Circular RNA circ_0006089 regulates the IGF1R expression by targeting miR-143-3p to promote gastric cancer proliferation, migration and invasion

ABSTRACT

Circular RNAs (circRNAs) figure prominently in regulating the progression of a variety of human malignancies. This study was performed to probe how circ_0006089 functioned in gastric cancer (GC). CircRNA expression profile GSE83521 was downloaded from Gene Expression Omnibus (GEO) database, and circRNAs and analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure circ_0006089, microRNA-143-3p (miR-143-3p) and insulin-like growth factor 1 receptor (IGF1R) mRNA expressions in GC tissues and cell lines. Kaplan-Meier curves were used to detect the relationship between circ_0006089 expression and overall survival time of GC patients. Cell counting kit-8 (CCK-8) and 5-bromo-2-deoxyuridine (BrdU) assays were employed to detect the proliferative ability of GC cells after circ_0006089 was overexpressed or knocked down. Wound healing assay and Transwell assay were executed to examine the migration and invasion ability of GC cells. Western blot was adopted to detect IGF1R protein expressions. Circ_0006089 expression was up-regulated in GC samples and cell lines. And high circ_0006089 expression was associated with shorter survival time in GC patients. Circ_0006089 overexpression in GC cells significantly accelerated GC cell proliferation, migration and invasion, whereas circ_0006089 knockdown resulted in the opposite effects. Additionally, miR-143-3p was validated as a downstream target of circ_0006089, and circ_0006089 could positively regulate IGF1R expression via repressing miR-143-3p. Circ_0006089 is highly expressed in GC, and it promotes the malignancy of GC cells via modulating miR-143-3p/IGF1R axis, suggesting that circ_0006089 may serve as a promising therapeutic target for GC.

Introduction

Gastric cancer (GC) is one of the most common tumors globally and ranks the second cause of cancer-related death [1]. Despite the progresses in clinical diagnoses and treatment strategies, patients with advanced GC suffer from poor prognosis, with an overall 5-year survival rate less than 20% for patients undergoing radical surgery and requiring long-term lymph node dissection [2–4]. In this context, clarifying the molecular mechanisms of GC progression and looking for molecular diagnostic markers and therapeutic targets are pivotal for improving the diagnosis and prognosis of GC patients.

Circular RNAs (CircRNAs), non-coding RNAs with covalently closed loop structure, are more stable than linear RNAs [5]. Previously, circRNAs were considered to be the by-products of genomic transcription [6]. However, recent studies have gradually revealed the biological functions of circRNAs, and there is growing evidence suggesting that circRNAs feature prominently in tumorigenesis [7]. Reportedly, circ_0004872 expression is markedly declined in GC tissues, and circ_0004872 overexpression inhibits the growth, migration and invasion of GC cells while circ_0004872 knockdown has opposite effects [8]. Another study finds that circ_0000745 expression in GC tissues is associated with tumor differentiation, and its expression in plasma is related to tumor-node-metastasis (TNM) stage of the patients [9]. By analyzing circRNA microarray dataset GSE83521, we found, in this study, that circ_0006089 expression in GC tissues was markedly higher than that in normal mucosal tissues. However, the function of circ_0006089 in GC remains unclear.

MicroRNAs (miRNAs) are small non-coding RNAs that are implicated in modulating the progression of a variety of cancers via restraining messenger RNA (mRNA) translation or promoting mRNA degradation [10,11]. For instance, miR-532 accelerates the migration and invasion of GC cells via restraining NKD1 expression and activating the Wnt/β-catenin pathway [12]. MiR-143-3p inhibits the progression of various cancers, including ovarian cancer, osteosarcoma and nasal squamous cell carcinoma [13–15]. Notably, miR-143-3p expression is reduced in GC, and miR-143-3p has tumor-suppressive effects [16]. However, its upstream mechanism remains unclear.

