circ_AKT3 knockdown suppresses cisplatin resistance in gastric cancer

Abstract Background Circular RNAs (circRNAs) are associated with cisplatin resistance in gastric cancer (GC). This study aims to explore the role of circRNA AKT serine/threonine kinase 3 (circ_AKT3) in the resistance of GC to cisplatin. Methods 42 sensitive and 23 resistant GC patients were recruited for tissue collection. The cisplatin-resistant GC cells MKN-7/DDP and HGC-27/DDP were used for in vitro study. circ_AKT3, microRNA-206 (miR-206) and protein tyrosine phosphatase non-receptor type 14 (PTPN14) levels were detected via quantitative reverse transcription real-time PCR (qPCR) and Western blot. Cisplatin resistance was assessed by detecting P-glycoprotein (P-gp) level, half maximal inhibitory concentration (IC50) of cisplatin and cell apoptosis. The target relationship between miR-206 and circ_AKT3 or PTPN14 was analyzed via dual-luciferase reporter and RNA pull-down assays. The role of circ_AKT3 in vivo was assessed using xenograft model. Results circ_AKT3 level was increased, but miR-206 was declined in cisplatin-resistant GC tissues and cells. circ_AKT3 knockdown or miR-206 overexpression decreased the level of P-gp and IC50 of cisplatin and increased apoptosis of MKN-7/DDP and HGC-27/DDP cells. Additionally, circ_AKT3 targeted miR-206, and regulated cisplatin resistance by interacting with miR-206. PTPN14 was regulated by circ_AKT3 through miR-206 as a bridge. Also, circ_AKT3 knockdown decreased xenograft tumor growth. Conclusion circ_AKT3 knockdown suppressed cisplatin resistance using miR-206/PTPN14 axis in cisplatin-resistant GC cells.


Introduction
Gastric cancer (GC) is one of the main malignancies with high mortality and incidence [1]. Surgery is the main therapeutic strategy of GC, while the clinical outcomes of advanced GC patients remain poor. The combined treatment of surgery and chemotherapy or radiotherapy has been implemented in GC [2]. Cisplatin (DDP) is a major known chemotherapy drug, whereas development of drug resistance limits its therapeutic efficacy [3]. Hence, it is necessary to urgently explore novel strategies to improve cisplatin sensitivity in GC treatment.
Noncoding RNAs, such as circular RNAs (circRNAs) and microRNAs (miRNAs), are not able to code proteins and play important roles in development and therapeutics of cancers [4]. circRNAs have a covalently closed continuous loop structure and play essential roles in cancers [5]. Moreover, circRNAs are associated with occurrence of chemoresistance in human cancers [6]. circRNA AKT serine/threonine kinase 3 (circ_AKT3) is derived from AKT3, which has been regarded to have a vital role in glioblastoma and clear cell renal cell carcinoma [7,8]. More importantly, circ_AKT3 could increase chemoresistance in GC by suppressing miR-198 [9]. However, the mechanism involving circ_AKT3 is complicated and requires further investigation, particularly with respect to cisplatin resistance. miRNAs regulate eukaryotic gene expression and exhibit pivotal roles in diagnosis, therapy and prognosis of GC [10]. Former works suggested that miR-206 could inhibit resistance to drugs, such as paclitaxel, euthyrox, gefitinib and cisplatin [11][12][13][14]. Moreover, miR-206 is reported to play an anti-cancer role and could inhibit cisplatin resistance in GC [15]. Nevertheless, whether miR-206 is responsible for circ_AKT3-meidated cisplatin resistance is unknown. Furthermore, previous studies report that protein tyrosine phosphatase non-receptor type 14 (PTPN14) is an oncogene and associated with drug resistance in GC [16,17]. Through bioinformatics analysis, we found the predicted complementary sites of miR-206 with circ_AKT3 and PTPN14. Thus, we hypothesized that miR-206 and PTPN14 might be associated with the mechanism addressed by circ_AKT3. In this study, we investigated the influence of circ_AKT3 on cisplatin resistance in GC and explored whether it was regulated by miR-206/PTPN14 axis.

Patients and tissues
42 cisplatin-sensitive and 23 cisplatin-resistant patients with advanced GC were recruited for this study. Patients had undergone two cycles of cisplatin-based chemotherapy and were divided into sensitive or resistant group according to the efficacy of the therapy [18]. The cancer tissues were collected and stored at −80°C for RNA extraction. Written informed consents were obtained. This study was approved by the ethics committee of the Changchun University of Chinese Medicine.

