CircRNA hsa_circ_0004781 promoted cell proliferation by acting as a sponge for miR-9-5p and miR-338-3p and upregulating KLF5 and ADAM17 expression in pancreatic ductal adenocarcinoma

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive types of solid tumor, and novel strategies must be developed for treating it. Previous studies predominantly utilized circular RNA (circRNA) expression plasmids incorporating Alu elements to facilitate the indirect expression of circRNA.

Methods: Public databases and bioinformatics tools were used to identify hsa_circ_0004781 that is highly expressed in PDAC and its potential microRNA (miRNA) targets and corresponding mRNA targets. Real hsa_circ_0004781, which is identical to the native form of hsa_circ_0004781 without any exogenous sequences, was prepared through in vitro transcription by using a ribozyme and ion-pair reversed-phase high-performance liquid chromatography (IP-RP HPLC). The biological functions of hsa_circ_0004781 were evaluated using loss-of-function and gain-of-function approaches with circRNA expression plasmids and real hsa_circ_0004781.

Results: Knockdown of hsa_circ_0004781 inhibited the proliferation and migration of PDAC cells, whereas its overexpression produced opposite effects. Hsa_circ_0004781 was identified as a sponge for miR-9-5p and miR-338-3p, and its expression was negatively correlated with that of these miRNAs. Among the targets of miR-9-5p and miR-338-3p, Kruppel-like factor 5 (KLF5) and a disintegrin and metalloproteinase domain 17 (ADAM17) were negatively correlated with survival in patients with PDAC and were inversely regulated by these miRNAs. Furthermore, real hsa_circ_0004781 exhibited the same effects as those of the circRNA expression plasmids.

Conclusions: This study is the first to use real circRNAs to validate results obtained using circRNA expression plasmids. The results suggest that hsa_circ_0004781 functions as an oncogene, promoting the proliferation of PDAC cells through the miR-9-5p/KLF5 and miR-338-3p/ADAM17 axes. Therefore, hsa_circ_0004781 might be a therapeutic target for PDAC.

Keywords: ADAM17; Hsa_circ_0004781; KLF5; Pancreatic ductal adenocarcinoma; Real circRNA; miR-338-3p; miR-9-5p.

Fig. 1

Identification of hsa_circ_0004781 in PDAC cells. (A) Schematic illustrating the canonical and back-splicing of PCNX1 pre-mRNA, presenting three paired primer annealing sites for PCNX1 mRNA and hsa_circ_0004781. (B) Total RNA from ASPC-1 and BXPC-3 cells was extracted, and hsa_circ_0004781 (c4781) was amplified through RT-PCR by using divergent primers, followed by agarose gel electrophoresis. (C) The hsa_circ_0004781 band was extracted from the gel, and the sequence of the BSJ region was determined through Sanger sequencing. F, forward strand; R, reverse strand. (D) cDNA and gDNA prepared from AsPC-1 and BxPC-3 cells were used to amplify the BSJ region of hsa_circ_0004781 and exon 2 of the PCNX1 gene by using divergent primers and convergent primer 1, respectively. (E) Total RNA extracted from AsPC-1 cells treated with or without RNase R was used to amplify the BSJ region of hsa_circ_0004781 and exon 2 of PCNX1 mRNA through RT-PCR with divergent primers and convergent primer 2, respectively

Fig. 2

Effects of the knockdown and overexpression of hsa_circ_0004781 on PDAC cell proliferation. (A) hsa_circ_0004781 siRNA (si-c4781) was designed to target the BSJ region of hsa_circ_0004781 (c4781), and si-c4781 sequences are presented. (B) The pcDNA3.1(+) ZKSCAN1 MCS Exon vector was used to construct the pcDNA-ZKSCAN-c4781 plasmid, which transcribed a pre-mRNA containing exons 2–5 of PCNX1 mRNA, the partial splicing acceptor (SA) and splicing donor (SD) intronic regions of the PCNX1 gene, and Alu sequences of the ZKSCAN1 gene. (C, D) AsPC-1 and BxPC-3 cells were transfected with (C) control siRNA (si-Ctr) or si-c4781 and (D) the control vector (OE-Ctr) or pcDNA-ZKSCAN-c4781 plasmid (OE-c4781) for 1–3 days. Total RNA was extracted, and the expression of hsa_circ_0004781 was determined through RT-PCR. Cell proliferation was examined using an MTT assay. Values are presented as mean ± SE, n > 3. ^##p < 0.0001 compared with si-Ctr or OE-Ctr

Fig. 3

Effects of the knockdown and overexpression of hsa_circ_0004781 on the cell migration and expression of EMT-related marker genes in PDAC cells. (A) AsPC-1 cells were transfected with control siRNA (si-Ctr) or hsa_circ_0004781 siRNA (si-c4781) for 24 h, and the number of migrating cells was determined by performing a wound healing assay. A representative photo and quantified data are presented. Each value represents the mean ± SE of three independent experiments. **p < 0.01 vs. si-Ctr. (B-E)AsPC-1 and BxPC-3 cells were transfected with (B, D) control siRNA (si-Ctr) or si-c4781 and (C, E) the control vector (OE-Ctr) or the pcDNA-ZKSCAN-c4781 plasmid (OE-c4781) for 24 h. (B, C) Total proteins were extracted, and protein expression was determined through a Western blot assay. (D, E) Total RNA was extracted, and mRNA expression was determined through RT-qPCR. Values are presented as the mean ± SE from three independent experiments. *p < 0.05, ^#p < 0.001, ^##p < 0.0001 vs. si-Ctr or OE-Ctr. E-cad, E-cadherin; Vim, vimentin