As reported, circRNAs can act as competitive endogenous RNA (ceRNA) to sponge miRNAs, thereby inhibiting the interaction between miRNAs and target mRNAs [17–20]. The circRNA-miRNA-mRNA regulatory network is pivotal in the progression of tumors [18]. For instance, circ_100395 raises TCF21 expression via adsorbing miR-1228, thus repressing lung cancer cell proliferation and metastasis [19]. In the present work, we report that circ_0006089 expression is raised in GC tissues and cell lines. Besides, circ_0006089 promotes the malignancy of GC cells via adsorbing miR-143-3p and increasing the expression of insulin-like growth factor 1 receptor (IGF1R).

Materials and methods

Tissue specimens

32 patients who were diagnosed with GC and underwent surgical resection in Chinese PLA General Hospital were enrolled in this study. GC tissues and adjacent normal tissues from patients were obtained during surgery and immediately preserved in liquid nitrogen. This study was endorsed by the Ethics Committee of Chinese PLA General Hospital and obtained written informed consent from all patients.

Bioinformatics analysis

The circRNA expression profile dataset GSE83521 was downloaded from Gene Expression Omnibus (GEO) database, and analyzed by GEO2R. P< 0.05 and∣log₂ (Fold Change)∣>1 were used as the thresholds for screening out the differential expressed circRNAs. The LinkedOmics database (http://www.linkedomics.org) is a public portal containing multi-omics data from 32 cancers in TCGA [21]. In the “LinkFinder” module, we performed co-expression statistical analysis of IGF1R using Spearman’s test, and the results were displayed as volcano and heat maps. In the “LinkInterpreter” module, we performed gene ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) pathway analysis by gene set enrichment analysis (GSEA). Screening criteria were set as false discovery rate (FDR) < 0.05, the minimum number of genes (size) was 3, and the number of simulations was 1000.

Cell culture and transfection

Humans GC cell lines (HGC-27, BGC-823, AGS and MKN45) and normal gastric epithelial immortalized cells (GES-1) were available from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in 5% CO₂ at 37°C. pcDNA-circ_0006089 (circ-OE), pcDNA-IGF1R (IGF1R-OE), their negative control (NC), circ_0006089 siRNA (si-circ_0006089#1 and si-circ_0006089#2), scramble siRNA (si-NC), miR-143-3p mimics/inhibitors and their negative controls (mimics NC/inhibitors NC) were purchased from Invitrogen (Carlsbad, CA, USA). Transfection was performed with Lipofectamine®2000 (Invitrogen) according to the manufacturer’s instruction.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were isolated from GC tissues and cells using TRIzol Reagent (Yeasen Biotech, Shanghai, China). For cellular fractionation, A NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA, USA) was used to separate the nuclear and cytoplasmic fractions. cDNA synthesis was conducted employing a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA) for miR-143-3p, and a PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan) was utilized for circ_0006089 and IGF1R, respectively. With cDNA as the template, qRT-PCR was conducted to examine the relative expressions of circ_0006089 and IGF1R with a SYBR® Premix Ex Taq^TM II kit (Takara, Tokyo, Japan). A stem-loop primer SYBR Green qRT-PCR kit (Synbio Tech, Suzhou, China) was used to examine miR-143-3p expression. U6 and GAPDH were used as internal references, and the relative expressions of circ_0006089, miR-143-3p and IGF1R were calculated by 2^{− ΔΔCt}. The primer sequences are shown in Table 1.

Table 1.

Primer sequences

Name	Primer sequences
circ_0006089		Forward: 5′-AGCTGGGGGTTCATTATTCC-3′
		Reverse: 5′-TCCTCAGGGTTGGTCTTCAC-3′
miR-143-3p		Forward: 5’-GGGGTGAGATGAAGCACTG-3’
		Reverse: 5′-CAGTGCGTGTCGTGGAGT-3′
IGF1R		Forward: 5′-GCCGGGTGTAAGCATCCTCGTG-3′
		Reverse: 5′-ATGTGCGTCGTGGAGTCCCG-3′
U6		Forward: 5’-GCTTCGGCAGCACATATACTAAAAT-3’
		Reverse: 5’-CGCTTCAGAATTTGCGTGTCAT-3’
GAPDH		Forward: 5’-AATTCCATGGCACCGTCAAG-3’
		Reverse: 5’-TGGACTCCACGACGTACTC-3’

Cell viability assay

A cell counting kit-8 (CCK-8) kit (Meilunbio, Shanghai, China) was used to measure the proliferative ability of GC cells. In brief, HGC-27 and BGC-823 cells were plated in 96 well plates (1 × 10³ cells/well) and cultured. At the 24^th h, 48^th h, 72^nd h and 96^th h, the cells were incubated with 10 µL of CCK-8 solution for 4 h at 37°C. The optical density (OD) at 450 nm wavelength was detected, respectively.