Western blot
MKN-7/DDP and HGC-27/DDP cells were lysed for total protein isolation using RIPA buffer with 1% phenylmethylsulfonyl fluoride (Solarbio, Beijing, China). After the high-speed centrifugation (12,000g, 5 min) at 4°C, the protein in supernatant was quantified and used for SDS-PAGE. Transmembrane was performed using nitrocellulose membranes (Solarbio) and western blot transfer buffer (10×, Solarbio). QuickBlock TM Blocking Buffer (Beyotime) was used for blocking the nonspecific binding sites. Subsequently, the membranes were incubated with primary antibodies overnight and then interacted with special secondary antibody. The antibodies against p-glycoprotein (P-gp) (ab235954), PTPN14 (ab204321), GAPDH (ab181602) and immunoglobulin G (ab205718) were obtained from Abcam (Cambridge, MA, USA). Protein signals were developed using BeyoECL Plus (Beyotime). The relative protein expression was half-quantified with Quantity One software (Bio-Rad) and normalized to GAPDH level.

Bioinformatics analysis, luciferase reporter analysis and RNA pull-down
Bioinformatics analysis was used to search potential binding sites of miR-206 and circ_AKT3 or PTPN14 by using starBase. For luciferase reporter assay, firefly luciferase-expressing pmirGLO vectors (Promega, Madison, WI, USA) were exploited for establishment of wild-type (Wt) or mutant (Mut) luciferase reporter vectors targeting circ_AKT3 or PTPN14, named as circ_AKT3-Wt, circ_AKT3-Mut, PTPN14 3′UTR-Wt or PTPN14 3′UTR-Mut, respectively. MKN-7/DDP and HGC-27/DDP cells were co-transfected with miR-206 mimic or miR-NC and these constructs, along with renilla vector. After 24 h post-transfection, luciferase activity was detected through a luciferase reporter assay kit (Promega).
For RNA pull-down, the Wt or Mut sequences of miR-206 and circ_AKT3 were labeled with biotin, and named as bio-miR-206, bio-miR-206-Mut, bio-circ_AKT3 and bio-circ_AKT3-Mut, respectively. The bio-miR-NC and bio-NC were used as the control. MKN-7/DDP and HGC-27/DDP cells were lysed in RIPA buffer with RNase inhibitor (Invitrogen, Carlsbad, CA, USA) and then incubated with biotinylated products for 2 h, followed by incubation with streptavidin beads for 1 h. After extraction of bound RNAs, the expressions of circ_AKT3 and miR-206 enriched in precipitates were detected by RT-qPCR analysis.

Xenograft model
The 5-week-old male BALB/c nude mice were obtained from Beijing Laboratory Animal Center (Beijing, China). The lentiviral vector of short hairpin RNA targeting circ_AKT3 (sh-circ_AKT3) or negative control (sh-NC) was generated by RiboBio. MKN-7/DDP cells (2 × 10 6 cells per mouse) stably transfected with sh-circ_AKT3 or sh-NC were subcutaneously injected into the flanks of the mice (n = 6/group). Tumor volume was examined every 4 days after 7 days and calculated as: 0.5 × length (mm) × width 2 (mm 2 ). After 31 days, the mice were killed by cervical dislocation. Tumor weight was measured and tumor samples were collected for the abundances of circ_AKT3, miR-206, PTPN14 and P-gp. The animal experiments were approved by the Animal Care and Use Committee of the Changchun University of Chinese Medicine.

Statistical analysis
Statistical analysis of every experiment was conducted by GraphPad Prism 6 (GraphPad Inc., La Jolla, CA, USA). The data from three independent experiments were shown as mean value ± standard deviation. Student's t test or Mann-Whitney test was employed for the comparison between the two groups, while ANOVA with Tukey test was employed for the comparison between three or more groups. The potential linear relationship between miR-206 and circ_AKT3 or PTPN14 level was analyzed by spearman's correlation assay. P < 0.05 was considered as significant difference.

Research involving human participants and/or animals:
This study was approved by the Institutional Animal Care and Use Committee of the Changchun University of Chinese Medicine. We have strictly carried out the relevant research work protecting the rights and interests of animals under the supervision of the Institutional Animal Care and Use Committee to ensure that the research is in line with the relevant provisions of the Institutional Animal Care and Use Committee.
Informed consent: Informed consent was obtained from all individual participants included in the study.  Ethics approval: All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

circ_AKT3 level is upregulated and miR-206 level is downregulated in cisplatinresistant GC
To probe the role of circ_AKT3 and miR-206 in cisplatin resistance, their levels were detected in cisplatin-sensitive or resistant GC tissues. Compared with sensitive samples (n = 42), circ_AKT3 level was evidently enhanced in cisplatin-resistant GC tissues (n = 23) (Figure 1a). On the contrary, the abundance of miR-206 was abnormally reduced in resistant tissues in comparison to that in sensitive group (Figure 1b). Moreover, the data from  (Figure 2c and d). In addition, the analysis of flow cytometry showed higher apoptotic rate in the two resistant cell lines after silencing circ_AKT3 as compared with the two sensitive cell lines (Figure 2e and f). The above findings manifested that circ_AKT3 knockdown improved sensitivity of GC cells to cisplatin.

