Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
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Cisplatin (CDDP) treatment is one of the most predominant chemotherapeutic strategies for patients with gastric cancer (GC). A better understanding of the mechanisms of CDDP resistance can greatly improve therapeutic efficacy in patients with GC. Circular RNAs (circRNAs) are a class of noncoding RNAs whose functions are related to the pathogenesis of cancer, but, in CDDP resistance of GC remains unknown.
circAKT3 (hsa_circ_0000199, a circRNA originating from exons 8, 9, 10, and 11 of the AKT3 gene) was identified by RNA sequencing and verified by quantitative reverse transcription PCR. The role of circAKT3 in CDDP resistance in GC was assessed both in vitro and in vivo. Luciferase reporter assay, biotin-coupled RNA pull-down and fluorescence in situ hybridization (FISH) were conducted to evaluate the interaction between circAKT3 and miR-198. Functional experiments were measured by western blotting, a cytotoxicity assay, clonogenic assay and flow cytometry.
The expression of circAKT3 was higher in CDDP-resistant GC tissues and cells than in CDDP-sensitive samples. The upregulation of circAKT3 in GC patients receiving CDDP therapy was significantly associated with aggressive characteristics and was an independent risk factor for disease-free survival (DFS). Our data indicated that circAKT3 promotes DNA damage repair and inhibits the apoptosis of GC cells in vivo and in vitro. Mechanistically, we verified that circAKT3 could promote PIK3R1 expression by sponging miR-198.
circAKT3 plays an important role in the resistance of GC to CDDP. Thus, our results highlight the potential of circAKT3 as a therapeutic target for GC patients receiving CDDP therapy.
KeywordsCisplatin resistance Gastric cancer circAKT3 Circular RNA miR-198 PIK3R1
Area under the receiver operating characteristic curve
Breast cancer type 1 susceptibility protein
Cell Counting Kit-8
Competitive endogenous RNAs
DNA damage repair
Fetal bovine serum
Fluorescence in situ hybridization
Minimum free energy
Quantitative real-time PCR
Quantitative reverse transcription PCR
Phosphorylated histone family member X
Gastric cancer (GC) is the most common malignant tumor of the digestive tract in East Asia and the third leading cause of cancer-related death worldwide [1, 2]. At present, the main treatments for advanced GC are systemic chemotherapy and palliative surgery, but the overall median survival after treatment is only 8 to 11 months . In patients with histologically confirmed advanced GC and who are chemotherapy-naïve, cisplatin (CDDP) and fluorouracil-based chemotherapies were deemed as first-line treatments . However, patients always acquired drug resistance after several cycles of CDDP-based treatment. Thus, chemotherapy resistance has limited overall clinical efficacy in patients [5, 6]. To improve GC patient survival, illuminating the molecular mechanism underlying CDDP resistance in GC is essential.
The cytotoxicity of CDDP is mediated by its interaction with DNA to form DNA adducts. Intracellular CDDP primarily binds to nuclear DNA with high affinity and can physically interact with mitochondrial DNA (mtDNA), which is involved in the activation of several signaling pathways and apoptosis [7, 8, 9]. In recent years, studies have shown that the PI3K/AKT signaling pathway could suppress cell apoptosis and facilitate cell survival. This PI3K/AKT signaling function is crucial in the regulation of chemotherapy resistance of cancer cells [10, 11]. Activated PI3K/AKT signaling promotes the phosphorylation of caspase-3 and prevents the activation of caspase-3 and the inhibition of apoptosis .
Circular RNAs (circRNAs), a category of noncoding RNAs (ncRNAs), play a crucial role in the process of transcriptional and posttranscriptional gene expression . Recently, circRNAs were found to function as competitive endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs) and then suppress their functions, indicating a novel mechanism for regulating miRNA activity and providing a promising mode of action for circRNAs. As miRNAs regulate a series of biological processes, circRNA sponge activity will affect these biological behaviors as well . miRNAs are a large class of short (~ 22 nt) ncRNAs that posttranscriptionally regulate gene expression through direct base pairing to target sites within mRNAs. circRNAs can affect miRNA activities by competing for miRNA-binding sites . However, the function of circRNAs as miRNA sponges has not been clearly elucidated in GC resistance to CDDP.
