Construction and identification of small hairpin RNA plasmids targeting neuropilin-1 gene and their inhibitory effect on human nasopharyngeal carcinoma CNE-2Z cell proliferation in vitro and in vivo

Abstract

We observed the effects of small hairpin RNA (shRNA) plasmids targeting neuropilin-1 (NRP-1) gene on human nasopharyngeal carcinoma (NPC) CNE-2Z cell growth in vitro and in vivo. Three fluorescein-labeled shRNA eukaryotic expression vectors targeting NRP-1 gene, including pSilencer-shRNA1, pSilencer-shRNA2 and pSilencer-shRNA3 were constructed. The three plasmids were, respectively, transfected into human NPC CNE-2Z cells. The most effective plasmid was injected into xenograft tumors in nude mice. The sequencing for these recombinant plasmids was consistent with that of designed shRNA templates. Green fluorescence was seen in the transfected CNE-2Z cells and xenograft tumors in nude mice. MTT assay indicated that CNE-2Z cell proliferation was significantly inhibited. PT-PCR and Western blot displayed that both mRNA and protein of NRP-1 gene were all decreased, particularly in the cells treated with shRNA3. At the end of the experiment, xenograft tumors in plasmid group (0.599±0.002cm(3)) were significantly inhibited with a tumor inhibition rate of 48.6%, as compared to those in negative (1.141±0.013cm(3)) and blank control groups (1.165±0.308cm(3)) (all P<0.05). shRNA targeting NRP-1 gene can effectively inhibit human NPC CNE-2Z cell proliferation in vitro and in vivo. This provides an experiment basis for NPC gene therapy.