5-bromo-2’-deoxyuridine (BrdU) assay

GC cells (1 × 10⁵ cells/ml) were inoculated in 35-mm diameter dishes, cultured for 1 d and then synchronized with medium with 0.4% FBS for 3 d. Then, 1.0 mg/ml BrdU reagent (BD Pharmingen, San Diego, CA, USA) was loaded and the cells were incubated at 37°C for 40 min. After that, the medium was discarded, and the slides were washed three times with phosphate buffer saline (PBS) and then fixed with methanol for 10 min. Besides, the slides were dried and blocked with 5% normal rabbit serum, and DNA was denaturized with 1.5 M HCl. Subsequently, the cells were rinsed with PBS and then incubated with antibody for 1 h (Beyotime, Shanghai, China). Subsequently, the cells were stained with DAPI staining solution (Beyotime). Finally, the number of BrdU-positive cells was counted under a fluorescence microscope.

Wound healing assay

Cell migratory abilities were detected using wound-healing assay. To be specific, the transfected HGC-27 and BGC-823 cells were plated in 6-well plate (1.2 × 10⁶ cells/well) and cultured until reaching a confluence of 90%. Then the monolayer cells were scratched with a 10 µL pipette tip, and then the cells were cultured with serum-free medium, and images were captured using an Olympus IX71 inverted microscope (Olympus Corporation, Tokyo, Japan; magnification, ×100) at 0 and 24 h. The wound area was quantified using ImageJ software.

Transwell assay

Transwell inserts (Corning Incorporated, Corning, NY, USA) were adopted to determine the migrative and invasive abilities of GC cells. For migration assay, the cells in each group were trypsinized with trypsin, and 100 μL (1 × 10⁵ cells/ml) of cells was transferred into the upper chamber containing serum-free medium. Then, 250 μL of medium with 10% FBS was dripped into the lower chamber, and subsequently the cells were cultured in 5% CO₂ at 37°C for 24 h. Subsequently, non-migrated cells were scraped off with a cotton swab, and the cells migrating below the microporous membrane of the inserts were fixed with 95% ethanol. Subsequently, the cells were stained with 0.05% crystal violet for 15 min, and five visual fields were randomly taken from each insert, and the number of cells was counted. In the invasion assay, the microporous membranes were coated with a layer of Matrigel, and the remaining steps were the same as in the migration assay.

Western blot

The cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China), and the supernatant was collected. Then, the protein concentration was measured by a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Next, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to PVDF membrane, and the membrane was blocked with 5% skimmed milk for 1 h at ambient temperature. After that, the membranes were incubated with anti-IGF1R antibody (1:1000, ab133542), anti-phosphoinositide 3 kinase (PI3K) antibody (1:1000, ab154598), anti-AKT antibody (1:1000, ab8805), anti-mammalian target of rapamycin (mTOR) antibody (1:1000, ab2732), anti-phosphorylated (p)-PI3K antibody (1:1000, ab182651), p-AKT antibody (1:1000, ab38449) and p-mTOR antibody (1:1000, ab109268) and anti-GAPDH antibody (1:2000, ab8245) overnight at 4°C. The next day, the membranes were washed by tris buffered saline tween (TBST), and incubated with horseradish peroxidase-conjugated secondary antibodies Goat Anti-Rabbit IgG H&L (1:5000, ab150077) and Goat Anti-Mouse IgG H&L (1:5000, ab205719) for 50 min at room temperature. All antibodies were purchased from Abcam (Cambridge, UK). Finally, the protein bands were developed by an Amersham Imager 600 (GEHealthcare, Chicago, IL, USA) with an electrochemiluminescence kit (Biosharp, Hefei, China).