To investigate the potential roles of circRNAs in the regulation of CDDP resistance in GC, we performed RNA sequencing (RNA-Seq) and verified thousands of distinct circRNAs in CDDP-sensitive and CDDP-resistant GC cells from humans. Through functional gain and loss experiments, we further observed that hsa_circ_0000199, which originates from exons 8, 9, 10, and 11 of the AKT3 gene and is termed circAKT3, was significantly upregulated in both CDDP-resistant GC tissues and CDDP-resistant cells. Furthermore, we found that circAKT3 modulates CDDP sensitivity by sponging miR-198 that suppresses PIK3R1 expression via activation of the PI3K/AKT pathway in GC.
Patients and samples
In total, 149 GC tissues (cohorts 1, 2) were obtained from the First Affiliated Hospital of Nanjing Medical University. All samples were collected in accordance with HIPAA guidelines and approved institutional protocols. Patients received treatment with standard CDDP-based therapeutic regimens after surgery. Disease-free survival (DFS) was defined as the time interval between gastrectomy (R0 excision) and the time of either disease recurrence or disease-associated death. CDDP resistance was defined as tumor relapse during CDDP-based chemotherapy after R0 excision, and CDDP sensitivity was defined as no tumor recurrence during CDDP-based therapy; both definitions followed standard CDDP response definitions published elsewhere . Forty-four samples (Cohort 1) were used for circRNAs validation, and another 105 samples (Cohort 2) were used to quantify circAKT3 levels and to analyze the correlation between circAKT3 expression and outcomes after R0 excision in patients undergoing CDDP-based chemotherapy. The samples from cohorts 1 and 2 were obtained in 2013–2016 and 2007–2011, respectively. The grouping of the ROC curve was based on the median relative expression of circAKT3. Detailed information is listed in Additional file 1: Table S1.
The CDDP-sensitive cell lines SGC7901 and BGC823 as well as their CDDP-resistant strains (SGC7901CDDP and BGC823CDDP, respectively) were maintained in RPMI 1640 medium (Wisent, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Wisent, Biocenter, China) (Additional file 2: Figure S1A). 293 T cells were cultured in DMEM with high glucose (Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% FBS. 293 T, SGC7901CDDP, BGC823 and SGC7901 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, and BGC823CDDP cells were established as previously described .
miRNA targets prediction of circAKT3
To predict the miRNA-binding sites of circAKT3, we used the bioinformatic databases miRanda, PITA and RNAhybrid. Filtering restrictions were as follows: (1) total score ≥ 140, total energy < 17 kcal/mol; (2) combined interaction energy (△△G) < 10; and (3) minimum free energy (MFE) ≤ 20 kcal/mol. Detailed information is listed in Additional 3: Dataset S1.
RNA preparation, treatment with RNase R, and PCR
Total RNA was extracted from GC cells or tissues using TRIzol Reagent (Invitrogen, 15,596,018). RNase R treatment was carried out for 15 min at 37 °C using 3 U/mg RNase R (Epicenter). For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China). The CT value was measured during the exponential growth phase. Relative gene expression levels were determined using the 2-△△CT method. The primers used are listed in Additional file 1: Table S2.
Isolation of nuclear and cytoplasmic fractions
SGC7901CDDP and BGC823CDDP cells were lysed on ice for 10 min in 0.3% NP-40/NIB-250 buffer (15 mM Tris–HCl (pH 7.5), 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 and 250 mM sucrose) supplemented with protease inhibitors. After centrifugation at 600 × g for 5 min at 4 °C, the resultant supernatant was collected as the cytoplasmic fraction and mixed with an equal volume of TRIsure reagent. After the pellet was washed with NIB-250, the nuclei were lysed in TRIsure reagent.
The method for overexpressing circRNAs was reported previously . For the construction of circAKT3 overexpression plasmids, human circAKT3 cDNA was amplified using PrimerSTAR Max DNA Polymerase Mix (Takara, RR036A, Japan) and inserted into the pCD5-ciR vector (Greenseed Biotech Co, Guangzhou, China). The pCD5-ciR vector contains a front circular frame and a back circular frame. Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The luciferase reporter containing the circAKT3 sequence in the 3′-UTR was constructed by subcloning the circAKT3 fragment into the region directly downstream of a cytomegalovirus promoter-driven firefly luciferase (FL) cassette in a pCDNA3.0 vector. Mutations of each miRNA-binding site in the circAKT3 sequence were created using a Mut Express II Fast Mutagenesis Kit (Vazyme, Nanjing, China). The mutations were introduced in both the circAKT3-expressing vector and the luciferase reporter containing the circAKT3 sequence.
siRNA and miRNA mimics and inhibitors were synthesized by GenePharma (Shanghai, China). The sequences used are listed in Additional file 1: Tables S3 and S4. Transfection was carried out using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions.
