KDM6B induces epithelial-mesenchymal transition and enhances clear cell renal cell carcinoma metastasis through the activation of SLUG

Abstract

Clear cell renal cell carcinoma (ccRCC) is one of the most common kidney cancers; epithelial-mesenchymal transition (EMT) is associated with carcinoma invasion and metastasis. There have been several studies about the molecular regulation of EMT, but the relationship between histone demethylase and EMT is little known. Here, we reported KDM6B has high expression level in ccRCC and is positively correlated with poor ccRCC prognosis. KDM6B, also known as JMJD3, is a histone demethylase, can remove repressive histone H3K27me3 marks from chromatin, thereby activating gene expression. We found that the knockdown of KDM6B could inhibit ccRCC tumorigenesis in vitro; furthermore, KDM6B could induce EMT in ccRCC cells by activating the expression of master transcription factor SLUG. ChIP assays revealed that KDM6B stimulated SLUG expression by demethylate histone H3K27me3. The knockdown of KDM6B strongly inhibited ccRCC cell invasion in vitro, while the overexpression of KDM6B shown the opposite trend. Meanwhile, our analysis of the ccRCC tissue found that KDM6B expression was significantly corresponded with lymph node metastasis. Together, our data provide a novel epigenetic mechanism regulating tumor cell invasion and EMT, and provide a biomolecule for ccRCC diagnosis and prognosis.

Keywords: EMT; KDM6B; SLUG; ccRCC.

Figure 1

KDM6B is frequently up-regulated in ccRCC and positively correlated with poor ccRCC prognosis. A. qRT-PCR was used to detect KDM6B mRNA in non-tumor tissue and tumor tissue from clear cell renal cell carcinoma (ccRCC) patients. B. Western blot was used to measure the protein expression of KDM6B in non-tumor tissue and tumor tissue from ccRCC. C. KDM6B protein levels between Human kidney epithelial cell line 293 and ccRCC cell lines Caki-2 or ACHN were detected by Western blot. D. The online tool was used to plot the Kaplan-Meier survival curves.

Figure 2

ccRCC tumorigenesis is inhibited by KDM6B knockdown in vitro. A. Western blot analysis verified shRNA-mediated interference of KDM6B expression in Caki-2 cells. B. The CCK-8 assay was use to draw the growth curves of Caki-2 cells, cells were transfected with shKDM6B or SCR. C. Graph representative inhibition of foci formation in monolayer culture due to the interference of KDM6B expression.

Figure 3

KDM6B promotes EMT by inducing SLUG. A. KDM6B was overexpressed in Caki-2 cells, qRT-PCR was used to measure mRNA levels of epithelial and mesenchymal markers. B. Western blot analysis revealed a significant decreased epithelial markers and increased mesenchymal marker in Caki-2/KDM6B cells. C. qRT-PCR was used to measure mRNA levels of epithelial and mesenchymal markers change in Caki-2/shKDM6B cells. D. In Caki-2/shKDM6B cells, western blot showed an opposite result compare with Caki-2/KDM6B cells, epithelial markers was increased and mesenchymal marker was decreased.

Figure 4

KDM6B promotes SLUG transcription via modulating H3K27 methylation. A. Caki-2 cells were transfected with KDM6B shRNA#2. ChIP analyses were performed with an H3K27me3 antibody. B. qRT-PCR was performed with specific primers for SLUG, SNAI1, TWIST or SIP-1 mRNA. Data represent the mean c primers for or dy. performed es were C. Reconstituted expression of RNAi-resistant KDM6B in Caki-2/shKDM6B cell, ChIP analyses were performed with an H3K27me3 antibody, subsequent qRT-PCR was performed with specific primers for SLUG, SNAI1, TWIST or SIP-1 mRNA. Data represent the mean ± SD of three independent experiments.

Figure 5

KDM6B promotes clear cell renal cell carcinoma cell invasion. A. 786-O cells were transfected with KDM6B shRNA and then subjected to the in vitro invasion assay 72 hr later. The graph indicates the average number of invaded cells per field. Three independent experiments were carried out, and the data are shown as the mean with SD (*P < 0.05). B. 786-O cells were transfected with KDM6B and then subjected to the in vitro invasion assay 72 hr later. Representative photos were shown. Three independent experiments were carried out, and the data are shown as the mean with SD (*P < 0.05). C. Caki-2 cells were transfected with shRNA and then subjected to the soft agar colony formation assay. The graph indicates the average number of colonies per field. Three independent experiments were carried out, and data are shown as the mean with SD (*P < 0.05).