Introduction
Renal cell carcinoma (RCC) accounts for at least 3% of malignant diseases [
1] and is the second leading cause of urological malignant neoplasm-related death [
2]. Clear cell renal cell carcinoma (ccRCC) comprises 80–90% of RCC and has a higher invasive ability and a higher relapse rate than other RCC subtypes. In the past few decades, the incidence and mortality of ccRCC appear to be increasing rapidly [
2]. Because of local recurrence and distant metastasis, overall patient survival is not satisfactory [
3,
4]. Therefore, it is essential that the molecular mechanisms that promote ccRCC development and progression be further studied.
In the last decades, due to the development of high-throughput sequencing, numerous circRNAs have been discovered in mammalian cells [
5‐
7]. And emerging evidence indicates that circRNAs may participate in the progression of many diseases, including cancers [
8‐
15]. For instance, hsa_circ_001783 promoted progression of breast cancer cells via sponging miR-200c-3p [
12]. circ-CPA4 regulated cell growth, mobility, stemness and drug resistance in NSCLC cells and inactivated CD8+ T cells in the tumor immune microenvironment through let-7 miRNA/PD-L1 axis [
9]. And Hsa_circ_0058124 promotes papillary thyroid cancer tumorigenesis and invasiveness through the NOTCH3/GATAD2A axis [
15]. circRNA hsa_circ_002577 accelerated EC progression by acting as a miR-625-5p sponge, upregulating IGF1R and activating the PI3K/Akt pathway [
14].
Although multiple circRNAs have been reported to regulate tumor progression [
16], the functional roles of circRNAs in ccRCC remain largely unknown. It has been shown that circTLK1 plays a critical role in RCC progression by sponging miR-136-5p, which increases CBX4 expression, and circTLK1 might act as a diagnostic biomarker and therapeutic target for RCC [
10]. hsa_circ_001895 might sponge miR-296-5p and promote SOX12 expression, which is the underlying mechanism of hsa_circ_001895-induced ccRCC progression [
8]. In this work, we discovered a novel circRNA, termed hsa_circ_0020303, which was upregulated in ccRCC specimens and associated with poor prognosis of patients with ccRCC. We determined that circCHST15 promotes proliferation and metastasis of ccRCC through directly binding to miR-125a-5p to attenuate miR-125a-5p-mediated suppression of EIF4EBP1. Taken together, our findings identify circCHST15 as a potentially novel prognostic biomarker and therapeutic target in ccRCC.
Methods
Microarray analysis
To identify differently expressed circRNAs in ccRCC, we obtained microarray expression data from the Gene Expression Omnibus (GEO) database (
https://www.ncbi.nlm.nih.gov/geo/; GSE100186 and GSE137836). In addition, to verify the pan-cancer expression of circRNA, we obtained microarray expression data from the GEO database for bladder cancer (GSE147984), breast cancer (GSE165884), rectal cancer (GSE121895), gastric cancer (GSE141977), glioma (GSE146463), and lung cancer (GSE101684). We also analyzed data from The Cancer Genome Atlas (TCGA) cohort, which contained 527 patients from the TCGA-KIRC project, and the corresponding gene expression data were obtained from the Genomic Data Commons Data Portal (
https://portal.gdc.cancer.gov).
Our study complied with the principles set forth in the Declaration of Helsinki, and access to the de-identified linked dataset was obtained from the TCGA and GEO databases in accordance with database policies. For analyses of de-identified data from the TCGA and GEO databases, institutional review board approval and informed consent were not required.
For all expression datasets from the GEO database, background correction and quartile normalization were performed for each series by applying the robust multi-array average algorithm [
17]. The average value of gene symbols with multiple probes was calculated as expression level. For datasets from the TCGA database, messenger RNA (mRNA) expression was quantified with fragments per kilobase of exon per million reads mapped (FPKM). The primary prognosis endpoint was overall survival (OS), and survival curves were estimated using the Kaplan–Meier method. Expression profiles of circRNAs, miRNAs, and mRNAs were retrieved from two circRNA microarrays and the TCGA database. We applied the limma and edgeR packages to identify differentially expressed RNAs in ccRCC compared to matched adjacent epithelial tissue and in primary compared to matched metastatic tumor tissue. Next, a competing endogenous RNA (ceRNA) network was established based on circRNA–miRNA and miRNA–mRNA intersections. Significantly differentially expressed transcripts were defined as characterized by fold change ≥2 or ≤ − 2 and
P ≤ 0.05.
