Elevated nuclear auto-antigenic sperm protein promotes melanoma progression by inducing cell proliferation

Abstract

Background: Nuclear auto-antigenic sperm protein (NASP) has been implicated in tumorigenesis. However, its role in melanoma is still unclear.

Materials and methods: In the present study, we detected the mRNA and protein level of NASP in melanoma cell lines and tissues. Then the role of NASP was investigated by transfecting with NASP siRNAs. Finally, the prognosis of NASP was analyzed in 100 melanoma patients through Cox regression and Kaplan-Meier analyses.

Results: We showed that NASP was significantly overexpressed in melanoma tissues, and unregulated NASP promoted melanoma cell proliferation via promoting cell cycle G1/S phase transition. Additionally, the expression of NASP was closely related to proliferating cell nuclear antigen, a widely accepted biomarker for cell proliferation. Clinically, we found that a high level of NASP predicated poor overall survival and high cumulative recurrence rates. Multivariate analysis revealed that NASP was a risk biomarker for predicting the prognosis of melanoma patients.

Conclusion: Elevated NASP plays an important role in melanoma cell proliferation and tumor progression, and it can be used as an independent prognostic biomarker for melanoma patients.

Keywords: NASP; melanoma; prognosis; proliferation.

Figure 1

The expression of NASP is significantly upregulated in melanoma. (A) NASP transcript levels in head-neck squamous cell carcinoma vs normal tissues. (B) NASP transcript levels in basal cell carcinoma vs normal skin tissues. (C) NASP mRNA expression in 22 pairs of melanoma tissues, shown as Log (T/P) (T, tumor; P, peritumor). (D) NASP protein expression in 22 pairs of melanoma tissues, representative bands are shown (T, tumor; P, peritumor). (E) Representative images of the TMA stained with H&E and IHC for NASP. Scale bar 500 μm, magnification 40×; 50 μm, 400×. (F) NASP protein expression in melanoma and peritumoral tissues was analyzed by densitometry (***P<0.001). Abbreviations: TMA, tissue microarray; IHC, immunohistochemistry.

Figure 2

NASP promotes the proliferation of melanoma cells. (A) NASP was knocked down in A2058 and A375 melanoma cells and was verified by Western blot and qRT-PCR. (B) Interference with NASP expression in A375 and A2058 cells inhibited cell proliferation as determined by CCK-8 assay. (C) Interference with NASP expression in A375 and A2058 cells inhibited cell proliferation as determined by colony formation assay. (D) Representative pictures of tumors derived from the A375-si-NC and A375-si-RNA2 cell lines. (E) Tumor growth curves of the subcutaneous xenografts. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Abbreviations: qRT-PCR, quantitative real-time reverse transcription PCR; CCK-8, Cell Counting Kit-8.

Figure 3

NASP promotes the proliferation of melanoma cells via cell cycle G1/S transition. (A) GSEA analyses were used for detecting the biological function of NASP. (B) Western blot analysis was performed to detect the expression of NASP, cyclin D1, and p21 in the indicated cells. (C) Interference with NASP expression in A375 and A2058 cells inhibited cell proliferation as determined by flow cytometric assay. *P<0.05, **P<0.01. Abbreviation: NES, normalized enrichment score.

Figure 4

The expression of NASP is positively correlated with PCNA. (A) The correlation analysis between NASP and PCNA was detected by GEPIA. (B) The correlation between NASP and PCNA was detected by IHC density. (C) Western blot analysis was performed to detect the expression of NASP and PCNA in the indicated cells. (D, E) Immunofluorescence staining was employed to detect the expression of NASP and PCNA in the indicated cells. Abbreviations: PCNA, proliferating cell nuclear antigen; IHC, immunohistochemistry.

Figure 5

The expression of NASP is related to OS and recurrence rate. (A) Patients with a high level of NASP had a lower OS than those with low NASP expression in our melanoma cohort. (B) Patients with a high level of NASP had a higher recurrence rate than those with low NASP expression. Abbreviation: OS, overall survival.