Fig. 4

Effects of the knockdown of hsa_circ_0004781 on the expression of miR-9-5p and miR-338-3p in PDAC cells. (A) Potential target miRNAs of hsa_circ_0004781 were predicted using ENCORI, circBank, and CircInteractome. miR-9-5p and miR-338-3p were selected on the basis of their downregulation in PDAC. Bioinformatics analysis predicted potential miR-9-5p and miR-338-3p binding sites on hsa_circ_0004781. (B) AsPC-1 and BxPC-3 cells were transfected with control siRNA (si-Ctr) or hsa_circ_0004781 siRNA (si-c4781) for 24 h. Total RNA was extracted, and the expression of miR-9-5p and miR-338-3p were determined through RT-qPCR. Values are presented as the mean ± SE from three independent experiments. ^#p < 0.001, ^##p < 0.0001 vs. si-Ctr

Fig. 5

Effects of miR-9-5p and miR-338-3p mimics on luciferase mRNA degradation and PDAC cell proliferation. (A) The 3′ UTR of luciferase mRNA contained predicted miR-9-5p and miR-338-3p target sequences from hsa_circ_0004781 (c4781). (B) AsPC-1 and BxPC-3 cells were cotransfected with the pmirGLO-c4781 reporter plasmid and either the control miRNA mimic (Ctr mimic), miR-9-5p mimic (miR9 mimic), or miR-338-3p mimic (miR338 mimic) for 24 h. Total cell lysates were collected. Luciferase activity was measured using a dual luciferase assay kit, and values were normalized to Renilla luciferase activity. Each value represents the mean ± SE of three independent experiments. ^##p < 0.0001 vs. the Ctr mimic. (C) AsPC-1 and BxPC-3 cells were transfected with the control miRNA mimic (Ctr mimic), miR-9-5p mimic (miR9 mimic), or miR-338-3p mimic (miR338 mimic) for 1–3 days. Cell proliferation was determined by performing an MTT assay. Values are presented as mean ± SE. ^##p < 0.0001, compared with the Ctr mimic

Fig. 6

Effects of hsa_circ_0004781 on miR-9-5p and miR-338-3p target gene expression in PDAC cells. (A, B) Potential target mRNAs of (A) miR-9-5p and (B) miR-338-3p were predicted using mirDIP, miRTarBase, and TargetScan. Five upregulated targets were selected on the basis of the findings of GEPIA2 database analysis. (C) AsPC-1 and BxPC-3 cells were transfected with control siRNA (si-Ctr) or hsa_circ_0004781 siRNA (si-c4781) for 24 h. (D) AsPC-1 and BxPC-3 cells were transfected with a control vector (OE-Ctr) or pcDNA-ZKSCAN-c4781 plasmid (OE-c4781) for 24 h. (C, D) Total RNA was extracted, and the expression of miR-9-5p and miR-338-3p target mRNAs was determined through RT-qPCR. Values are presented as the mean ± SE from three independent experiments. *p < 0.05, **p < 0.01, ^##p < 0.0001 vs. si-Ctr or OE-Ctr

Fig. 7

Effects of miR-9-5p and miR-338-3p on target gene expressions in PDAC cells. (A, B) AsPC-1 cells were cotransfected with the pcDNA-ZKSCAN-c4781 plasmid (OE-c4781) and either the control mimic (Ctr mimic), miR-9-5p mimic (miR9 mimic), or miR-338-3p mimic (miR338 mimic) for 24 h. Total RNA was extracted, and the expression of (A) miR-9-5p and (B) miR-338-3p target mRNAs was determined through RT-qPCR. Target gene expression levels were normalized to those of the control (no treatment). Values are presented as the mean ± SE from three independent experiments. *p < 0.05, **p < 0.01 vs. the Ctr mimic. (C) AsPC-1 cells were transfected with mimics or inhibitors of miR-9-5p and miR-338-3p for 24 h. Total cell protein lysates were collected, and the protein expression of KLF5 and ADAM17 was determined through Western blot analysis

Fig. 8

Generation and purification of real hsa_circ_0004781. (A) The sequences of hsa_circ_0004781 are presented, and the ribozyme target sequence is highlighted in blue. (B) Schematics for the primary and secondary structures of the IVT RNA precursor containing the Tetrahymena group I intron ribozyme and hsa_circ_0004781 (c4781). The 3′ target sequence (TS) is complementary to the 5′ internal guide sequence (IGS), forming a G·U wobble base pair. GMP induces self-splicing ligation through two transesterification reactions, forming real hsa_circ_0004781. (C) The self-splicing junction (SSJ) formed during ligation differs from the natural BSJ. (D, F) IVT products were (D) subjected to denaturing urea–PAGE and stained with GelRed dye or (F) separated through IP-RP HPLC. M1, DNA markers; M2, RNA markers; lane 1, IVT products; lane 2, peak C from HPLC. (E) Band C from urea–PAGE was extracted, and RNA was amplified at the SSJ region through RT-PCR. Sequences of forward and reverse strands were determined through Sanger sequencing

Fig. 9

Effects of real hsa_circ_0004781 on cell proliferation and the expression of miR-9-5p, miR-338-3p, KLF5, and ADAM17 in PDAC cells. AsPC-1 cells were transfected with control RNA (Ctr) or real hsa_circ_0004781 (real c4781) for 24 h. (A) Cell proliferation was determined using a CCK-8 assay. (B, C) Total RNA was extracted, and the expression of (B) miR-9-5p and miR-338-3p as well as (C) KLF5 and ADAM17 mRNA was detected through RT-qPCR. Values are presented as the mean ± SE from three independent experiments. *p < 0.05, **p < 0.01, ^#p < 0.001, ^##p < 0.0001 vs. Ctr