Kelch-like protein 14 promotes proliferation and migration of ovarian cancer cells

Abstract

Kelch-like protein 14 (KLHL14) belongs to the Kelch gene family, which interacts with TorsinA and is associated with dystonia symptoms. However, the effect of KLHL14 on tumorigenesis remains unclear; thus, we aimed to explore the effects of KLHL14 on ovarian cancer cells. By analyzing information regarding ovarian cancer patients obtained from The Cancer Genome Atlas (TCGA)-Ovarian Cancer Database, we found that the KLHL14 gene is highly expressed in ovarian cancer, and patients with high KLHL14 expression had lower survival than those with low expression. qRT-PCR and western blot results revealed that the mRNA and protein levels of KLHL14 in ovarian cancer cells were higher in A-2780 cells than in KGN cells. After constructing cell lines with a knocked down KLHL14 gene, we used the MTT assay, flow cytometry with propidium iodide (PI), Annexin V-FITC/PI, and transwell assay and found that knockdown of KLHL14 gene inhibited proliferation of A-2780 cells, caused cell G0/G1 phase arrest, promoted apoptosis, and inhibited migration and invasiveness. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that KLHL14 may promote development of ovarian cancer by regulating signaling pathways such as mTOR, WNT, and TGF-beta. In short, the KLHL14 gene plays an important role in ovarian cancer development and may be a target for ovarian cancer detection and treatment.

Keywords: Kelch-like protein 14; The Cancer Genome Atlas; cell invasiveness; cell migration; ovarian cancer.

Figure 1

The mRNA level of KLHL14 gene is high in ovarian cancer, indicating a low survival rate. A. Volcanic maps about TCGA database of ovarian cancer tissue samples shows that KLHL14 mRNA was highly expressed in ovarian cancer. B. Scatter diagram shows that KLHL14 mRNA was highly expressed in ovarian cancer tissue. C. Survival curve shows that the overall survival rate of ovarian cancer patients with high expression of KLHL14 gene was lower than that of ovarian cancer patients with low expression of KLHL14 gene. ****: P < 0.0001.

Figure 2

KLHL14 expression is significantly increased in ovarian cancer. (A) qRT-PCR results show KLHL14 mRNA levels in normal ovarian granulosa cell KGN and several ovarian cancer cell strains HEY, A2780, SK-OV-3, and COC1. (B) Western blot analysis results show protein levels of KLHL14 in KGN, HEY, A2780, SK-OV-3, and COC1. (C, D) Two shRNA vectors targeting KLHL14 were constructed, and qRT-PCR was used to detect the knock-down efficiency of shRNA vectors in A2780 and SK-OV-3 cells (C); the most efficient shKLHL14-2 was used for transfection of A2780 and SK-OV-3 cell strains, and western blot analysis was used to detect the knock-down efficiency of KLHL14 protein (D). ns: not significant; ***: P < 0.001; **: P < 0.01.

Figure 3

Knockdown of KLHL14 gene inhibits the proliferation of ovarian cancer cells and blocks cell cycle. A. After KLHL14 gene knock-down in A-2780 and SK-OV-3 cell strains, cell proliferation was detected through the MTT assay. B, C. PI staining and flow cytometry were used to examine cell cycles. ***: P < 0.001.

Figure 4

Knockdown of KLHL14 gene can promote the apoptosis of ovarian cancer cells. After knock-down of KLHL14 gene in A-2780 and SK-OV-3 cell lines, annexin V-FITC/PI double-staining method and flow cytometry were used to detect the proportion of apoptosis. **: P < 0.01.

Figure 5

Knockdown of KLHL14 gene inhibits the migration and invasiveness of ovarian cancer cells. A, B. After knockdown of KLHL14 gene in A-2780 and SK-OV-3 cell lines, Transwell without Matrigel was used to examine cell migration. C, D. After knock-down of KLHL14 gene, Transwell with Matrigel was used to detect cell invasiveness. ***: P < 0.001.

Figure 6

KEGG analysis was used to predict the signaling pathways that KLHL14 gene may regulate. A. KEGG enrichment analysis shows that KLHL14 may regulate mTOR, WNT, TGF-beta, and other tumor-related signaling pathways. B. Genetic correlation analysis shows that KLHL14 level is positively associated with many genes’ expression levels in these tumor-related signaling pathways (BMPR1B, FGFR2, PAX8, and TGFB2). KEGG: Kyoto Encyclopedia of Genes and Genomes.