Dual-reporter luciferase gene assay

The binding sites of circ_0006089 to miR-143-3p and miR-143-3p to IGF1R 3′-UTR were predicted by bioinformatics, respectively, and the binding fragments of circ_0006089 and IGF1R 3′-UTR to miR-143-3p were amplified by PCR. The amplified products were respectively inserted into the pGL3-Promoter plasmid vector (Promega, Madison, WI, USA) to construct circ_0006089 and IGF1R wild-type (wt) dual-luciferase reporter gene vector plasmids; the construction of the dual-luciferase reporter vectors containing mutant type (mut) circ_0006089 sequence or IGF1R sequence was performed with site-directed mutagenesis. Subsequently, the recombinant plasmid was co-transfected with miR-143-3p mimic or mimics NC into HEK-293 T cells. After 48 h, the cells were collected. According to the instructions, the fluorescence value was detected by dual-luciferase reporter gene assay system (Promega, Madison, WI, USA).

RNA immunoprecipitation (RIP) assay

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) was used for RIP assay. Briefly, HGC-27 and BGC-823 cells were collected and lysed in complete RIPA buffer containing a protease inhibitor cocktail and RNase inhibitor. Next, the cell lysates were incubated with RIP buffer containing magnetic bead conjugated with human anti-Ago2 antibody (Abcam, Cambridge, UK) or control anti-IgG antibody (Abcam, Cambridge, UK). Subsequently, the samples were incubated with proteinase K to remove the protein in the immunoprecipitate. Finally, qRT-PCR was performed to verify the existence of the potential target.

Statistical analysis

Each experiment was repeated three times. All data were expressed as “mean ± standard deviation” and statistical analysis was performed with SPSS version 20.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, US). Independent samples t-test was adopted for the comparison between the two groups, and one-way analysis of variance and Tukey’s post hoc test were utilized for the comparison among multiple groups. Overall survival analysis was conducted with Kaplan-Meier plots and log-rank tests. Gene expression correlations were analyzed with Pearson’s correlation analysis, and statistically, P< 0.05 was meaningful.

Results

Circ_0006089 expression is upregulated in GC tissues and cells

The dataset GSE83521 was obtained from the GEO database, and significantly up-regulated or down-regulated circRNAs expressions were screened out by │log₂ (Fold Change)│>1 and P< 0.05 (Figure 1a). We found 70 down-regulated circRNAs and 80 up-regulated circRNAs in GC tissues. Among them, the fold change of circ_0006089 was the largest, so we choose it for subsequent studies (Figure 1a). Circ_0006089 expression in GC tissues and normal tissues, GC cell lines and gastric immortalized cells (GES-1) was quantified by qRT-PCR, showing that circ_0006089 expression in GC tissues was remarkably higher than that in normal tissues (Figure 1b); circ_0006089 expression was markedly higher in GC cell lines as against that in GES-1 cells (Figure 1c). In addition, high circ_0006089 expression was associated with shorter overall survival and higher TNM stage in GC patients (Figure 1d-e). Further analysis showed that the high expression of circ_0006089 was associated with larger tumor size and positive lymph node metastasis in GC (Table 2). This suggested that the abnormal upregulation of circ_0006089 was relevant to the progression of GC.

Figure 1.

Circ_0006089 is highly expressed in GC. (a) Volcano plot showed the up-regulated and down-regulated circRNAs in GC (v.s. normal tissues). Highly expressed cricRNAs are indicated by “red”, and lowly expressed circRNAs are indicated by “green”. (b) qRT-PCR was used to detect the expression of circ_0006089 in human GC tissues and adjacent tissues. (c) qRT-PCR was used to detect the expression of circ_0006089 in normal gastric epithelial cells (GES-1) and GC cell lines (HGC-27, BGC-823, AGS and MKN45). (d) Kaplan-Meier curve was used to analyze the relationship between the expression of circ_0006089 and the overall survival time of patients with GC. (e) The relationship between the expression of circ_0006089 and TNM stage of GC patients.

**P< 0.01 and ***P< 0.001.

Table 2.

