hsa_circ_0003596, as a novel oncogene, regulates the malignant behavior of renal cell carcinoma by modulating glycolysis

Abstract

Background: This research was planned to analyze hsa_circ_0003596 (circCOL5A1) and glycolysis-focused mechanisms in renal cell carcinoma (RCC).

Methods: circCOL5A1, miR-370-5p, and PRKCSH levels were determined in RCC tissues and selected cell lines by RT-qPCR and/or Western blot. RCC cells after corresponding transfection were tested by colony formation assay, EdU assay, Transwell assay, and flow cytometry to analyze cell proliferation, invasion, migration, and apoptosis. Meanwhile, glycolysis in cells was evaluated by measuring glucose consumption, lactic acid, and ATP production, as well as immunoblotting for HK2 and PKM2. In addition, circCOL5A1 knockdown was performed in animal experiments to observe tumor growth and glycolysis. Finally, the ceRNA network between circCOL5A1, miR-370-5p, and PRKCSH was studied by luciferase reporter assay and RIP experiment.

Results: circCOL5A1 and PRKCSH were highly expressed and miR-370-5p was poorly expressed in RCC. circCOL5A1 knockdown depressed RCC proliferation, invasion, migration, and glycolysis, and enhanced apoptosis. circCOL5A1 competitively adsorbed miR-370-5p. Artificial upregulation of miR-370-5p saved the pro-tumor effect of circCOL5A1 on RCC cells, as evidenced by suppression of tumor malignancy and glycolysis. miR-370-5p targeted PRKCSH. PRKCSH overexpression contributed to a reversal of the anti-tumor effect of circCOL5A1 silencing. Silencing circCOL5A1 inhibited RCC tumor growth and glycolysis.

Conclusions: circCOL5A1 regulates the malignant behavior of RCC by modulating glycolysis.

Keywords: Glycolysis; Renal cell carcinoma; hsa_circ_0003596; miR-370-5p; PRKCSH.

Fig. 1

Abnormally high expression of circCOL5A1 in RCC. A Heat map of differential expression of circRNAs in normal tissue and RCC tissue; B volcanic maps of different circRNAs; C top 10 circRNAs with highest expression and lowest expression; D changes of circCOL5A1 expression; E genetic information of circCOL5A1; F circCOL5A1 in RCC tissues and normal tissues analyzed by RT-qPCR. G circCOL5A1 in HK-2 and human RCC cell lines analyzed by RT-qPCR; H, I the stability of circCOL5A1 evaluated by actinomycin D assay and RNAse R experiment; J gel electrophoresis to examine the products of RT-qPCR amplification using polymeric and diverging primers; data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 2

circCOL5A1 knockdown impairs RCC progression. circCOL5A1 targeted siRNA was transfected into ACHN cells. A circCOL5A1 expression analyzed by RT-qPCR; B, C cell proliferation detected by clonal assay and EdU assay; D apoptosis evaluated by flow cytometry; E invasion and migration evaluated by Transwell assays; F–H glucose consumption, lactic acid production, and ATP levels; I HK2 and PKM2 detected by Western blot; data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 3

Competitive adsorption of miR-370-5p by circCOL5A1 in RCC. A RT-qPCR detection of miRNA expression after circCOL5A1 knockdown; B subcellular localization of miR-370-5p and circCOL5A1 assayed by FISH; C potential binding sites of miR-370-5p and circCOL5A1; D, E the targeting relationship between miR-370-5p and circCOL5A1 detected by dual luciferase reporter assay and RIP experiment; F miR-370-5p expression in RCC tissues and normal tissues analyzed by RT-qPCR; G: miR-370-5p expression in HK-2 and human RCC cell lines analyzed by RT-qPCR; data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 4

miR-370-5p is involved in the process of circCOL5A1 regulation of RCC. pcDNA 3.1-circCOL5A1 and miR-370-5p mimic were co-transfected into ACHN cells. A circCOL5A1 and miR-370-5p expression analyzed by RT-qPCR; B, C cell proliferation detected by clonal assay and EdU assay; D apoptosis evaluated by flow cytometry; E invasion and migration evaluated by Transwell assays; F–H glucose consumption, lactic acid production, and ATP levels; I HK2 and PKM2 detected by Western blot; Data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 5

Targeted regulation of PRKCSH by miR-370-5p. A RT-qPCR detection of mRNA expression after overexpressing miR-370-5p; B potential binding sites of miR-370-5p and PRKCSH; C, D the targeting relationship between miR-370-5p and PRKCSH detected by dual luciferase reporter assay and RIP experiment; E PRKCSH expression in RCC tissues and normal tissues analyzed by Western blot; F PRKCSH expression in HK-2 and human RCC cell lines analyzed by Western blot; data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 6

CircCOL5A1 affects RCC progression via miR-370-5p/PRKCSH axis. si-circCOL5A1 and pcDNA 3.1-PRKCSH were co-transfected into ACHN cells. A PRKCSH expression analyzed by Western blot; B, C cell proliferation detected by clonal assay and EdU assay; D apoptosis evaluated by flow cytometry; E invasion and migration evaluated by Transwell assays; F–H glucose consumption, lactic acid production, and ATP levels; I HK2 and PKM2 detected by Western blot; Data were expressed as mean ± SD (N = 3). * P < 0.05

Fig. 7

CircCOL5A1 silencing delays the growth of RCC tumors. A representative pictures of tumors; B, C tumor volume and weight; D HK2 and PKM2 in tumors detected by IHC staining; E PRKCSH and Ki-67 in tumors detected by Western blot. Data were expressed as mean ± SD (n = 5). * P < 0.05