The NF-κB modulated miR-194-5p/IGF1R/PPFIBP axis is crucial for the tumorigenesis of ovarian cancer

Abstract

miRNAs are involved in the tumorigenesis of various malignancies. In the current study, we found that miR-194-5p expression is downregulated in ovarian cancer tissues, and downregulation of miR-194-5p expression promotes proliferation, invasion and migration of human ovarian cancer cells in vitro and ovarian tumor growth in nude mice. We further found that IGF1R and PPFIBP are targets of miR-194-5p, and downregulation of miR-194-5p expression increases IGF1R and PPFIBP expression, resulting in increased proliferation, invasion and migration of ovarian cancer cells. Moreover, we showed that NF-κB can bind to the promoter region of miR-194-5p, and negatively regulate the expression of miR-194-5p in ovarian cancer cells. Taken together, our results suggested a NF-κB modulated miR-194-5p/IGF1R/ PPFIBP axis that is crucial for the tumorigenesis of ovarian cancer, which provides a new insight into the development of ovarian cancer.

Keywords: IGF1R; NF-κB; PPFIBP; miR-194-5p; ovarian cancer.

Figure 1

miR-194-5p is a tumor suppressor in ovarian cancer. (a) qRT-PCR detected the expression of miR-194-5p in 12 ovarian cancer tissues and their corresponding non-tumor tissues. U6 was used for normalization. (b) and (c) After transfection with Lv-hsa-miR-194-5p up or Lv-hsa-miR-194-5p inhibitor, the proliferation of SKOV3 or ES-2 cells was examined by CCK-8 assay. (d) The colony formation rate was calculated with the following equation: colony formation rate= (number of colonies/ number of planted cells) × 100%. The colony formation rate of cells infected with empty lentiviral vector was defined as 1. (e) and (f) Transwell migration or invasion assay of SKOV3 and ES-2 cells. Cells in three random fields of view at 100x magnification were counted and expressed as the average number of cells per field. The average cell number of its corresponding control group was defined as 1 (*, p<0.05, **, p<0.01).

Figure 2

miR-194-5p suppresses the tumor growth of ovarian cancer in the xenograft model. (a) SKOV3 cells with miR-194-5p overexpress or knockdown were injected subcutaneously into nude mice. (b) Tumor size was measured after the mice were sacrificed. (c) Tumor size was monitored at different time points. (d) The average size of the tumors was calculated (**, p<0.01).

Figure 3

IGF1R and PPFIBP1 are direct targets of miR-194-5p. (a) and (b) Predicted miR-194-5p binding sites in the 3'UTR of IGF1R and PPFIBP1. HEK293T cells were transfected with pGL3-WT or pGL3-MT in the presence or absence of Lv-hsa-miR-194-5p up or Lv-hsa-miR-194-5p inhibitor, the luciferase assay examined the luciferase activity of 3'UTR of IGF1R and PPFIBP1 respectively. (c) and (d) HEK293T cells were transfected with Lv-hsa-miR-194-5p up or Lv-hsa-miR-194-5p inhibitor, western blot analysis detected the protein expression of IGF1R and PPFIBP1 respectively. The results are representative of three independent experiments (*, p<0.05).

Figure 4

IGF1R promotes the proliferation, migration and invasion of ovarian cancer cells. (a) Western blot detected the expression of IGF1R after transfection with IGF1R overexpression or knock down. The results are representative of three independent experiments. (b) and (c) After IGF1R was overexpressed or knocked down, CCK-8 assay examined proliferation of SKOV3 and ES-2 cells respectively. (d) The colony formation rate was calculated with the following equation: colony formation rate= (number of colonies/ number of planted cells) × 100%. The colony formation rate of cells transfected with its control vector was defined as 1. (e) and (f) Transwell migration and invasion assay of SKOV3 and ES-2 cells. Cells in three random fields of view at 100x magnification were counted and expressed as the average number of cells per field. The average cell number of its corresponding control was defined as 1 (*, p<0.05, **, p<0.01).

Figure 5

PPFIBP1 promotes the proliferation, migration and invasion of ovarian cancer cells. (a) Western blot examined the expression of PPFIBP1. The results are representative of three independent experiments. (b) and (c) CCK-8 assay examined proliferation of SKOV3 and ES-2 cells when PPFIBP1 was overexpressed or knocked down. (d) The colony formation rate was calculated with the following equation: colony formation rate= (number of colonies/ number of planted cells) × 100%. The colony formation rate of cells transfected with its control vector was defined as 1.(e) and (f) Transwell migration and invasion assay of SKOV3 and ES-2 cells. Cells in three random fields of view at 100x magnification were counted and expressed as the average number of cells per field. The average cell number of its corresponding control was defined as 1 (*, p<0.05, **, p<0.01).

Figure 6

miR-194-5p, IGF1R and PPFIBP1 expression were inhibited by NF-κB. (a) The Expression of miR-194-5p in ES-2 cells was determined after pyrrolidine dithiocarbamate (PDTC) treatment. U6 snRNA was used for normalization. (b) qRT-PCR examined the regulation of NF-κB1 on miR-194-5p expression. U6 was used for normalization. (c) and (d) The correlation of protein expression of IGF1R or PPFIBP1 with NF-κB1 expression were analyzed by western blotting(*, p<0.05, **, p<0.01).

Figure 7

NF-κB directly binds to miR-194 promoter. (a) Schematic representation of the miR-194 mRNA and the NF-κB1 binding site. (b) ChIP-PCR analyses of NF-κB binding to the miR-194 promoter using NF-κB1 antibodies. ChIP primers are located at -2001bp ~ -2150bp upstream of the miR-194 gene. (c) The luciferase reporter assay showed that the fragment upstream of the miR-194 gene has strong promoter activity. (d) The luciferase intensity of miR-194 in ES-2 cells after PDTC treatment. (e) The promoter activity of miR-194 in ES-2 cells when NF-κB1 was overexpressed or knocked down. (f) Gel-shift signals by EMSA for the binding site probe incubated with nuclear extracts from ES-2 cells or PDTC treated ES-2 cells. (g) Gel-shift signals by EMSA for the binding site probe incubated with nuclear extracts of ES-2 cells with modified NF-κB1 expression (*, p<0.05, **, p<0.01).