The relationship between circ_0006089 expression and GC clinicopathological parameters

Characteristics	Numbers (32)	Circ_0006089 expression High (16) Low (16)		P-Value
Age (years)				0.723
< 61	15	7	8
≥ 61	17	9	8
Gender				0.465
Male	20	11	9
Female	12	5	7
Drinking alcohol				0.280
Yes	19	8	11
No	13	8	5
Tumor size (cm)				0.033*
≥5	18	12	6
<5	14	4	10
Distant metastasis				0.288
Yes	17	7	10
No	15	9	6
Lymph node invasion				0.003**
Negative	12	2	10
Positive	20	14	6

Note: *P < 0.05, and **P < 0.01.

Circ_0006089 promotes GC cells growth, migration and invasion

To explore the impacts of circ_0006089 on GC progression, we transfected pcDNA-circ_0006089 into HGC-27 cells with the lowest expression of circ_0006089, and transfected circ_0006089 siRNAs into BGC-823 cells with the highest expression of circ_0006089, respectively, and the transfection efficiency determined by qRT-PCR (Figure 2a). CCK-8, BrdU, wound healing and Transwell assays showed that circ_0006089 overexpression markedly promoted the proliferation, migration and invasion of HGC-27 cells (Figure 2b-e). Consistently, circ_0006089 knockdown significantly inhibited the proliferation, migration and invasion of BGC-823 cells (Figure 2b-e). These results further supported that circ_0006089 promoted the progression of GC.

Figure 2.

Circ_0006089 promotes the proliferation, migration and invasion of GC cells. (a) qRT-PCR was employed to confirm that the circ_0006089 overexpression and circ_0006089 knockdown cell models were constructed successfully. B-C. CCK-8 and BrdU assays were used to detect the proliferation of GC cells. Scale bar, 500 μm (c). The original figure of the BrdU assay were shown in Supplementary material 2: Figure S1. Wound healing assay was used to detect the migration of GC cells. Scale bar, 100 μm. Representative photomicrographs of the wound healing assay were shown in Supplementary material 3: Figure S1. (e) Transwell assay was used to detect the migration and invasion of GC cells. Scale bar, 250 μm. Representative photomicrographs of the Transwell assay were shown in Supplementary material 4: Figure S1.

**P< 0.01 and ***P< 0.001.

Circ_0006089 directly interacts with miR-143-3p

StarBase database (http://starbase.sysu.edu.cn) prediction suggested the possible binding sites of miR-143-3p in circ_0006089 sequence (Figure 3a). Dual-luciferase reporter gene assay showed that up-regulated miR-143-3p expression could suppress the luciferase activity of HEK-293 T cells co-transfected with circ_0006089-wt, but had no significant impact on that of cells with circ_0006089-mut (Figure 3b). In addition, RIP experiments showed that compared with anti-IgG group, circ_0006089 and miR-143-3p were significantly enriched in anti-Ago2 group of HGC-27 and BGC-823 cells (Figure 3c). Besides, circ_0006089 was mainly distributed in the cytoplasm of HGC-27 and BGC-823 cells (Figure 3d). qRT-PCR showed that circ_0006089 overexpression inhibited miR-143-3p expression in HGC-27 cells while knockdown of circ_0006089 induced the expression of miR-143-3p in BGC-823 cells (Figure 3e). In addition, miR-143-3p expression in GC tissues was markedly lower than that in normal tissues (figure 3f). Furthermore, Pearson’s correlation analysis showed that there was a negative relation between circ_0006089 expression and miR-143-3p expression in GC tissues. (Figure 3g). All these results suggested that miR-143-3p was a target of circ_0006089.

Figure 3.

MiR-143-3p is the target of circ_0006089. (a) Online bioinformatics tool was used to predict the binding site between circ_0006089 and miR-143-3p. (b) Dual-luciferase reporter gene assay showed that the binding site between circ_0006089 miR-143-3p was functional. (c) The direct targeting relationship between circ_0006089 and miR-143-3p was validated by RIP assay. (d) qRT-PCR was used to detect the subcellular distribution of circ_0006089 in GC cells. (e) qRT-PCR was used to detect the effects of overexpression or knockdown of circ_0006089 on miR-143-3p expression. (f) The expression of miR-143-3p in GC tissues/adjacent tissues was detected by qRT-PCR. (g) The correlation between the expressions of circ_0006089 and miR-143-3p in GC tissues was analyzed by Pearson’s correlation analysis.

ns: not significant, ***P< 0.001.