A pull-down assay was performed as described previously [17, 18]. The biotin-labeled circAKT3 probe was synthesized by RiboBio (Guangzhou, China). In brief, 1 × 107 circAKT3-overexpressing GC cells were harvested, lysed, and sonicated. The circAKT3 or oligo probe was incubated with streptavidin-coupled Dynabeads (Invitrogen) at 30 °C overnight to generate probe-bound Dynabeads. After the treated beads were washed with wash buffer, the RNA complexes bound to the beads were eluted and disrupted with lysis buffer and proteinase K prior to RT-PCR or RT-qPCR. Biotinylated probes sequences used in this study (see Additional file 1: Table S5).
Luciferase reporter assay
293 T, SGC7901CDDP and BGC823CDDP cells were seeded in 24-well plates and cotransfected with corresponding plasmids and miRNA mimics in triplicate. At 48 h after transfection, luciferase reporter assays were conducted using a dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s instructions. Relative luciferase activity was normalized to Renilla luciferase activity.
Fluorescence in situ hybridization (FISH)
The double FISH assay was performed in SGC7901CDDP cells and GC tissues as previously described [16, 19]. Biotin-labeled probes specific to circAKT3 and Dig-labeled locked nucleic acid miR-198 probes were used in the hybridization (Exiqon, Vedbaek, Denmark). The sequences are listed in Additional file 1: Table S6, FISH probes sequences used in this study. The signals of the biotin-labeled probes were detected using Cy5-conjugated streptavidin (Life Technologies), and the signals of the Dig-labeled miR-198 probes were detected using a tyramide-conjugated Alexa 488 fluorochrome TSA kit. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Images were acquired on a Leica TCS SP2 AOBS confocal microscope (Leica Microsystems, Mannheim, Germany). CircAKT3 and miR-198 expression levels were evaluated by the proportions and intensities of the positive cells detected within 5 fields of view on every slide (400-fold magnification). Proportion scores were assigned as follows: < 10% = 0, 10–25% = 1, 26–50% = 2, 51–75% = 3 and > 75% = 4. Intensity scores were assigned as follows: 0 = no staining, 1 = weak, 2 = moderate, 3 = strong and 4 = significantly strong.
Western blot analysis
For western blot analysis, cells were extracted using a protein extraction kit (Key Gene, KGP9100). Lipid proteins were added into 8, 10, 12% or 15% gels, subjected to 120 V to promote migration, and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA in TBST buffer and incubated with specific primary antibodies at 4 °C overnight. The next day, membranes were washed 3 times for 15 min in TBST and incubated with secondary antibodies for 2 h at room temperature. HRP substrate (WBKL0100, Millipore, USA) was used to detect the protein bands (Molecular Imager, ChemiDoc XRS+, BIO-RAD, USA), and the band intensities were quantified using Image-Pro Plus software (Mediacy, USA). Detailed information of antibody used in this study (see Additional file 1: Table S7).
The cytotoxicity assay was performed as previously described . Cell viability was measured using Cell Counting Kit-8(CCK8)following the manufacturer’s directions (Dojindo, Kumamoto, Japan).
A clonogenic assay was performed as previously described . At 48 h after transfection, BGC823CDDP, SGC7901CDDP, BGC823 and SGC7901 cells were cultured with CDDP at the indicated concentrations for 3 h. Then, the cells were harvested, seeded into six-well plates (500 cells per well) and cultured for an additional 2 (BGC823CDDP and SGC7901CDDP cells) or 3 weeks (BGC823 and SGC7901 cells). For scoring the colony-forming units, we fixed cells in 1 ml of methanol for 10 min and then stained the cells with crystal violet for 15 min.
Cell apoptosis was detected using a PI/Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, 556,547) according to the manufacturer’s instructions. Briefly, after GC cells were treated with CDDP at the indicated concentrations for 48 h in 6-well plates, they were harvested and resuspended in 300 ml of binding buffer. Next, 5 μl of Annexin V-FITC and 5 μl of PI were added to the suspensions, and the cells were incubated in the dark at 4 °C for 15 min. The samples were subsequently analyzed with a flow cytometer (Gallios, Beckman, USA).
Actinomycin D assay
The Actinomycin D assay was performed as previously described . SGC7901CDDP and BGC823CDDP cells were seeded in 5 wells in 24-well plates (5 × 104 cells per well). Twenty-four hours later, the cells were exposed to Actinomycin D (2 μg/ml, Abcam, ab141058) for 0 h, 6 h, 12 h, 18 h and 24 h. The cells were then harvested, and the relative RNA levels of circAKT3 and AKT3 mRNA were analyzed by RT-qPCR and normalized to the values measured in the group in the 0 h group (mock treatment).