Patients and tumor tissue collection
We obtained 175 paired pathologically diagnosed ccRCC tissues and matched adjacent normal epithelial tissues from Sun Yat-sen University Cancer Center (Guangzhou, China). And all tissue specimens were immediately frozen in liquid nitrogen and stored at − 80 °C. The collection of tissue samples was approved by the Ethics Committee of Sun Yat-sen University Cancer Center. Moreover, we verified the identified circRNA expression levels in the Sun Yat-sen University (SYSU) patient cohort for prediction of OS and progression-free survival (PFS) by Kaplan–Meier analysis and compared them to clinicopathological characteristics and algorithms for clinical prognostic scores (stage, size, grade, necrosis [SSIGN] score).
Cell culture and treatments
Human RCC cell lines 786-O, 769P, ACHN, CAKI-1, A498, and OSRC2, and the human renal proximal tubular epithelial cell line HK2 were purchased from the American Type Culture Collection. 786-O and 769P cells were cultured, in a humidified atmosphere of 5% CO2 maintained at 37 °C, in RPMI-1640 (Invitrogen-Gibco); CAKI-1 cells in McCoy’s 5A (Gibco); ACHN, OSRC2, HK2, and HEK293T cells in DMEM (Gibco); and A498 cells in MEM (Gibco). All cell culture media contained 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco). When we performed the actinomycin D assay, we added 2 μg/ml of actinomycin D (Sigma-Aldrich) to 786-O and CAKI-1 cells, and incubated ccRCC cells for 4, 8, 16, and 24 h.
RNase R treatment and quantitative real-time polymerase chain reaction
Genomic DNA and total RNA were extracted with MiniBEST Universal Genomic DNA Extraction Kit, version 5.0 (Takara) and RNAiso Plus (Takara), respectively. RNase R treatment was carried out for 30 min at 37 °C using 3 U/μg of RNase R (Epicenter Technologies). The nuclear and cytoplasmic fractions were isolated using a PARIS Kit (Life Technologies), according to the manufacturer’s instructions. RNA was reverse transcribed using PrimeScript RT Reagent Kit (Takara). TB Green Premix Ex Taq II (Takara) was used for qRT-PCR. The circRNA and mRNA levels were normalized by GAPDH. The miRNA level was normalized by small nuclear U6. Primers are listed in Additional file
1: Table S1.
Oligonucleotide transfection
SiRNAs were synthesized by RiboBio (Guangzhou, China). Sequences used are listed in Additional file
2: Table S2. Transfection was carried out using RNAiMAX(Life Technologies) according to the manufacturer’s instructions.
Plasmid construction and stable transfection
To construct circCHST15 over-expression plasmids, human circCHST15 cDNA was synthesized and cloned into a pLenti-CMV-GFP-Puro vector by GeneCreate (Wuhan, China), which was confirmed by sequencing. An empty plasmid served as the negative control. HEK-293 T cells were cotransfected with pLenti-CMV-GFP-Puro- circCHST15 or empty plasmid by Lipofectamine 2000 (Invitrogen). Forty-eight hours later, lentiviruses were harvested. ccRCC cells were infected with lentiviruses with 8 mg/mL polybrene by ViraPower Packaging Mix (ThermoFisher). Stable cell lines were obtained by treatment with 2 μg/mL puromycin (Sigma Aldrich) for 3 days. For silencing of circCHST15, sh-circCHST15 and sh-control were purchased from GenePharma.
Cell proliferation, wound healing, migration, and invasion assays
For cell proliferation assay, 2 × 103 cells were seeded in 100 μl of complete culture media in 96-well plates for various time periods. Cell Counting Kit-8 assay (APExBio) was performed to measure cell viability according to manufacturer’s instructions.
For colony formation assays, 1 × 103 cells were seeded in 6-well plates. Approximately 7–10 days later, the clones were then imaged and quantified.
For 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assays, we obtained the EdU kit from RiboBio to detect cell proliferation of 786-O, CAKI-1, HK2, or A498 cells. Cultured RCC cells (200 μl of 2 × 104 cells/ml) were incubated with 50 μmol/L of EdU for 8 h. After fixation with 70% alcohol and permeabilization with Triton X-100, the cells were then incubated with Apollo staining reaction liquid (Click-iT EdU Apollo stain kit, Invitrogen) to label the cells. Nuclei were stained with DAPI. Immunostaining was visualized and photographed under a fluorescence microscope (Olympus inverted microscope IX71).
For wound healing assay, cells were seeded in 6-well plates with 5 × 105 cells per well. Then, a wound was made by using a 200 μl pipette tip on the cell monolayer and photographs were taken at the appropriate time to estimate the area occupied by migratory cells.