Circ_0006089 promotes the malignant phenotype of GC cells via miR-143-3p

Next, miR-143-3p mimics were transfected into HGC-27 cells with circ_0006089 overexpression. The results showed that the transfection of miR-143-3p mimics weakened the inhibitory effects of circ_0006089 overexpression on miR-143-3p expression in HGC-27 cells (Figure 4a). CCK-8, BrdU, wound healing and Transwell assays showed that the promotional effects of circ_0006089 overexpression on HGC-27 cell proliferation, migration and invasion were partially reversed by the transfection of miR-143-3p mimics (Figure 4b-e). The above data showed that circ_0006089 promoted the proliferation, migration and invasion of GC cells via regulating miR-143-3p.

Figure 4.

Up-regulation of miR-143-3p offsets the promotion of circ_0006089 overexpression on the proliferation, migration and invasion of GC cells. (a). HGC-27 cells with circ_0006089 overexpression were transfected with miR-143-3p mimics, and the expression of miR-143-3p in GC cells was detected by qRT-PCR. B-C. CCK-8 and BrdU assays were used to detect the proliferation of HGC-27 cells after the transfection. Scale bar, 500 μm (c). The original figure of the BrdU assay were shown in Supplementary material 2: Figure S2. (d) Wound healing assay was used to detect the migration of HGC-27 cells after the transfection. Scale bar, 100 μm. Representative photomicrographs of the wound healing assay were shown in Supplementary material 3: Figure S2. (e) Transwell assay was used to detect the migration and invasion of HGC-27 cells after the transfection. Scale bar, 250 μm. Representative photomicrographs of the Transwell assay were shown in Supplementary material 4: Figure S2.

**P< 0.01 and ***P< 0.001.

IGF1R is a direct target of miR-143-3p

MiRNAs regulate tumor progression mainly by suppressing the translation of mRNAs [22]. StarBase database suggested that IGF1R 3’-UTR had a sequence which could probably bind with miR-143-3p (Figure 5a). Dual-luciferase reporter gene assay showed that the transfection of miR-143-3p mimics could significantly inhibit the activity of IGF1R-wt luciferase but exerted no significant effect on that of IGF1R-mut (Figure 5b). qRT-PCR showed that compared with normal tissues, the expression of IGF1R mRNA in GC tissues was significantly higher (Figure 5c). Furthermore, the results showed that IGF1R mRNA was positively correlated with circ_0006089 expression in GC tissues and negatively correlated with the expression of miR-143-3p (Figure 5d-e). Western blot assay showed that the transfection of miR-143-3p mimics or circ_0006089 knockdown significantly inhibited IGF1R protein expression in HGC-27 and BGC-823 cells while the transfection of miR-143-3p inhibitors or overexpression of circ_0006089 increased the expression of IGF1R protein in HGC-27 and BGC-823 cells (figure 5f-g). Additionally, co-transfection of miR-143-3p mimics could reverse the promoting effects of circ_0006089 overexpression on the expression of IGF1R protein in HGC-27 cells (Figure 5h). In addition, we found that overexpression of circ_0006089 significantly increased the protein levels of p-PI3K, p-Akt and p-mTOR in HGC-27 cells, and the transfection of miR-143-3p mimics reversed these effects (Figure 5i). Collectively, these results indicated that circ_0006089/miR-143-3p axis could promote the development of GC by regulating the expression of IGF1R and activating the PI3K/Akt/mTOR signaling pathway.

Figure 5.

MiR-143-3p directly targeted the 3’-UTR of IGF1R in GC cells. (a) StarBase database was used to predict the binding site between miR-143-3p and IGF1R 3’-UTR. (b) The interaction between miR-143-3p and IGF1R was confirmed by dual-luciferase reporter gene assay. (c) qRT-PCR was used to detect the expression of IGF1R mRNA in human GC tissues and adjacent tissues. D-E. Pearson’s correlation analysis was adopted to detect the correlation between IGF1R mRNA and circ_0006089 or miR-143-3p expressions in GC tissues. F-I. Western blot assay was used to detect the expressions of IGF1R, p-PI3K, p-Akt, p-mTOR, PI3K, AKT and mTOR protein in GC cells. The original Western blot images with maker were shown in Supplementary material 1: Figures S1-.4

ns: not significant, **P< 0.01 and ***P< 0.001.

