GPC6 Promotes Cell Proliferation, Migration, and Invasion in Nasopharyngeal Carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is a highly metastatic tumor that occurs frequently in Southeast Asia, particularly including southern China. Epstein-Barr virus infection is well established as a primary cause of NPC; nevertheless, the mechanisms underlying NPC pathogenesis remain largely unknown. In our previous study, we conducted whole-genome sequencing to screen for genomic variations that were associated with NPC. Of the resultantly identified variations, glypican-6 (GPC6), was shown, for the first time, to be frequently mutated in NPC. In the present study, we verified this finding and conducted a series of functional experiments, which demonstrated that GPC6 promotes the migration, invasion, and proliferation of NPC cells in vitro. Thus, the present study identified novel biological functions for GPC6 in NPC, and thus, showed that GPC6 may be a promising potential therapeutic target for this disease.

Keywords: GPC6; NPC; genomic variations; invasion; migration; proliferation.

Figure 1

Validation of somatic mutations in nasopharyngeal carcinoma (NPC). A combination of polymerase chain reaction (PCR) and Sanger-sequencing techniques was used to validate previously identified somatic mutations in tumor and blood (negative control) samples collected from patients with NPC. The depicted GPC6 G-C mutation was found to occur more frequently in the analyzed tumor than in blood samples.

Figure 2

GPC6 is highly expressed in nasopharyngeal carcinoma (NPC). (A) GPC6 expression was shown to be significantly increased in NPC biopsies via the analysis of publicly available datasets GSE68799 (Normal, n = 4; Tumor, n = 42; p = 0.0006) and GSE12452 (Normal, n = 10; Tumor, n = 31; p = 0.041) that were sourced from the Gene Expression Omnibus database. (B) GPC6 expression was measured by RT-qPCR and was shown to be significantly increased in 37 NPC compared to 19 non-tumor tissues collected from newly enrolled patients (left), and this increase in GPC6 expression was shown to be positively associated with higher TNM stages (right). N, normal tissue sample; T, tumor tissue sample.

Figure 3

GPC6 overexpression promotes nasopharyngeal carcinoma (NPC) cell proliferation. (A) Transfection of an GPC6 overexpression construct, pIRESneo3-GPC6, dramatically increased GPC6 expression in 5-8F and HNE2 cells, as shown via qRT-PCR. (B) Cells were seeded in 6-well plates, transfected with pIRESneo3-GPC6 or an empty vector (negative control, NC), and allowed to proliferate for a further 24 h. They were then digested, counted, suspended (800 cells/200 µl), added to a 96-well plate, and subjected to an MTT assay. The results of this analysis showed that GPC6 overexpression significantly promoted the proliferation of NPC cells.

Figure 4

GPC6 increased the capacity of nasopharyngeal carcinoma (NPC) cells to form colonies and become invasive in vitro. 5-8F and HNE2 cells were transfected with pIRESneo3-GPC6 or an empty vector (negative control, NC), and allowed to proliferate for 24 h before being subjected to (A) a colony formation and (B) a Matrigel invasion assay. The results of these analyses showed that GPC6 overexpression increased both the number of colonies that were formed by the cells, and the invasive capacity of the cells, respectively.

Figure 5

Overexpression of GPC6 promoted nasopharyngeal carcinoma (NPC) cell migration. 5-8F and HNE2 cells were transfected with pIRESneo3-GPC6 or an empty vector (negative control, NC), and cultured to 100% confluency before being subjected to a wound healing assay. After scratching, images were captured at 0, 12, 24, and 36 h with an inverted phase contrast microscope. Data are presented as the width ratio of migratory promotion.