HNF4A Regulates the Proliferation and Tumor Formation of Cervical Cancer Cells through the Wnt/ β-Catenin Pathway

Abstract

Hepatocyte nuclear factor 4 alpha (HNF4A) is a transcriptional factor which plays an important role in the development of the liver, kidney, and intestines. Nevertheless, its role in cervical cancer and the underlying mechanism remain unknown. In this study, both immunohistochemistry and western blotting revealed that the expression of HNF4A was downregulated in cervical cancer. Xenograft assays suggested that HN4A could inhibit tumorigenic potential of cervical cancer in vivo. Functional studies illustrated that HNF4A also inhibited the proliferation and viability of cervical cancer cells in vitro. In addition, FACS analysis implied that HNF4A could induce cell cycle arrest from the G0/G1 phase to S phase. Further studies suggested that HNF4A downregulated the activity of the Wnt/β-catenin pathway. Altogether, our data demonstrated that HNF4A inhibited tumor formation and proliferation of cervical cancer cells through suppressing the activity of the Wnt/β-catenin pathway.

Figure 1

The expression of HNF4A is downregulated in cervical cancer. (a) Immunohistochemical staining of HNF4A in clinical samples, including the normal cervix (NC, n = 17) and cervical carcinomas (CC, n = 37) (original magnification, 1000x). (b) The immunohistochemical staining intensity was classified into negative, weak positive, and strong positive, and the percentage of each group was shown. (c) The scatter plots showed the IHC scores obtained for the staining of HNF4A in different cervix lesion samples (points represent the IHC score per specimen, and Student's t-test is performed). (d) HNF4A expression was detected by western blot in 8 normal cervix samples and 8 cervical carcinoma samples. GAPDH was used as a loading control. (e) The quantitative illustration of the levels of HNF4A protein using densitometry to measure the density of the corresponding bands in (d). Student's t-test was carried out. (f) The relationship between relapse-free survival (RFS) probability of CESC patients (n = 304) and the expression level of HNF4A in their tumors was shown by the Kaplan–Meier estimator in TCGA database. ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 2

HNF4A inhibits tumor formation and tumor growth of cervical cancer cells in vivo. The expression of HNF4A in human cervical cancer cell lines was detected using immunocytochemistry (a) and western blotting (b). Stably transfected HNF4A-modified cervical cancer cells were identified by western blotting (c, d). (e) Xenograft tumor formation of HeLa-GFP and HeLa-HNF4A cells. The tumor growth curve (f), tumor weight (g), and tumor-free survival (h) of HeLa-GFP and HeLa-HNF4A cells, respectively. (i) Xenograft tumor formation of SiHa-GFP and SiHa-HNF4A cells. The tumor growth curve (j), tumor weight (k), and tumor-free survival (l) of SiHa-GFP and SiHa-HNF4A cells, respectively. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

HNF4A inhibits tumor formation and tumor growth of cervical cancer cells by inhibiting cell proliferation. (a, b) Immunohistochemical staining of HNF4A and Ki67 in xenograft tumor tissues derived from HeLa-GFP cells, HeLa-HNF4A cells, and SiHa-GFP and SiHa-HNF4A cells, respectively. (c, d) Immunoreactivity scores of HNF4A and Ki67 in xenograft tumor tissues derived from HeLa-GFP cells, HeLa-HNF4A cells, SiHa-GFP, and SiHa-HNF4A cells. Data were statistically analyzed by Student's t-test, and values are shown as mean ± SD. The proliferation was detected using growth curves in HeLa-GFP and HeLa-HNF4A cells (e) and SiHa-GFP and SiHa-HNF4A cells (f). The viability was detected by the MTT assay in HeLa-GFP and HeLa-HNF4A cells (g) and SiHa-GFP and SiHa-HNF4A cells (h). ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 4

HNF4A inhibited cell proliferation through inducing cell cycle arrest from the G0/G1 phase to S phase. In the flow cytometry figures, the y-axis shows the count of effective cells and the x-axis shows the DNA content. Each colored area represents the cells of different phases of the cell cycle: blue area refers to the cells in the G0/G1 phase, green area refers to the cells in the S phase, and pink area refers to the cells in the G2/M phase. The cell cycles of HeLa-GFP (a) and HeLa-HNF4A (b) cells were analyzed using flow cytometry, and a quantitative analysis of the cell cycle is shown (c). The cell cycles of SiHa-GFP (d) and SiHa-HNF4A (e) cells and the quantitative analysis (f) are shown. The data were shown as the mean ± SD of three independent experiments. Data were statistically analyzed by Student's t-test, and values are shown as mean ± SD. ^∗∗∗p < 0.001.

Figure 5

HNF4A downregulated the activity of the Wnt/β-catenin pathway. (a) Heatmap of the data from RNA-seq. (b) The result of gene set enrichment analysis. (c) The significantly changed genes in GSEA. (d, e) TOP/FOP-Flash reporter assays were carried out in HNF4A-modified cervical cancer cells. (f, g) Real-time PCR analysis is shown for the mRNA levels of the Wnt/β-catenin pathway key genes in HNF4A-modified cervical cancer cells. (h, i) The expression of Wnt/β-catenin pathway key proteins in HNF4A-modified cervical cancer cells was determined by western blotting. (j, k) The quantitative analysis of the western blotting in (h) and (i). Data represent mean ± SD of triplicate experiments, and statistical analysis was done by Student's t-test. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 6

HNF4A suppressed the Wnt/β-catenin pathway in a mouse xenograft. (a) Expression of HNF4A, β-catenin, c-Myc, and cyclin D1 in tumor xenografts derived from HeLa-GFP cells and HeLa-HNF4A cells. (b) Immunoreactivity scores of HNF4A, β-catenin, c-Myc, and cyclin D1 in xenograft tissues derived from HeLa-GFP cells and HeLa-HNF4A cells. (c) Expression of HNF4A, β-catenin, c-Myc, and cyclin D1 in xenografts derived from SiHa-GFP and SiHa-HNF4A cells. (d) Immunoreactivity scores of HNF4A, β-catenin, c-Myc, and cyclin D1 in xenograft tissues derived from SiHa-GFP cells and SiHa-HNF4A cells. Representative images were shown. Scale bar: 10 μm. Data represent mean ± SD of triplicate experiments, and statistical analysis was done by Student's t-test. ^∗p < 0.05, ∗∗∗p < 0.001.