efficiently reduced endogenous NRP‑1 expression in HCCLM6 cells and significantly inhibited cell growth in vitro. In vivo, NRP‑1 knockdown in tumor tissues resulted in decreased vasculature. NRP‑1 promotes the growth of HCC in vitro and in vivo, and therefore may be considered as a novel therapeutic target for HCC.

View Figures

View References

1	Tang ZY: Hepatocellular carcinoma-cause, treatment and metastasis. World J Gastroenterol. 7:445–454. 2001.PubMed/NCBI
2	Parkin DM, Bray F, Ferlay J and Pisani P: Global cancer statistics, 2002. CA Cancer J Clin. 55:74–108. 2005. View Article : Google Scholar
3	Fujisawa H, Takagi S and Hirata T: Growth-associated expression of a membrane protein, neuropilin, in Xenopus optic nerve fibers. Dev Neurosci. 17:343–349. 1995. View Article : Google Scholar : PubMed/NCBI
4	Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ and Ginty DD: Neuropilin is a semaphorin III receptor. Cell. 90:753–762. 1997. View Article : Google Scholar : PubMed/NCBI
5	He Z and Tessier-Lavigne M: Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell. 90:739–751. 1997. View Article : Google Scholar : PubMed/NCBI
6	Rossignol M, Gagnon ML and Klagsbrun M: Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble isoforms. Genomics. 70:211–222. 2000. View Article : Google Scholar : PubMed/NCBI
7	Bernatchez PN, Rollin S, Soker S and Sirois MG: Relative effects of VEGF-A and VEGF-C on endothelial cell proliferation, migration and PAF synthesis: role of neuropilin-1. J Cell Biochem. 85:629–639. 2002. View Article : Google Scholar : PubMed/NCBI
8	Lee P, Goishi K, Davidson AJ, Mannix R, Zon L and Klagsbrun M: Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish. Proc Natl Acad Sci U S A. 99:10470–10475. 2002. View Article : Google Scholar : PubMed/NCBI
9	Soker S, Takashima S, Miao HQ, Neufeld G and Klagsbrun M: Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell. 92:735–745. 1998. View Article : Google Scholar : PubMed/NCBI
10	Kitsukawa T, Shimono A, Kawakami A, Kondoh H and Fujisawa H: Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs. Development. 121:4309–4318. 1995.PubMed/NCBI
11	Takashima S, Kitakaze M, Asakura M, et al: Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci U S A. 99:3657–3662. 2002. View Article : Google Scholar : PubMed/NCBI
12	Hansel DE, Wilentz RE, Yeo CJ, Schulick RD, Montgomery E and Maitra A: Expression of neuropilin-1 in high-grade dysplasia, invasive cancer, and metastases of the human gastrointestinal tract. Am J Surg Pathol. 28:347–356. 2004. View Article : Google Scholar : PubMed/NCBI
13	Bachelder RE, Lipscomb EA, Lin X, et al: Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells. Cancer Res. 63:5230–5233. 2003.PubMed/NCBI
14	Stephenson JM, Banerjee S, Saxena NK, Cherian R and Banerjee SK: Neuropilin-1 is differentially expressed in myoepithelial cells and vascular smooth muscle cells in preneoplastic and neoplastic human breast: a possible marker for the progression of breast cancer. Int J Cancer. 101:409–414. 2002. View Article : Google Scholar
15	Ochiumi T, Kitadai Y, Tanaka S, Akagi M, Yoshihara M and Chayama K: Neuropilin-1 is involved in regulation of apoptosis and migration of human colon cancer. Int J Oncol. 29:105–116. 2006.PubMed/NCBI
16	Kreuter M, Woelke K, Bieker R, et al: Correlation of neuropilin-1 overexpression to survival in acute myeloid leukemia. Leukemia. 20:1950–1954. 2006. View Article : Google Scholar : PubMed/NCBI
17	Lu L, Zhang L, Xiao Z, Lu S, Yang R and Han ZC: Neuropilin-1 in acute myeloid leukemia: expression and role in proliferation and migration of leukemia cells. Leuk lymphoma. 49:331–338. 2008. View Article : Google Scholar : PubMed/NCBI
18	Hu B, Guo P, Bar-Joseph I, et al: Neuropilin-1 promotes human glioma progression through potentiating the activity of the HGF/SF autocrine pathway. Oncogene. 26:5577–5586. 2007. View Article : Google Scholar : PubMed/NCBI
19	Hong TM, Chen YL, Wu YY, et al: Targeting neuropilin 1 as an antitumor strategy in lung cancer. Clin Cancer Res. 13:4759–4768. 2007. View Article : Google Scholar : PubMed/NCBI
20	Fukasawa M, Matsushita A and Korc M: Neuropilin-1 interacts with integrin beta1 and modulates pancreatic cancer cell growth, survival and invasion. Cancer Biol Ther. 6:1173–1180. 2007. View Article : Google Scholar : PubMed/NCBI
21	Lois C, Hong EJ, Pease S, Brown EJ and Baltimore D: Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 295:868–872. 2002. View Article : Google Scholar : PubMed/NCBI
22	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-DeltaDeltaC(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
23	Weidner N, Semple JP, Welch WR and Folkman J: Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. New Engl J Med. 324:1–8. 1991. View Article : Google Scholar : PubMed/NCBI
24	Miao HQ and Klagsbrun M: Neuropilin is a mediator of angiogenesis. Cancer Metastasis Rev. 19:29–37. 2000. View Article : Google Scholar : PubMed/NCBI
25	Miao HQ, Lee P, Lin H, Soker S and Klagsbrun M: Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression. Faseb J. 14:2532–2539. 2000. View Article : Google Scholar : PubMed/NCBI
26	Bachelder RE, Crago A, Chung J, et al: Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res. 61:5736–5740. 2001.PubMed/NCBI
27	Barr MP, Byrne AM, Duffy AM, et al: A peptide corresponding to the neuropilin-1-binding site on VEGF(165) induces apoptosis of neuropilin-1-expressing breast tumour cells. Br J Cancer. 92:328–333. 2005.PubMed/NCBI