Circ_0035483 Functions as a Tumor Promoter in Renal Cell Carcinoma via the miR-31-5p-Mediated HMGA1 Upregulation

Abstract

Background: Renal cell carcinoma (RCC) that originates from the proximal renal tubules is the most common cancer of the human kidney. Increasing circRNA/miRNA/mRNA networks have been found in RCC regulation. This study will explore the regulatory relation of circular RNA (circRNA) circ_0035483, microRNA-31-5p (miR-31-5p) and high mobility group A1 (HMGA1).

Methods: The levels of circ_0035483, miR-31-5p and HMGA1 were measured by real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell proliferation was determined using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cell migration and invasion were assessed by transwell assay. HMGA1 and epithelial-mesenchymal transition (EMT)-related protein levels were quantified using Western blot. Glycolytic metabolism was evaluated by glucose consumption and lactate production. The interaction between targets was confirmed via dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. In vivo experiment was performed through the establishment of xenograft models in mice.

Results: Circ_0035483 expression was upregulated in RCC tissues and cells. The inhibitory effects on RCC cell proliferation, migration, invasion, EMT and glycolysis were induced after circ_0035483 was downregulated. MiR-31-5p was identified as a target of circ_0035483 and miR-31-5p upregulation was related to the function of circ_0035483 knockdown in RCC cells. Additionally, miR-31-5p targeted HMGA1 and inhibited the malignant behaviors of RCC cells by negatively regulating HMGA1. Moreover, HMGA1 expression was regulated by circ_0035483 via targeting miR-31-5p. Circ_0035483 also affected tumor growth in vivo by relying on the miR-31-5p/HMGA1 axis.

Conclusion: These findings clarified that the tumor-promoting function of circ_0035483 in RCC was partly achieved by regulating the miR-31-5p/HMGA1 axis.

Keywords: HMGA1; circ_0035483; miR-31-5p; renal cell carcinoma; RCC.

Figure 1

Circ_0035483 level was overexpressed in RCC tissues and cells. (A and B) The relative circ_0035483 expression was examined by qRT-PCR in RCC tissues and cells (786-O and Caki-1), as well as the normal tissues and HK-2 cells. *P < 0.05.

Figure 2

Silencing circ_0035483 retarded proliferation, migration, invasion and glycolysis of RCC cells. Si-NC, si-circ_0035483#1 or si-circ_0035483#2 was transfected into 786-O and CaKi-1 cells. (A) The qRT-PCR was used to measure the expression level of circ_0035483. (B–D) CCK-8 assay (B and C) and colony formation assay (D) were performed for detecting the proliferative ability. (E and F) Transwell assay was used for migration/invasion detection. (G) Western blot was used for the detection of EMT markers. (H and I) Glucose consumption and lactate production were determined for the glycolytic evaluation. *P < 0.05.

Figure 3

MiR-31-5p was a miRNA target of circ_0035483. (A) The mutual miRNAs were screened by Veen diagram after the target prediction of circ_0035483 using circinteractome, circBank and Starbase3.0. (B) The detection of miR-31-5p, miR-432-5p, miR-582-5p was carried out by qRT-PCR in RCC tissues. (C) The binding sites of miR-31-5p were presented in the sequence of circ_0035483 by circinteractome. (D) The transfection efficiency of miR-31-5p mimic was analyzed using qRT-PCR. (E–I) The binding between miR-31-5p and circ_0035483 was performed using the dual-luciferase reporter assay (E and F), RIP (G and H) and RNA pull-down assay (I). (J) The level of miR-31-5p was assayed via qRT-PCR in si-NC or si-circ_0035483#2 transfected 786-O and CaKi-1 cells. (K and L) The qRT-PCR was applied for detecting the miR-31-5p expression in RCC tissues (K) and cells (L). *P < 0.05.

Figure 4

Circ_0035483 affected the malignant behaviors of RCC cells via negatively regulating miR-31-5p. (A) The miR-31-5p expression was quantified by qRT-PCR after transfection of anti-miR-NC or anti-miR-31-5p. (B–H) The cellular analysis was performed by CCK-8 assay (B and C) and colony formation assay (D) for cell proliferation, transwell assay for cell migration (E) and invasion (F), Western blot for EMT protein detection (G and H) in si-NC, si-circ_0035483#2, si-circ_0035483#2+anti-miR-NC or si-circ_0035483#2+anti-miR-31-5p. (I and J) Glycolysis was analyzed by examining the glucose consumption (I) and lactate production (J) in the above four groups. *P < 0.05.

Figure 5

HMGA1 was a downstream target of miR-31-5p. (A) Starbase3.0 showed the binding sites of miR-31-5p in HMGA1 3ʹUTR sequence. (B–E) Dual-luciferase reporter assay (B and C) and RIP assay (D and E) were conducted for the analysis of interaction between miR-31-5p and HMGA1. (F and G) The qRT-PCR and Western blot were adopted for assessing the effects of miR-31-5p inhibition or overexpression on HMGA1 mRNA and protein levels in 786-O and CaKi-1 cells. (H–K) HMGA1 expression was assayed by mRNA level via qRT-PCR and protein level via Western blot in RCC/Normal tissues (H and I) and RCC/HK-2 cells (J and K). *P < 0.05.

Figure 6

MiR-31-5p downregulated HMGA1 to impede the progression of RCC. (A and B) The mRNA and protein levels of HMGA1 were measured by qRT-PCR and Western blot in 786-O and CaKi-1 cells transfected with pcDNA or pcDNA-HMGA1. (C–I) Functional experiments were conducted by cell proliferation using CCK-8 assay (C and D) and colony formation assay (E), cell migration (F) and invasion (G) using transwell assay, EMT protein detection (H and I) using Western blot after transfection of miR-NC, miR-31-5p, miR-31-5p+pcDNA or miR-31-5p+pcDNA-HMGA1. (J and K) The assessment of glycolytic metabolism was carried out by glucose consumption (J) and lactate production (K) in four transfection groups. *P < 0.05.

Figure 7

Circ_0035483 could regulate HMGA1 by serving as a sponge of miR-31-5p (A and B) After 786-O and CaKi-1 cells were transfected with si-circ_0035483#2, si-circ_0035483#2+anti-miR-31-5p or the corresponding controls, the detection of HMGA1 mRNA and protein expression was performed using qRT-PCR and Western blot. (C–E) The linear correlations among circ_0035483, miR-31-5p and HMGA1 expression in RCC tissues were analyzed by Pearson’s correlation coefficient. *P < 0.05.

Figure 8

RCC tumor growth in vivo was reduced by downregulation of circ_0035483 via the miR-31-5p/HMGA1 axis. (A and B) Tumor growth was assessed by measuring tumor volume (A) and weight (B) in xenograft model of sh-NC or sh-circ_0035483 group. (C and D) The qRT-PCR was applied to analyze the relative levels of circ_0035483 (C) and miR-31-5p (D) in tumor tissues. (E and F) The quantification of HMGA1 was performed by qRT-PCR and Western blot in tumor tissues. *P < 0.05.