For transwell invasion assays, a 24-well transwell chamber (Costar, USA) with Matrigel (BD Biosciences) was used to detect cell invasive ability according to the manufacturer’s protocol. Cells suspended in 0.2 ml serum-free medium (5 × 104/well) were added to the upper chambers, and media supplemented with 10% FBS was applied to the lower chambers. After incubating the cells for 6 h (for 786-O and HK2), 20 h (for CAKI-1) and 24 h (for A498), at 37 °C; and 5% CO2, cells that invaded to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 1% crystal violet in PBS. Invaded cells were counted in five randomly selected fields. Three independent experiments were performed in triplicate.
Western blot analysis
Proteins extracted from ccRCC tissues or cells (30 μg) were separated by SDS-PAGE, and then transferred to PVDF membrane. After blocking for 1 h with 5% skim milk powder at room temperature, membranes were incubated with primary antibodies specific to EIF4EBP1 (1:1000, Absin), vimentin (1:1000, Cell Signaling Technology), N-cadherin (1:1000, Cell Signaling Technology), E-cadherin (1:1000, Cell Signaling Technology), PCNA (1:1000, Cell Signaling Technology), p-AKT (1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), mTOR (1:1000, Cell Signaling Technology), p-mTOR (1:1000, Cell Signaling Technology), PI3K (1:1000, Cell Signaling Technology), p-PI3K (1:1000, Cell Signaling Technology), or GAPDH (1:5000, Abcam) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1:5000, Cell Signaling Technology) and visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore).
Biotin-coupled probe pull-down assay
The biotinylated probe was specifically designed to bind to the junction area of circCHST15, while the oligo probe was taken as a control. Approximately 1 × 107 cells were harvested and lysed. The circCHST15 probe (GenePharma, Suzhou, China) was incubated with streptavidin magnetic beads (Life Technologies, USA) at room temperature for 2 h to generate probe-coated beads. The cell lysates were incubated with probe-coated beads at 4 °C overnight. The beads were washed and the bound miRNAs in the pull-down materials were extracted using Trizol reagent and analyzed by qRT-PCR assay.
Biotin-coupled miRNA capture
The 3′ end biotinylated miRNA mimics or control RNA (Ribio, Guangzhou, China) were transfected into ccRCC cells for 48 h before harvest. Then biotin-coupled RNA complex was pulled down by incubating the cell lysates with streptavidin-coated magnetic beads (Life Technologies). The abundance of circCHST15 in bound fraction was evaluated by qRT-PCR analysis.
Fluorescence in situ hybridization
The ccRCC cells were first fixed in 4% formaldehyde solution, and then incubated with 0.5% Triton X-100. The Cy5-labeled circCHST15 probe and Cy3-labeled miR-125a-5p probe (GenePharma, Suzhou, China) were hybridized at 37 °C with cells in the dark for 5 h. The cells were then photographed by laser scanning confocal microscopy (Carl Zeiss). The sequences of the probes are listed in Additional file
3: Table S3.
Luciferase reporter assay
Wild-type or mutant circCHST15 or 3′-UTR EIF4EBP1 was synthesized and then subcloned into psiCHECK-2 vector. HEK293T cells were seeded in 24-well plates at a concentration of 1.5 × 105 per well and cotransfected miR-125a-5p mimics or miR-NC with psiCHECK-2-wt-circCHST15, psiCHECK-2-mut-circCHST15,
psiCHECK-2-wt-EIF4EBP1 or psiCHECK-2-mut-EIF4EBP1. Two days later, luciferase activities were measured by Lucifer Reporter Assay System (Promega) and normalized to Renilla luciferase activity.
Hematoxylin and eosin staining and immunohistochemical analysis
The primary antibodies specific for EIF4EBP1(Absin), vimentin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), and E-cadherin (Cell Signaling Technology) were used at the appropriate dilution in the experiments. Tissue samples of 5-μm-thick paraffin sections were stained with hematoxylin and eosin and subjected to immunohistochemical analysis. Images were captured using a Nikon Eclipse 80i system with NIS-Elements software.
Animal experiments
BALB/c nude mice (4–6 weeks old) were purchased from Charles River Laboratories. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University and were performed in accordance with established guidelines.
For the tumor growth study, eight mice were included in each group, and 786-O cells with stable knockdown of circCHST15 or control cells (5 × 106 cells per mouse) were injected subcutaneously into the left side of the axilla. The size of the tumor was measured every week. Four weeks later, the mice were sacrificed, and tumor weight was recorded.
An in vivo metastasis model was established by intravenous injection through the tail vein of 2 × 106 ccRCC cells that stably expressed firefly luciferase into 4-week-old BALB/c nude mice. Six weeks after injection, bioluminescence of lung metastases of tumor was detected using an in vivo bioluminescence imaging system. Then, the mice were sacrificed. The lungs were removed and fixed with phosphate-buffered formalin. Subsequently, consecutive tissue sections were made for each block of the lung. The numbers of pulmonary metastatic nodules in the lung were recorded.