Cells seeded onto coated cover slips growth for 24 h, then treated with CDDP, and harvested the cells at 0, 2, and 8 h. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min and then permeabilized with PBS containing 0.25% Triton X-100 for 10 min. Next, the cells were blocked with 1% BSA for 20 min before incubation with primary antibodies at room temperature for 2 h. After the cells were washed with PBS, they were incubated with appropriate secondary antibodies (FITC-conjugated goat anti-rabbit, Molecular Probes, USA) at room temperature for 2 h. Following a final wash with PBS, cells were mounted with antifading mounting medium containing DAPI. The images were captured with a Leica DMI3000B (Germany) fluorescence microscope.
Transduction with lentivirus
SGC7901CDDP cells stably expressing circAKT3 siRNA (si-circ-1) and its negative control siRNA (si-NC) were generated by infection with lentiviruses as previously described . Transfection was carried out according to the manufacturer’s instructions. The lentiviral expressing vectors were purchased from HanBio Co. Ltd. (Shanghai, China).
Nude mouse xenograft model
Six-week-old female BALB/c nude mice were purchased from the Laboratory Animal Center of Nanjing Medical University and maintained under pathogen-free conditions. A total of 5 × 106 SGC7901CDDP cells infected with lentivirus containing si-circ-1 or si-NC (2 μl of 109 viral genomes μl− 1, HanBio) in 100 μl of PBS were subcutaneously injected into different sides of the groin of each mouse. One week after injection, we intraperitoneally injected mice with cisplatin (5 mg/kg) in PBS or PBS alone three times per week. The xenograft tumors were harvested after 5 weeks. The entire experimental protocol was conducted in accordance with the guidelines of the local institutional animal care and use committee.
Immunohistochemical staining (IHC)
Xenografts and GC tissues exposed to the indicated concentrations of CDDP were prepared for IHC as previously described . Sections were identified by IHC Imager (DM4000B, LEIKA, Germany), and target protein expression levels were evaluated by the proportions and intensities of positive cells detected within 5 fields of view on every slide (400-fold magnification). Proportion scores were assigned as follows: < 10% = 0, 10–25% = 1, 26–50% = 2, 51–75% = 3 and > 75% = 4. Intensity scores were assigned as follows: 0 = no staining, 1 = weak, 2 = moderate, 3 = strong and 4 = significantly strong.
All experiments were performed in triplicate. Data were analyzed with SPSS 19.0 software (IBM, USA) and presented as the mean ± SEM. The statistical significance of the results was calculated using an unpaired Student’s t-test. DFS analysis was performed using the Kaplan-Meier method and log-rank test. Clinicopathological features were analyzed by a χ2 test. A Cox proportional hazards regression model was used to identify independent prognostic factors associated with DFS. Linear correlation analyzes were performed to determine correlations between circAKT3, miR-198 and PIK3R1 expression levels. A P value< 0.05 was defined as statistically significant.
Ectopic circAKT3 expression levels are observed in CDDP-resistant GC cells and tissues and are correlated with poor prognosis in GC patients receiving CDDP therapy
Correlation of relative circAKT3 expression with the clinicopathological characteristics of 105 patients accepted cisplatin-based chemotherapy with gastric cancer
No. of patients
circAKT3 facilitates CDDP resistance in vitro
circAKT3 exerts its function by sponging miR-198
PIK3R1 is a direct target of miR-198
circAKT3 regulates PIK3R1 expression, activates the PI3K/AKT signaling pathway and ultimately facilitates CDDP resistance by targeting miR-198 in vitro
circAKT3 promotes CDDP resistance of GC cells in vivo
CDDP treatment is one of the most predominant chemotherapeutic strategies for patients with GC . In this study, using RNA-Seq analysis, we determined that circRNA expression is associated with CDDP resistance in GC. We found a novel circular RNA termed circAKT3 that was upregulated in tissue samples from patients with CDDP-resistant GC and in CDDP-resistant cell lines and was correlated with five-year DFS. Moreover, circAKT3 was expressed at higher levels than other candidate circRNAs in CDDP-resistant GC patients, which meant that it may play a more important role than other circRNAs in GC.
