Discovery of key genes as novel biomarkers specifically associated with HPV-negative cervical cancer

Abstract

Cervical cancer is a common female malignancy that is mainly caused by human papillomavirus (HPV) infection. However, the incidence of HPV-negative cervical cancer has shown an increasing trend in recent years. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative cervical cancer and verify the underlying potential mechanism. Differentially expressed genes were compared among HPV-positive cervical cancer, HPV-negative cervical cancer, and normal cervical tissues retrieved from TCGA. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical cancer tissues and HPV-negative cell lines were validated by qRT-PCR, western blotting, and immunohistochemical staining. We found seventeen highly expressed genes that were particularly associated with HPV-negative cervical cancer from analysis of TCGA database. Among the 17 novel genes, 7 genes (preferentially expressed antigen in melanoma [PRAME], HMGA2, ETS variant 4 [ETV4], MEX3A, TM7SF2, SLC19A1, and tweety-homologs 3 [TTYH3]) displayed significantly elevated expression in HPV-negative cervical cancer cells and HPV-negative cervical cancer tissues. Additionally, higher expression of MEX3A and TTYH3 was associated with a shorter overall survival of patients with HPV-negative cervical cancer. Our study implies that these seven genes are more likely to provide novel insights into the occurrence and progression of HPV-negative cervical cancer.

Keywords: ETV4; HMGA2; HPV-negative cervical cancer; HPV-positive cervical cancer; MEX3A; PRAME; TTYH3; prognosis; therapy.

Graphical abstract

Figure 1

Differentially expressed genes in HPV-negative cervical cancer (A) Workflow of the analysis of differentially expressed genes used in the study. (B) Differentially expressed genes specifically associated with HPV-negative cervical cancer.

Figure 2

The mRNA expression of seventeen upregulated genes in HPV-negative cervical cancer cells C33A is an HPV-negative cervical cancer cell line. SiHa, Caski, MS751, and HeLa cells are HPV-positive cervical cancer cell lines. Ect1 is a normal cervical squamous cell line. (A and B) The mRNA expression of 17 upregulated genes, including PRAME, HMGA2, SBK1, ETV4, MEX3A, PLS1, TM7SF2, NIPSNAP1, PYCR1, SLC19A1, TTYH3 (A), MT1G, SLC7A5, RHPN1, CHD7, HSPH1, and SLC25A13 (B) was detected in different cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns, not significant. Data are presented as the means ± SD from triplicate experiments.

Figure 3

The protein levels of eleven upregulated genes in HPV-negative cervical cancer cells C33A is an HPV-negative cervical cancer cell line. SiHa, Caski, MS751, and HeLa cells are HPV-positive cervical cancer cell lines. Ect1 is a normal cervical squamous cell line. Levels of the PRAME, HMGA2, ETV4, MEX3A, TM7SF2, SLC19A1, TTYH3, NIPSNAP1, SBK1, PYCR1, and PLS1 proteins in different cells were determined by western blotting.

Figure 4

The protein levels of eleven upregulated genes in HPV-negative cervical cancer tissues The protein expression level was determined by calculating immunohistochemical staining score. Immunohistochemical staining scores for 11 proteins, PRAME, HMGA2, SBK1, MEX3A, TM7SF2, SLC19A1, ETV4, PYCR1, TTYH3, PLS1, and NIPSNAP1, in HPV-negative cervical cancer tissues, HPV-positive cervical cancer tissues, and normal cervical tissues are shown. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Data are presented as the means ± SD from triplicate experiments.

Figure 5

Representative images of immunohistochemical staining for several genes The images of PRAME, HMGA2, SBK1, MEX3A, TM7SF2, and SLC19A1 were presented in HPV-negative cervical cancer tissues, HPV-positive cervical cancer tissues, and normal cervical tissues.

Figure 6

Representative image of immunohistochemical staining for several genes The images of ETV4, PYCR1, TTYH3, PLS1, and NIPSNAP1 were presented in HPV-negative cervical cancer tissues, HPV-positive cervical cancer tissues, and normal cervical tissues.

Figure 7

The correlations between five genes and survival rates are shown in cervical cancer patients Analysis of the correlations between PRAME, HMGA2, SBK1, ETV4, and MEX3A expression and survival rates in patients with HPV-negative cervical cancer (22 samples), patients with HPV-positive cervical cancer (281 samples), and all patients with cervical cancer (304 samples). All samples information was obtained from the TCGA.

Figure 8

The associations between six genes and survival rates are shown in cervical cancer patients Analysis of the correlation between PLS1, TM7SF2, PYCR1, SLC19A1, TTYH3, and NIPSNAP1 expression and survival rates in patients with HPV-negative cervical cancer (22 samples), patients with HPV-positive cervical cancer (281 samples), and all patients with cervical cancer (304 samples). All samples information was obtained from the TCGA.