Overexpression of IGF1R attenuates the inhibitory effects of upregulated miR-143-3p expression on GC cell proliferation, migration and invasion

To further validate the biological function of miR-143-3p in GC cells was dependent on IGF1R, we transfected miR-143-3p mimics or/and IGF1R overexpression plasmid into HGC-27 cells. Western blot showed that the transfection of IGF1R overexpression plasmid could attenuate the inhibitory effects of upregulated miR-143-3p expression on IGF1R protein expression in HGC-27 cells (Figure 6a). Subsequently, CCK-8, BrdU, wound healing and Transwell assays were performed to detect the role of miR-143-3p/IGF1R axis in regulating GC cell proliferation, migration and invasion. Compared with the mimics NC group, the transfection of miR-143-3p mimics inhibited the proliferation, migration and invasion of HGC-27 cells while restoration of IGF1R reversed these effects (Figure 6b-e).

Figure 6.

MiR-143-3p regulated the behaviors of GC cell by targeting IGF1R. (a) MiR-143-3p mimic and IGF1R overexpression plasmid were transfected into HGC-27 cells, and the expression of IGF1R protein in HGC-27 cells was detected after transfection by Western blot assay. The original Western blot images with maker were shown in Supplementary material 1: Figures S5. B-C. CCK-8 and BrdU assays were used to evaluate the proliferation of HGC-27 cells after the transfection. Scale bar, 500 μm (c). The original figure of the BrdU assay were shown in Supplementary material 2: Figure S3. D-E. Wound healing assay and Transwell assay were used to evaluate the migration and invasion ability of HGC-27 cells after transfection. Scale bars, 100 μm (d) and 250 μm (e). Representative photomicrographs of the wound healing assay were shown in Supplementary material 3: Figure S3. Representative photomicrographs of the Transwell assay were shown in Supplementary material 4: Figure S3.

*P< 0.05, **P< 0.01 and ***P< 0.001.

In addition, we analyzed genes co-expressed with IGF1R in the TCGA-STAD cohort using the LinkFinder module in the LinkedOmics database (http://www.linkedomics.org/admin.php). The results showed that a total of 6811 genes (red dots) were positively correlated with IGF1R, while 4200 genes (dark green dots) were negatively correlated with IGF1R in GC (FDR<0.05) (Supplementary Fig. 1A). The top 50 genes correlated with IGF1R (positively and negatively) were displayed in the heatmap (Supplementary Fig. 1B-C). Next, GSEA was performed on IGF1R and its co-expressed genes by the LinkInterpreter module. GO term indicated that IGF1R and its co-expressed genes were mainly involved in biological processes such as axon development, cell-substrate adhesion, synapse organization, oligosaccharide-lipid intermediate biosynthetic process, antibiotic metabolic process and mitochondrial transport (Supplementary Fig. 1D). KEGG analysis revealed that these genes were mainly enriched in focal adhesion, axon guidance, Hippo signaling pathway, neuroactive ligand-receptor interaction, MAPK signaling pathway, spliceosome, peroxisome, carbon metabolism, Huntington disease and ribosome, some of which are crucial cancer-related pathways/cancer-related biological processes (Supplementary Fig. 1E). These data suggested that IGF1R was an oncogene, and collectively, circ_0006089/miR-143-3p axis modulated the proliferation, migration and invasion of GC cells by targeting IGF1R.

