KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1

Abstract

Background: Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines.

Objective: Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines.

Method: Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates.

Result: After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene.

Conclusion: These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

Keywords: EMT; KLF5; SNAI1; cervical cancer cells.

Figure 1.

The expression of KLF5 gene in the C33A, SiHa and HeLa cell lines. (A) Western blot analysis of the endogenous KLF5 protein expression level in the C33A, SiHa and HeLa cell lines. (B) Real-time PCR showing the KLF5 gene mRNA expression levels in the C33A, SiHa and HeLa cell lines.

Figure 2.

The effects of KLF5 on the proliferation and motility of HeLa cells. (A) MTT assays assessing the effect of KLF5 overexpression on the proliferation of HeLa cells. (B) Scratch wound-healing assays evaluating the effect of KLF5 overexpression on the motility of HeLa cells. (C) Transwell assay evaluating the effect of KLF5 overexpression on the migration and invasion abilities of HeLa cells. The bar graphs show the numbers of migrating and invading cells from triplicate experiments (right).

Figure 3.

The effect of KLF5 overexpression on the EMT phenotype in HeLa cells. (A) Western blot analysis of the exogenous KLF5 protein expression level in HeLa cells. (B) Real-time PCR showing the effect of KLF5 overexpression on the mRNA level of E-cadherin, N-cadherin and MMP9 in HeLa cells. The bar graphs show the fold changes in relative mRNA levels normalized against GAPDH. (C) Real-time PCR showing the effect of KLF5 overexpression on the mRNA level of SNAI1, SLUG, ZEB1, ZEB2 and TWIST1 in HeLa cells. The bar graphs show the fold changes in relative mRNA levels normalized against GAPDH. (D) Western blot analysis of the effect of KLF5 overexpression on the protein expression levels of E-cadherin, N-cadherin and SNAI1 in HeLa cells.

Figure 4.

The effect of silencing the KLF5 gene on the EMT phenotype in SiHa cells. (A) The efficacy of KLF5 silencing as validated by real-time PCR. (B) Real-time PCR showing the effect of KLF5 knockdown on the mRNA levels of E-cadherin, N-cadherin and MMP9 in SiHa cells. (C) Real-time PCR showing the effect of KLF5 knockdown on the mRNA levels of SNAI1, SLUG, ZEB1 and ZEB2 in SiHa cells. (D) Western blot analysis of the effect of silencing KLF5 on the protein expression levels of E-cadherin, N-cadherin and SNAI1 in SiHa cells.

Figure 5.

Assessment of the effects of KLF5 on the activity of SNAI1 promoter. (A) JASPER database analysis of potential KLF5 binding sites in the SNAI1 promoter region. (B) Schematic illustrating the construction of a series of human SNAI1 promoter reporter plasmids. (C) Luciferase reporter gene experiment assessing the effect of the KLF5 on the activities of the SNAI1 constructs with different promoter truncations. (D) Luciferase reporter gene experiment assessing the effect of the KLF5 on the activity of the SNAI1-Luc-200 promoter at different concentrations.

Figure 6.

The effects of overexpressing the SNAI1 gene on EMT phenotypes in HeLa cells. (A) Real-time PCR showing the effect of SNAI1 overexpression on the mRNA levels of E-cadherin, N-cadherin and MMP9 in HeLa cells. The bar graphs show the fold changes in relative mRNA levels normalized against GAPDH. (B) Real-time PCR showing the effect of SNAI1 overexpression on the mRNA levels of SNAI1, SLUG, ZEB1, ZEB2 and TWIST1 in HeLa cells. The bar graphs show the fold changes in relative mRNA levels normalized against GAPDH. (C) Western blot showing the effect of SNAI1 overexpression on the expression levels of E-cadherin, N-cadherin and GFP-SNAI1 in HeLa cells.

Figure 7.

Prognostic value of the SNAI1 gene in cervical cancer. (A) A heatmap showing SNAI1 gene expression profiles in cervical cancer from the TCGA databases. (B) The prognosis values of SNAI1 gene in TCGA data. (C) The prognosis values of SNAI1 gene in GBM in the TCGA data.