Functional Role of NRF2 in Cervical Carcinogenesis

Abstract

Nuclear factor erythroid-2-related factor 2 (NFE2L2) is a transcription factor associated with resistance to chemotherapy and increased tumor growth. NRF2 is repressed by the inhibitor Keap1. The Keap1-NRF2 pathway is dysfunctional in multiple tumor types. Among Uighur women, the incidence of cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia (CIN) was associated with elevated nuclear expression of NRF2 and decreased cytoplasmic expression of Keap1. Up-regulation of nuclear NRF2 was significantly associated with reduced cytoplasmic Keap1 expression. NRF2 positivity and Keap1 negativity were frequently associated with more advanced tumors (i.e., higher histological grade, lymph node involvement, and higher tumor stages) (p<0.05 for all). Methylated CpG islands in the Keap1 gene promoter in cervical cancer tissue were identified using MassARRAY. Moreover, promoter hypermethylation of this gene was significantly associated with decreased protein expression and increased nuclear NRF2 expression in cervical cancer tissues. Overexpression and knockdown of NRF2 in CSCC cell lines showed that NRF2 promotes proliferation, inhibits apoptosis, and enhances migration and invasion. These studies support the concept that epigenetic changes regulate expression of Keap1 in cervical cancer tissues. The association of NRF2 expression with aggressive tumor behavior suggests that NRF2 may be a marker of poor prognosis in patients with cervical cancer.

Fig 1. Detection of NRF2 and Keap1 protein expression assessed by immunohistochemical staining in representative specimens of normal cervical epithelia, CIN and CSCC, respectively.

A: Expression of NRF2 in normal cervical epithelia with weak cytoplasm staining; B: Moderate expression of NRF2 protein in CIN tissue; C: Strong nucleus expression of NRF2 proteins in CSCC tissue; D: Expression of Keap1 in normal cervical epithelia with Strong cytoplasm staining; E: Moderate expression of Keap1 protein in CIN tissue; F: weak expression of Keap1 proteins in CSCC tissue. (original magnification, × 200).

Fig 2. The detection of NRF2 protein transfection with mimics and inhibitor.

Morphology of transfected Siha cells for 48 h under microscopy (magnification ×200). A. Transfection with Short interfering RNA (SiRNA); B. transfection with mimics; C. The levels of NRF2 protein detected by Western blotting after transfection for 72 h.1 and 2 were normal control; 3 and 4 were Knockdown group; 5 and 6 were normal control; 7 and 8 were overexpression group; D The relative expression of NRF2 was displayed, which normalized to b-tubulin. There is a statistically significant difference between the group transfected with NRF2 mimics, NRF2 inhibitor and normal control.

Fig 3. NRF2 positively modulates CSCC cellular malignant phenotypes.

A, D, G and J: Cell apoptosis, Proliferation, Migration and invasion in Siha cells, respectively (Normal controls). B, E, H and K: a Knockdown of NRF2 increased cell apoptosis, decreased cell proliferation, migration and invasion, which significantly decreased malignant phenotypes of Siha cells. C, F, I and L: Overexpression of NRF2 sharply decreased cell apoptosis, increased cell proliferation, migration and invasion, which significantly enhanced cell proliferation and migration in Siha cell line. All experiments were performed at least three times.

Fig 4. Effect of siRNA-expressing vectors and over-expressing vectors on the cell migration and invasion of Siha cells following transfection.

For overexpression studies, NRF2 was overexpressed using a pcDNA3.1 vector and inhibition of NRF2 was achieved using siRNA vectors. A and B: regulates migration of Siha cells; C and D: regulates invasion of Siha cells. *P < 0.05; **P < 0.01.