Statistical analysis
Statistical analyses were conducted using SPSS 19.0 or GraphPad Prism 8.0 software. Student’s t-test (two tailed) was applied to assess the statistical significance between two groups. The chi-square test was used to analyze the correlation between circCHST15 expression levels and clinicopathological features in ccRCC. OS curves were calculated with the Kaplan–Meier method and analyzed with the log-rank test. Data were presented as the mean ± standard error of the mean. P < 0.05 was considered statistically significant.
Discussion
With the increasing application of bioinformatics analysis and high-throughput sequencing, plenty of circRNAs were identified and proven to take an important role in the development and progression of various cancers [
9,
10,
12,
14,
15,
26‐
29], including ccRCC [
8,
10,
29]. However, the functions of circRNAs in ccRCC, and how they might be leveraged for clinical applications, remain largely unknown.
In this study, we identified a new circRNA, circCHST15, originating from exons 3, 4, 5, 6, 7, 8, and 9 of its host gene
CHST15, which was upregulated in ccRCC cells and tissue. The existing literature suggests that CHST15 exerts oncogenic effects in pancreatic cancer stroma [
30,
31], esophageal cancer [
32], breast cancer [
33], and ovarian cancer [
34]. Additionally, these studies suggest that CHST15 can promote tumor progression by driving the proliferation and invasion of cancer cells, suggesting that high CHST15 might be associated with poor prognosis. According to our study, it was concluded that silencing of circCHST15 significantly suppressed the progression of ccRCC cells, whereas overexpression of circCHST15 had the opposite effect. And it was increasingly implied that circRNAs act as miRNA sponges in many cancer development18,25,34, thereby regulating downstream target genes. For instance, Circular RNA circ-ZKSCAN1 inhibits bladder cancer progression through miR-1178-3p/p21 axis and acts as a prognostic factor of recurrence [
35]. According to our study, it was concluded that silencing of circCHST15 significantly suppressed the progression of ccRCC cells, and overexpressed circCHST15 acts inversely. And it was increasingly implied that circRNAs serve as miRNA sponges in cancer development [
19,
26,
36], thereby regulating downstream target genes. For instance, circ-ZKSCAN1 inhibits bladder cancer progression through miR-1178-3p/p21 axis and acts as a prognostic factor of recurrence. Moreover, because circRNA is enriched in the cytoplasm, it was likely to play a role of ceRNA [
9,
19,
37]. Our study confirmed that circCHST15 was mainly located in the cytoplasm through Cytoplasmic & Nuclear RNA Extraction assays and FISH assay. Besides, we performed RNA pull-down and dual-luciferase reporter assays, the results showed that there is an interaction between circCHST15 and miR-125a-5p in ccRCC cells. Subsequently, the functional effects of miR-125a-5p were assessed by regulating its’ expression in ccRCC cells. It was showed that miR-125a-5p exerted an anti-tumor role in ccRCC cells. Additionally, overexpression of circCHST15 alleviated miR-125a-5p-mediated suppression of cell proliferation, migration, and invasion in ccRCC cells. Taken together, circCHST15 performed its’ biological function by interacting with miR-125a-5p.
It was reported that miRNAs can bind to the 3′ UTR of target mRNAs to reduce expression level of this target gene [
38]. And recently, it was indicated that circRNAs can regulate gene expression by directly binding to miRNAs to prevent them from interacting with target genes [
8,
10,
28,
29,
37]. Our study identified that circCHST15 promoted ccRCC cells proliferation and metastasis through miR-125a-5p-mediated EIF4EBP1. Although EIF4EBP1 had been proven to act as an oncogene in some tumors [
22‐
25], its biological effect in ccRCC has not been discovered. Thus, our study just delves into this in ccRCC cells.
Recent studies have indicated that circRNAs play a crucial role in the progression and prognosis of human cancer [
36,
39], and the involvement of circRNAs in ccRCC has been investigated in several studies. For instance, circ-AKT3 inhibits ccRCC metastasis via altering miR-296-3p/E-cadherin signals, and patients with ccRCC with low circ- AKT3 had shortened survival [
13]. circPTCH1 promotes RCC metastasis via the miR-485-5p/MMP14 axis and activation of the epithelial–mesenchymal transition process, and upregulated expression of circPTCH1 was positively correlated with advanced stage and worse survival in patients with ccRCC [
11]. In our study, we showed that high expression of circCHST15 was associated with advanced pathological stage and poor survival, including OS and PFS.
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