Discussion

CircRNAs were previously considered to be byproducts of aberrant splicing [23]. With advances in high-throughput sequencing technology, the extensive biological functions of circRNAs have been gradually explored, and circRNA is gradually becoming one of the research hot spots [24,25]. CircRNA, a highly abundant type of RNA with a long half-life, is a promising tumor biomarker [23]. Additionally, circRNAs exert their biological functions through a variety of mechanisms. Reportedly, circRNAs can interact with RNA-binding proteins to regulate the subcellular distribution and biological function of proteins [26,27]. However, the most concerned mechanism by which circRNAs regulates cancer progression, is the ceRNA mechanism, and it suggests that circRNAs can specifically recognize miRNA response elements, bind complementarily to miRNAs, and inhibit the ability of miRNA to target mRNA [28]. For instance, circ-PRMT5 works as a tumor-promoter in GC by modulating miR-145/miR-1304/MYC axis [29]. Herein, by analyzing the circRNA microarray data, we found that circ_0006089 expression was markedly elevated in GC. Further experiments confirmed that circ_0006089 was in high expression in GC tissues and cell lines, and its high expression predicted a poor prognosis of GC patients. In addition, in vitro experiments highlighted that circ_0006089 promoted GC cell proliferation, migration and invasion. The above findings suggest that circ_0006089 acts as a tumor-promoter in GC to regulate the malignant progression of GC.

MiRNAs are a class of short non-coding RNAs that usually inhibit the translation and stability of mRNAs and participate in regulating cellular life activities such as inflammation, stress response, cell cycle progression, proliferation, apoptosis, differentiation and migration [30]. MiRNAs are implicated in modulating various intracellular signaling pathways, and their dysregulation is closely associated with the development and progression of cancers including GC [31–33]. MiR-143-3p is aberrantly expressed and plays a regulatory role in a variety of malignancies. For example, miR-143-3p restrains proliferation and invasion of hepatocellular carcinoma cells via modulating its target FGF1 [34]. MiR-143-3p targets MAPK7 to inhibit osteosarcoma cell proliferation, migration and invasion [35]. In addition, miR-143-3p represses the growth and induces the apoptosis of GC cells [16]. In this study, we discovered that miR-143-3p expression was declined in GC tissues, which was consistent with the findings of the previous study [16]. MiR-143-3p was identified as a downstream target of circ_0006089, and its expression could be negatively modulated by circ_0006089. Additionally, miR-143-3p could reverse the promoting impacts of overexpression of circ_0006089 on the growth, migration and invasion of GC cells. These results illustrate that circ_0006089 accelerates the malignant progression of GC via targeting miR-143-3p.

IGF1R is a transmembrane receptor tyrosine kinase and its gene is located on chromosome 15q26. IGF1R contains 1367 amino acid residues that is constitutively expressed in most cells [36]. The IGF signaling participates in modulating the differentiation and growth of cells [37]. IGF signaling also has an effect on hypoxic signaling, tumor cell motility, protease secretion and cell adhesion, thus influencing the migration. invasion and metastasis of cancer cells [36]. IGF1R can promote tumor progression in multiple cancers, esophageal squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, prostate cancer and GC included [38–42]. For example, high IGF1R expression is correlated with disease-free survival time of the patients with stage III colonic cancer [43]. We proved in this study that IGF1R was the target of miR-143-3p, which inhibited IGF1R expression, and circ_0006089 increased IGF1R expression by inhibiting miR-143-3p expression. Reportedly, PI3K/Akt/mTOR signal is related to cell proliferation, migration and invasion, and it is frequently activated in GC [44,45]. In addition, IGF1R mainly performs its function through the PI3K/Akt/mTOR signaling pathway [46,47]. The results of this study also showed that circ_0006089 could up-regulate IGF1R expression and activate the PI3K/Akt/mTOR pathway to promote GC progression through sponging miR-143-3p. However, there were some limitations of this study, such as the lack of in vivo experiments. This study only explored the regulatory mechanism of circ_0006089 in vitro. Our conclusions need to be validated in vivo. In addition, other downstream targets of circ_0006089 need to be further explored.

Conclusion

Circ_0006089 is highly expressed in GC tissues and cell lines, and its high expression implies poor prognosis of the patients. Functionally and mechanistically, circ_0006089 increases IGF1R expression and activates the PI3K/Akt/mTOR pathway via absorbing miR-143-3p, thereby promoting GC cell growth, migration and invasion.

Funding Statement

This study was supported by general projects of the Hainan Natural Science Foundation (Grant No. 819MS113).

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethics statement

Our study was approved by the Ethics Review Board of Chinese PLA General Hospital.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2022.2075197.