CircPVT1 promotes progression in clear cell renal cell carcinoma by sponging miR-145-5p and regulating TBX15 expression

Abstract

Emerging evidence revealed that circular RNAs (circRNAs) play significant roles in regulating tumorigenesis and cancer progression. However, few circRNAs were well characterized in clear cell renal cell carcinoma (ccRCC). We found that circPVT1 was significantly upregulated in ccRCC tissues and positively associated with the clinical stage. The Area Under Curve of tissue and serum circPVT1 expression in ccRCC were 0.93 and 0.86, respectively. Importantly, we demonstrated that circPVT1 promoted ccRCC growth and metastasis in vitro and in vivo. We also found that circPVT1 directly binds to miRNA-145-5p via the Biotin-labelled miRNA pulldown assay and dual-luciferase reporter assay, and miR-145-5p inhibitor significantly attenuated the effect of circPVT1 knockdown on ccRCC cells. Moreover, through RNA sequencing and bioinformatics analysis, we demonstrated that TBX15 was regulated by the circPVT1/miR-145-5p axis and predicted poor prognosis in ccRCC. These findings suggest that circPVT1 promotes ccRCC growth and metastasis through sponging miR-145-5p and regulating downstream target TBX15 expression. The circPVT1/miR-145-5p/TBX15 axis might be a potential diagnostic marker and therapeutic target in ccRCC.

Keywords: ccRCC; circPVT1; invasion; miR-145-5p; proliferation.

FIGURE 1

Characterization and expression of circPVT1 in clear cell renal cell carcinoma (ccRCC). A, Heat map for differentially expressed circular RNAs (circRNAs) in seven pairs of ccRCC tissues and adjacent normal tissues from GSE108735. B, Relative expression of circPVT1 in ccRCC tissues (n = 7) and adjacent normal tissues (n = 7) from GSE108735. C, Schematic illustration of circPVT1 produced from exon 3 of PVT1 gene. D, circPVT1 junction site in circBase was validated by Sanger sequencing. E, circPVT1 is resistant to RNase R in ccRCC cell lines. F, RNA FISH was performed and confocal microscopy was used to detect the localization of circPVT1 in the ccRCC cells. G, Subcellular fractionation and qRT‐PCR were performed to detect the localization of circPVT1 in ccRCC cells. H, Expression of chromosome interval containing circPVT1 between ccRCC tissues (n = 448) and normal tissues (n = 67) in TCGA database. I, Expression of circPVT1 in 90 ccRCC tissues and 90 adjacent normal tissues quantified by qRT‐PCR. J, Expression of circPVT1 in ccRCC cell lines quantified by qRT‐PCR. *P < .05, **P < .01, ***P < .001

FIGURE 2

Diagnostic value of circPVT1 for clear cell renal cell carcinoma (ccRCC) patients. A, circPVT1 expression in serum samples of 60 ccRCC patients and 40 healthy volunteers quantified by qRT‐PCR. B, Receiver operating characteristic (ROC) curve of tissue circPVT1 and serum circPVT1 expression to distinguish ccRCC from normal controls. C, The correlation between 31 serum and paired tissue expressions of circPVT1 of ccRCC patients was determined by Pearson correlation coefficients (r = .680, n = 31, P < .001). ***P < .001

FIGURE 3

circPVT1 knockdown suppressed the proliferative and invasive phenotype in clear cell renal cell carcinoma (ccRCC) cell lines. A, Quantification of circPVT1 expression assessed by qRT‐PCR in ccRCC cell lines treated with si‐circPVT1 or negative control. B, MTS assay was performed to detect the proliferation of ccRCC cell lines treated with si‐circPVT1 or negative control. C, Edu assay was performed to detect the proliferation of ccRCC cell lines treated with si‐circPVT1 or negative control. Representative images and quantification results are shown. D, Cell cycle of ccRCC cell lines treated with si‐circPVT1 or negative control was assessed by flow cytometry analysis. Quantification results are shown. E, Representative images and quantification results of cell migration and invasion of ccRCC cell lines treated with si‐circPVT1 or negative control. *P < .05, **P < .01, ***P < .001

