LncRNA RCAT1 promotes tumor progression and metastasis via miR-214-5p/E2F2 axis in renal cell carcinoma

Abstract

Renal cell carcinoma is the second malignant tumors in the urinary system with high mortality and morbidity. Increasing evidence suggests that long non-coding RNAs (lncRNAs) play critical roles in tumor development and progression. In the current study, based on the publicly available data obtained from GEO and TCGA database, we identified five prognosis-related lncRNAs with the ability to predict the prognosis of patients with renal cell carcinoma. Among them, the uncharacterized and upregulated lncRNA RCAT1 (renal cancer-associated transcript 1) was identified as the key lncRNA. Our data further revealed that the expression of lncRNA RCAT1 was significantly upregulated in renal cell carcinoma tissues and cells. Gain-of-function and loss-of-function studies showed that lncRNA RCAT1 promoted cell proliferation, migration, and invasion in vitro and in vivo. Furthermore, we verified that lncRNA RCAT1 could abundantly sponge miR-214-5p, which served as a tumor suppressor in renal cell carcinoma. Significantly, miR-214-5p overexpression could attenuate the promotion of cell proliferation and metastasis induced by lncRNA RCAT1. Moreover, we found that E2F2 was a direct target of miR-214-5p, and lncRNA RCAT1 could protect E2F2 from miR-214-5p-mediated degradation. Taken together, our findings suggested that lncRNA RCAT1 could enhance the malignant phenotype of renal cell carcinoma cells by modulating miR-214-5p/E2F2 axis, and lncRNA RCAT1 might be a novel prognostic biomarker and a potential therapeutic target for renal cell carcinoma.

Fig. 1. Identification of differentially expressed lncRNAs.

a, b Hierarchical clustering heatmap (a) and volcano plots (b) of TCGA and GSE96574 showing the differentially expressed lncRNAs between ccRCC samples and normal samples. c Comparison of the differentially upregulated or downregulated lncRNAs in the two datasets. d A Circos plot was used to show the differentially expressed lncRNAs (log₂ fold change (FC) | > 1, P < 0.05). The inner circle shows the downregulated lncRNAs (purple dots) and the second circle identifies the upregulated lncRNAs in ccRCC tissues (red triangles) according to GSE96574. The third circle shows the downregulated lncRNAs (purple dots) and the fourth circle identifies the upregulated lncRNAs in ccRCC tissues (red triangles) according to TCGA. The outside circle represents the overlap of upregulated (pink dot) or downregulated lncRNAs (blue dots).

Fig. 2. The lncRNA prognostic model.

a Lasso regression analysis of 94 DElncRNAs. Those with P < 0.05 were showed. b A forest plot illustrating the HR and P-value from the multivariate cox regression analysis of 24 DElncRNAs. Those with P < 0.05 were showed. c The bar plot shows coefficients of five lncRNAs in the prognostic model. d Risk score system of the prognostic model. The above scatterplot exhibits the risk scores of each ccRCC patient with survival data. The middle scatterplot showed the relationship between the risk scores and the survival status/survival time. The below heatmap displays the expression profiles of the five lncRNAs in the prognostic model. e The time-dependent ROC curve of OS suggests the reliability of the prognostic model. f Kaplan–Meier overall survival curves for ccRCC patients exhibited that the OS of high-risk group was shorter than the OS of low-risk group.

Fig. 3. High lncRNA RCAT1 expression predicts worse prognosis of patients with ccRCC.

a The differential expression of lncRNA RCAT1 in ccRCC tissues and normal tissues. b The differential expression of lncRNA RCAT1 in ccRCC cells and normal cells. c The Kaplan–Meier analysis was used to evaluate the relationship between lncRNA RCAT1 expression and overall survival time of ccRCC patients. d–g The differential expression of lncRNA RCAT1 in indicated ccRCC tissues according to TCGA database (^*P < 0.05, ^***P < 0.001).

