Long non-coding RNA UCA1 promotes gallbladder cancer progression by epigenetically repressing p21 and E-cadherin expression

Abstract

A growing number of studies indicated that long non-coding RNAs (lncRNAs) determine some cellular processes in cancer, such as proliferation, metastasis and differentiation. Urothelial carcinoma associated 1 (UCA1), an lncRNA, had been reported for its overexpression and oncogenic effect on various human cancers. In this study, we found that UCA1 was significantly overexpressed in gallbladder cancer (GBC) and positively correlated with tumor size, lymph node metastasis, TNM stage and short survival time. Moreover, UCA1 promoted GBC cell proliferation and metastasis in vitro and tumor growth in vivo. Mechanically, we identified that UCA1 promoted GBC progression through recruiting enhancer of zeste homolog 2 (EZH2) to the promoter of p21 and E-cadherin, and epigenetically suppressing their transcript.

Keywords: E-cadherin; EZH2; gallbladder cancer; lncRNA UCA1; p21.

Figure 1. Relative expression of UCA1 in GBC and its clinical significance

(A and B) Relative expression of UCA1 in GBC tissues and neighboring noncancerous tissues was detected by qRT-PCR (n = 45). Relative expression of UCA1 was normalized to GAPDH. (C) Relative expression of UCA1 in GBC cell lines (EH-GB1, GBC-SD, NOZ, OCUG-1 and SGC-996) and human gallbladder epithelium cell line H69 was detected by qRT-PCR. (D) Kaplan–Meier method with the log-rank test was used to analyze the overall survival curves of patients in high and low UCA1 expression groups (log-rank=4.97, p<0.05). The mean ± SD of triplicate experiments were plotted, **p<0.01, ***p<0.001.

Figure 2. Effect of UCA1 on GBC cell growth in vitro

(A) Relative expression of UCA1 in si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells was detected by qRT-PCR. (B) The cell viability of si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells was determined by CCK8 assays. (C) The coloning ability of si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells was determined by colony formation assays. (D) The cell viability of si-UCA1-transfected NOZ cells was determined by EdU retention assay. (E) Flow cytometric analyses were performed to determine the cell cycle progression in si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells. The mean ± SD of triplicate experiments were plotted, *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Effect of UCA1 overexpression on tumor growth in vivo

(A) The nude mice carrying tumors from GBC-SD/LV-UCA1 and GBC-SD/LV-NC groups were shown. Tumor growth curves were calculated per week. (B) Tumor weight from GBC-SD/LV-UCA1 and GBC-SD/LV-NC groups was shown. (C) Relative expression of UCA1 in tumors from GBC-SD/LV-UCA1 and GBC-SD/LV-NC groups was detected by qRT-PCR. (D) The Ki-67 expression and positive cell numbers was determined by immunohistochemical staining. The mean ± SD of triplicate experiments were plotted, *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Effect of UCA1 on GBC cell metastasis and EMT in vitro

(A) The cell migration and invasion ability of si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells were determined by wound healing and transwell invasion assays, respectively. (B and C) The protein levels of E-cadherin and Vimentin in si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells were determined by immunofluorescenceand western blot assays. (D) Relative expression of UCA1 in TGF-β1 treated NOZ and GBC-SD cells was detected by qRT-PCR. The mean ± SD of triplicate experiments were plotted, **p<0.01, ***p<0.001.

Figure 5. UCA1 bound with EZH2 to epigenetically repress p21 and E-cadherin transcription

(A) Relative expression of UCA1 in cell cytoplasm or nucleus of NOZ and GBC-SD cells was detected by qRT-PCR. (B) Amount of UCA1 bound to SNRNP70 (a positive control), EZH2 or IgG (a negative control) was detected by qRT-PCR after RIP in NOZ and GBC-SD cells. (C) RNA pull-down assay was conducted using biotin-labeled UCA1 probe and detected the EZH2 expression by western blot assay. Antisense of the UCA1 probe was used as negative control. (D) Relative expression of p15, p16, p21, p53, p57, KLF2, E-cadherin, FBXO32, BRCA1, TRAIL, NKD2, DDIT3, DAB2IP, PLK3, CLDN14, LATS2 and PTEN mRNA in si-UCA1-transfected NOZ cells, relative expression of p21 and E-cadherin mRNA in pcDNA-UCA1-transfected GBC-SD cells was determined by qRT-PCR. (E) The protein levels of p21 in si-UCA1-transfected NOZ cells, or pcDNA-UCA1-transfected GBC-SD cells were determined by western blot assay. (F) Relative expression of p21 and E-cadherin mRNA, protein levels of p21 and E-cadherin in EZH2 inhibitors treated NOZ and GBC-SD cells were determined by qRT-PCR and western blot assay. (G) ChIP-qRT-PCR analysis of EZH2 occupancy, H3K27me3 binding to the p21 or E-cadherin promoter regions in NOZ and SGC-996 cells, and IgG as a negative control. The mean ± SD of triplicate experiments were plotted, *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Effect of p21 on GBC cell growth in vitro

(A) The protein levels of p21 in si-p21-transfected NOZ and GBC-SD cells were determined by western blot assay. (B) The cell viability of si-p21-transfected NOZ and GBC-SD cells was determined by CCK8 assays. (C) The coloning ability of si-p21-transfected NOZ and GBC-SD cells was determined by colony formation assays. (D) Flow cytometric analyses were performed to determine the cell cycle progression in si-p21-transfected NOZ and GBC-SD cells. The mean ± SD of triplicate experiments were plotted, *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Effect of UCA1/EZH2/p21 and UCA1/EZH2/E-cadherin axes on GBC progression

(A) The cell viability of si-NC-, si-UCA1- or si-UCA1 and si-p21-co-transfected NOZ cells was determined by CCK8 assays. (B) The coloning ability of si-NC-, si-UCA1- or si-UCA1 and si-p21-co-transfected NOZ cells was determined by colony formation assays. (C) Flow cytometric analyses were performed to determine the cell cycle progression in si-NC-, si-UCA1- or si-UCA1 and si-p21-co-transfected NOZ cells. (D) The protein levels of E-cadherin in pcDNA-NC-, pcDNA-UCA1- or EZH2 inhibitors treated and pcDNA-UCA1-transfected GBC-SD cells were determined by western blot assay. (E) The cell invasion ability in pcDNA-NC-, pcDNA-UCA1- or EZH2 inhibitors treated and pcDNA-UCA1-transfected GBC-SD cells was determined by transwell invasion assay. The mean ± SD of triplicate experiments were plotted, *p<0.05, **p<0.01, ***p<0.001.