FIGURE 4

circPVT1 overexpression promoted the proliferation and invasion of clear cell renal cell carcinoma (ccRCC) in vitro and in vivo. A, Representative images of quantification results of ccRCC cells transfected with circPVT1‐overexpressing vector and negative control. B, MTS assay was performed to assess the proliferation ability of ccRCC cells transfected with circPVT1‐overexpressing vector and negative control. C, Transwell migration assay was performed to detect the migratory ability of ccRCC cells transfected with circPVT1‐overexpressing vector and negative control. D, Transwell invasion assay was performed to detect the invasive ability of ccRCC cells transfected with circPVT1‐overexpressing vector and negative control. E, Xenograft tumor models showed that circPVT1 overexpression promoted ccRCC growth in vivo. F, Analysis of tumor weight in the circPVT1 overexpression group and negative control group (n = 5). G, Analysis of tumor volume in the circPVT1 overexpression group and negative control group (n = 5). H, Hematogenous metastasis model showed that circPVT1 overexpression promoted ccRCC metastasis in vivo. I, Analysis of tumor number in the circPVT1 overexpression group and negative control group (n = 5). *P < .05, **P < .01, ***P < .001

FIGURE 5

circPVT1 directly binds to miR‐145‐5p. A, A bioinformatics tool CircInteractome was used to predict miRNA targets of circPVT1. B‐D, Relative miRNA expression in clear cell renal cell carcinoma (ccRCC) cell line treated with si‐circPVT1 or negative control. E, Biotin‐labeled miRNA pulldown assay was performed to detect the direct binding of circPVT1 and miR‐145‐5p in ccRCC cell lines. F, 293T cell was cotransfected with circPVT1‐wt or circPVT1‐mut with miR‐145‐5p mimic. *P < .05, **P < .01, ***P < .001

FIGURE 6

miR‐145‐5p inhibitor attenuated the effect of circPVT1 knockdown on clear cell renal cell carcinoma (ccRCC) cells. A, Edu assay was performed to detect the proliferation of ccRCC cell lines treated with mimic‐NC or miR‐145‐5p mimic. B, Edu assay was performed to detect the proliferation of ccRCC cell lines treated with inhibitor‐NC and miR‐145‐5p inhibitor. C, Transwell assay was performed to detect the migratory ability of ccRCC cell lines treated with mimic‐NC or miR‐145‐5p mimic. D, Transwell assay was performed to detect the migratory ability of ccRCC cell lines treated with inhibitor‐NC and miR‐145‐5p inhibitor. E, MTS assay was performed to detect the proliferative effect of miR‐145‐5p on circPVT1‐knockdown ccRCC cells. F, Transwell assay was performed to detect the migratory effect of miR‐145‐5p on circPVT1‐knockdown ccRCC cells. *P < .05, **P < .01, ***P < .001

FIGURE 7

circPVT1 regulated the miR‐145‐5p/TBX15 axis in clear cell renal cell carcinoma (ccRCC). A, Volcano plot visualizes the differentially expressed mRNAs in circPVT1‐knockdown and negative control cells. The red and blue dots represent upregulated and downregulated mRNAs with fold change > 2, respectively. B, KEGG pathway analysis of differentially expressed mRNAs in circPVT1‐knockdown and negative control cells. C, Venn diagram showing 18 miR‐145‐5p‐regulated genes predicted by RNA‐seq, TargetScan, and MiRPathDB. D, Kaplan‐Meier analysis was performed to assess the relationship between TBX15 expression and overall survival of ccRCC patients. E, Kaplan‐Meier analysis was performed to assess the relationship between TBX15 expression and disease‐free survival of ccRCC patients. F, Relative expression of TBX15 mRNA between ccRCC tissues and adjacent normal tissues from TCGA database. G, Relative expression of TBX15 mRNA between 20 pairs of ccRCC tissues and adjacent normal tissues. H, 293T cell was cotransfected with TBX15‐Wild or TBX15‐Mut with miR‐145‐5p mimic. I, Relative expression of TBX15 mRNA in ccRCC cell lines treated with Si#circPVT1 or Si#NC. J, Relative expression of TBX15 mRNA in ccRCC cell lines treated with mimic‐NC or miR‐145‐5p mimic. K, Western blot analysis of TBX15 in ccRCC cell lines treated with circPVT1 knockdown or miR‐145‐5p mimic. Representative images of TBX15 in ccRCC tissues relative to normal adjacent tissues are shown. L, Representative immunohistochemistry (IHC) images of TBX15 in ccRCC tissues and normal adjacent tissues. *P < .05, **P < .01, ***P < .001