DNA is the recognized target for CDDP cytotoxicity in cancer therapy. The resultant biological process in response to CDDP and other DNA-damaging therapies is the activation of apoptosis and the destruction of malignant cells. The most favorable evidence is the hypersensitivity of both eukaryotic and prokaryotic cells deficient in DNA repair to CDDP . An enhanced DNA repair mechanism can induce the survival of damaged or mutated tumor cells, resulting in resistance and subsequent tumor recurrence . BRCA1, a tumor-suppressor gene, is widely involved in cellular metabolism , transcriptional regulation [29, 30], and epigenetic modification . A growing body of evidence revealed that BRCA1 has a great effect on the modulation of CDDP resistance [32, 33, 34]. Alterative BRCA1 expression can regulate the mitochondrial fission program, which could modulate CDDP sensitivity . Some microRNAs contribute to DNA repair and CDDP sensitivity through BRCA1 deregulation, including miR-9  and miR-638 . A previous study demonstrated that BRCA1 mRNA levels were negatively associated with CDDP sensitivity in GC . Currently, there is a lack of research on circRNAs that regulate the BRCA1 gene in tumor cells, including GC. The underlying mechanisms require further exploration.
PIK3R1 encodes the regulatory subunit of PI3K (p85α). Some studies have reported that p85α positively regulates PI3K signaling in CDDP resistance. Because the PI3K pathway is a critical player in tumorigenesis and the ubiquitously hyperactivated signaling pathway in neoplasms, its inhibition both pharmacologically and genetically is considered to be the most promising strategy for targeted cancer treatment . The PI3K pathway has been revealed as a mediator of platinum resistance [39, 40] For instance, AKT activation mediated resistance to caspase-independent CDDP-induced apoptosis through inhibiting the apoptosis-inducing factor-associated pathway . One study reported that PI3K/AKT activation induced the upregulation of BRCA1 in tamoxifen-resistant breast cancer cells and resensitized them to CDDP treatment . Nevertheless, the underlying mechanism of how PIK3R1 mediates resistance to chemotherapy and whether this involves canonical PI3K signaling and downstream BRCA1 activity remains to be investigated in GC.
Based on the abovementioned studies, we conducted a series of experiments and demonstrated that circAKT3 reduced CDDP-induced activation of caspase-3 and apoptosis, leading to enhanced DDR and resistance to CDDP chemotherapy. Mechanistically, circAKT3 functions as a ceRNA by sponging miR-198 to abolish the suppressive effect of this miRNA on its target gene PIK3R1, which activated the PI3K/AKT signaling pathway in GC cells. The study showed that PI3K/AKT pathway activation contributes to upregulation of the DNA repair molecule BRCA1 and leads to resistance to CDDP-based DNA-damaging chemotherapy . In the current study, circAKT3 influenced DDR in GC cells, implying that circAKT3 might enhance CDDP resistance through the PI3K/AKT pathway and DDR mechanisms in GC cells. However, there are some drawbacks in this study, and we have not yet identified the specific mechanism by which BRCA1 regulates DDR in GC. At the same time, the reasons for circAKT3 shear formation and the upstream regulatory mechanism were not discussed. For the in vivo experiments, we did not use animal models of GC in situ, nor did we fully simulate the process of drug resistance. These approaches should be further pursued in subsequent studies.
In conclusion, we show that circAKT3 is upregulated in human GC and that it can efficiently sponge miR-198 to restore PIK3R1 expression. We also demonstrate that downregulation of circAKT3 can effectively promote CDDP sensitivity in GC cells by targeting the miR-198/PIK3R1 axis. Our results provide novel evidence that circRNAs function as “microRNA sponges” and highlight a promising therapeutic target for the CDDP resistance of GC patients.
Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University.
This work was supported by the National Natural Science Foundation of China (81572362); the National Natural Science Foundation Project of International Cooperation (NSFC-NIH, 81361120398); the Primary Research & Development Plan of Jiangsu Province (BE2016786); the Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU; the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801); 333 Project of Jiangsu Province (BRA2015474); Jiangsu Key Medical Discipline (General Surgery)(ZDXKA2016005); University Natural Science Research Project of Anhui Province(KJ2018A0247); Wannan Medical College Cultivation Fund of the Key Scientific Project(WK2017ZF10).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its Additional files.
ZKX, ZL and WZW conceived of the study and XXH carried out its design. XXH, QZ, BWL, LW, ZPX and XZ performed the experiments. XXH, XZ and ZYH collected clinical samples. XXH, ALZ, QL, GLS and QL analyzed the data and wrote the paper. LJW, LZ and HX revised the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Hospital’s Protection of Human Subjects Committee.
Consent for publication
We have received consents from individual patients who have participated in this study. The consent forms will be provided upon request.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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