Fig. 4. lncRNA RCAT1 knockdown suppresses malignant phenotypes in RCC cells.

a The lncRNA RCAT1 mRNA levels in 786-O and 769-P cells transfected with lncRNA RCAT1 or negative control siRNAs. b The cell proliferation of 786-O and 769-P cells in response to lncRNA RCAT1 knockdown was measured using MTT assay. c Colony-formation assays performed with the 786-O and 769-P cells transfected with lncRNA RCAT1 or negative control siRNAs. d EdU assay was used to evaluate the effect of lncRNA RCAT1 knockdown on cell proliferation. Scale bar, 200 μm. e Apoptosis was assayed by flow cytometry in 786-O and 769-P cells after lncRNA RCAT1 knockdown. f The wound-healing assay was performed to examine the migration abilities after lncRNA RCAT1 knockdown. Scale bar, 200 μm. g The effects of lncRNA RCAT1 knockdown on migration and invasive abilities of 786-O and 769-P cells were evaluated by the transwell assays. Scale bar, 200 μm (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Fig. 5. MiR-214-5p was one target of lncRNA RCAT1.

a The subcellular location of lncRNA RCAT1 in 786-O cells. U6 and GAPDH were used as internal controls. b FISH assay was used to detect the subcellular location of lncRNA RCAT1 in 786-O cells. Scale bar, 2000 μm. c The lncRNA RCAT1-centric ceRNA network. d Predicted binding sites of miR-214-5p in lncRNA RCAT1 sequence. e Dual-luciferase assay was performed to confirm the interaction between miR-214-5p and lncRNA RCAT1. f The expression levels of lncRNA RCAT1 and miR-214-5p were detected in the substrate of RIP assay by qRT-PCR. g lncRNA RCAT1 knockdown led to increased expression of miR-214-5p (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Fig. 6. miR-214-5p negatively regulates the function of lncRNA RCAT1.

a The efficiency of miR-214-5p mimics was detected by qRT-PCR. b Overexpression of miR-214-5p led to decreased cell proliferation. c Overexpression of miR-214-5p led to increased cell apoptosis. d Overexpression of miR-214-5p led to decreased cell migration. Scale bar, 200 μm. e Overexpression of miR-214-5p attenuated the effect on promoting proliferation of lncRNA RCAT1 by MTT assay. f Overexpression of miR-214-5p effectively reverses lncRNA RCAT1-induced promotion of cell migration using transwell assay. Scale bar, 200 μm (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Fig. 7. lncRNA RCAT1 promoted RCC cell progression through protecting E2F2 from miR-214-5p-induced degradation.

a The differential expression of E2F2 in ccRCC tissues and normal tissues based on TCGA and GEO databases. b The differential expression of E2F2 in ccRCC tissues with or without metastasis. c The Kaplan–Meier analysis was used to evaluate the relationship between E2F2 expression and overall survival time of ccRCC patients. d, e Overexpression of miR-214-5p (d) or lncRNA RCAT1 (e) knockdown led to decreased mRNA and protein levels of E2F2 in RCC cells. f Overexpression of miR-214-5p effectively reverses lncRNA RCAT1-induced increased mRNA and protein levels of E2F2. g The schematic illustration showing the predicted binding sites of miR-214-5p in 3′UTR of E2F2. h Luciferase assay was used to show the regulatory relationship between miR-214-5p and E2F2. i The efficiency of E2F2 knockdown was detected by qRT-PCR and western blot. j Cell proliferation was evaluated after E2F2 knockdown using MTT assay. k Cell apoptosis was evaluated after E2F2 knockdown using flow cytometry assay. l Cells migration and invasion abilities were detected after E2F2 knockdown by transwell assays. Scale bar, 200 μm (^**P < 0.01, ^***P < 0.001).

Fig. 8. lncRNA RCAT1 knockdown suppresses tumor proliferation and metastasis in vivo.

a–c The photos (a), growth curve (b), and weight (c) of the xenograft tumors. Scale bar, 1 cm. d H&E staining of the xenograft tumors. IHC results of Ki67 level and E2F2 level in xenograft tumor tissues. Scale bar, 100 μm. e Representative images of lung metastatic nodules and H&E staining of lungs isolated from nude mice (n = 5 for each group). Scale bar, 100 μm. f The numbers of metastatic nodules in the lungs of nude mice were calculated and compared. Scare bar = 50 μm (^***P < 0.001).