The Effects of circ_000558/miR-1225-5p/ARL4C on Regulating the Proliferation of Renal Cell Carcinoma Cells

Abstract

Renal cell carcinoma (RCC) is one of the top ten tumors over the world. RCC is not sensitive to radiotherapy and chemotherapy. Therefore, it is necessary to find new targets for the treatment. CircRNAs are a special type of noncoding RNAs, which play important roles in many types of cancer. In this study, we found circ_000558 was upregulated in RCC cells, and it elevated the proliferation ability of RCC cells. The relationship between miR-1225-5p and circ_000558 or ARL4C was predicted via circBank and circular RNA interactome and confirmed by dual-luciferase reporter assay. Then, the effects of circ_000558/miR-1225-5p/ARL4C on RCC cell proliferation and apoptosis were assessed by CCK-8 assay. The results revealed that the knockdown of ARL4C significantly reduced RCC cell proliferation and overexpression of circ_000558 could significantly induce RCC cell proliferation after miR-1225-5p treatment further promoted the inhibitory ability of ARL4C knockdown. Overall, our study suggested that circ_000558/miR-1225-5p/ARL4C network was related to the RCC cell proliferation. This finding could provide new targets for the treatment and prognosis of RCC.

Figure 1

The expression of circ_000558 in RCC cells. (a) Hot map showed the differential expressed circRNAs between RCC tissues and matched nontumor tissues in GSE100186. (b) The expression of circ_000558 in RCC cells (ACHN, 786-O, 769-P, and Caki-1) and human renal epithelial cells (HK2) was tested by qPCR. ^∗p < 0.05 and ^∗∗∗p < 0.001 compared to HK2 cells. (c, d) The expression location of circ_000558 in 769-P (c) and ACHN cells (d) was tested by qPCR analysis. (e, f) The expression of circ_000558 in 769-P cells (e) and ACHN cells (f) after RNase R digested was tested by qPCR assay. ^∗∗p < 0.01; ns, no significant difference.

Figure 2

The effects of circ_000558 on cell proliferation and apoptosis in RCC cells. (a) The expression of circ_000558 in 769-P and ACHN cells after transfected with si-NC and si-circ_000558 (si-circ) was tested by qPCR analysis. (b) The inhibitor rate of cell proliferation in 769-P and ACHN cells after si-NC and si-circ transfection was measured using CCK-8 assay. (c) The cell apoptosis in 769-P and ACHN cells after si-NC and si-circ transfection was detected by flow cytometry. (d) The protein expression of apoptosis related protein (Bcl-2, Bax, and cleaved caspase-3) was analyzed by western blot assay (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Figure 3

circ_000558 regulated RCC cell proliferation by inhibiting miR-1225-5p. (a) Venn diagram showed the overlapped potential target miRNAs in GSE23085, GSE16441, circBank, and circular RNA interactome. The binding site between miR-1225-5p and circ_000558 was shown. (b) The expression of miR-1225-5p in renal carcinoma cells (ACHN, 769-P, and Caki-1) and human renal epithelial cells (HK2) was tested by qPCR analysis. (c) The expression of miR-1225-5p in 769-P and ACHN cells after control and circ_000558 overexpression plasmid transfection was measured by qPCR. (d) The relative luciferase intensity in 769-P and ACHN cells after wild-type of circ_000558 (circ-WT) or mutation type of circ_000558 (circ-Mut) and miR-1225-5p mimics (miR-mimics) cotransfect was tested by dual-luciferase reporter assay. (e) The cell viability of 769-P and ACHN cells after miR-mimics, miR-inhibitor, or miR-mimics + ov-circ transfection was tested by CCK-8 assay. (f) The cell apoptosis of 769-P and ACHN cells after miR-mimics, miR-inhibitor, or miR-mimics + ov-circ transfection was tested by flow cytometry. (^∗∗p < 0.01; ^∗∗∗p < 0.001).

Figure 4

The effect of circ_000558/miR-1225-5p on RCC tumor growth in vivo. (a) The tumor tissues of control, si-circ, and si-circ + miR-mimics groups. (b) Tumor volume was measured every two days (n = 6). (c) Tumor weight was measured (^∗∗p < 0.01, ^∗∗∗p < 0.001).

Figure 5

ARL4C was a potential target gene of miR-1225-5p. (a) Venn diagram showing the overlapped potential target genes in TargetScan7.2, Targetprofiler, GSE16441, and GSE100666. The binding site between miR-1225-5p, ARL4C, and SEL1L3. (b, c) The expression profile of ARL4C (b) and SEL1L3 (c) in RCC samples by TCGA analysis. Blue, normal samples; red, tumor samples. (d, e) Effect of ARL4C (d) and SEL1L3 (e) expression levels on renal carcinoma patients' survival; red, higher expression; blue, lower expression. (f) The relative luciferase intensity in 769-P and ACHN cells after wild-type of ARL4C (ARL4C-WT) or mutation type of ARL4C (ARL4C-Mut) and miR-1225-5p mimics (miR-mimics) cotransfect was tested by dual-luciferase reporter assay (^∗∗∗p < 0.001).

Figure 6

circ_000558/miR-1225-5p/ARL4C regulated RCC cell proliferation. (a) The protein expression of ARL4C in RCC cells (ACHN, 769-P, and Caki-1) and human renal epithelial cells (HK2) was tested by qPCR. (b) qPCR analysis was applied to assess the expression of ARL4C. (c) Cell inhibitor ratio of 769-P and ACHN cells after si-ARL4C, si-ARL4C + ov-circ, si-ARL4C + miR-mimics, or si-ARL4C + miR-mimics + ov-circ transfection was tested by CCK-8 assay. (d) The cell apoptosis of 769-P and ACHN cells after si-ARL4C, si-ARL4C + ov-circ, si-ARL4C + miR-mimics, or si-ARL4C + miR-mimics + ov-circ transfection was tested by flow cytometry. (^∗∗p < 0.01 and ^∗∗∗p < 0.001).