Overexpression of miR-206 represses cisplatin resistance of resistant GC cells
The role of miR-206 in cisplatin resistance was also explored in MKN-7/DDP and HGC-27/DDP cells by overexpressing miR-206 using miRNA mimic. After the transfection of miR-206 mimic, miR-206 abundance was markedly increased in MKN-7/DDP and HGC-27/DDP cells (Figure 3a). Then, miR-206 overexpression induced obvious reduction in IC 50 of cisplatin in the two types of resistant cells (Figure 3b). Meanwhile, the results of Western blot demonstrated that miR-206 overexpression led to a decline in P-gp protein production (Figure 3c and d). Besides, the apoptotic rate of MKN-7/DDP and HGC-27/DDP cells was significantly increased by miR-206 overexpression (Figure 3e and f). Taken together, these results explained that miR-206 was able to improve cisplatin sensitivity in GC cells.

circ_AKT3 regulates cisplatin resistance of resistant GC cells by sponging miR-206
Results of bioinformatics analysis displayed that circ_AKT3 carried the complementary sites of miR-206 (Figure 4a), suggesting the potential correlation between them. To validate this interaction, the Wt or Mut luciferase reporter vectors of circ_AKT3 were constructed and transfected into MKN-7/DDP and HGC-27/DDP cells. Results first showed that miR-206 overexpression induced decrease in luciferase activity in circ_AKT3-Wt-transfected cells, but the luciferase activity of circ_AKT3-Mut had no response to miR-206 overexpression (Figure 4b and c). Moreover, RNA pull-down assay revealed the binding ability of circ_AKT3 and miR-206 by biotinylated miR-206 or circ_AKT3, respectively (Figure 4d and e). According to spearman's correlation analysis, miR-206 expression in GC tissues was inversely associated with circ_AKT3 level (r = −0.629 and P < 0.001) (Figure 4f). Subsequently, the effect of circ_AKT3 on miR-206 level was investigated in MKN-7/DDP and HGC-27/DDP cells by overexpressing or silencing circ_AKT3. As exhibited in Figure 4g and h, miR-206 abundance was obviously inhibited by circ_AKT3 overexpression, but enhanced by circ_AKT3 interference. To explore whether the effect of circ_AKT3 on cisplatin resistance was modulated via miR-206, MKN-7/DDP and HGC-27/DDP cells were transfected with si-NC, si-circ_AKT3, si-circ_AKT3 + anti-miR-NC or anti-miR-206. The data of RT-qPCR demonstrated that transfection of anti-miR-206 obviously abolished the promoting impact of circ_AKT3 interference on miR-206 expression level (Figure 5a). Besides, the rescue experiments displayed that miR-206 deficiency attenuated the inhibitive role of circ_AKT3 silence in IC 50 of cisplatin and P-gp protein level as well as the promoting effect of circ_AKT3 on apoptosis (Figure 5b-f). Thus, all these findings explained that circ_AKT3 regulated cisplatin resistance by interacting with miR-206 in GC cells.

miR-206 inhibits cisplatin resistance of resistant GC cells via targeting PTPN14
The mRNA and protein levels of PTPN14 were evidently enhanced in cisplatin-resistant GC tissues and cells ( Figure 6a-d). Meanwhile, PTPN14 level was negatively associated with miR-206 level in GC tissues (Figure 6e). To further elucidate the mechanism, bioinformatics analysis showed that miR-206 was able to bind to PTPN14 (Figure 6f). Subsequent results displayed that the luciferase activity was notably reduced in PTPN14 3′UTR-Wt + miR-206 group in MKN-7/DDP and HGC-27/DDP cells, but it was not changed in PTPN14 3′UTR-Mut + miR-206 group (Figure 6f). Moreover, restoration of PTPN14 weakened the effect of miR-206 overexpression on IC 50 of cisplatin, P-gp expression and cell apoptosis in MKN-7/DDP and HGC-27/ DDP cells (Figure 6g-i). By large, all the above observations demonstrated that PTPN14 could regulate cisplatin resistance through interaction with miR-206.

circ_AKT3 modulates PTPN14 expression by miR-206
To explore whether circ_AKT3 could regulate PTPN14 in DDP-resistant GC cells, MKN-7/DDP and HGC-27/DDP cells were transfected with si-NC, si-circ_AKT3, si-circ_AKT3 + anti-miR-NC or anti-miR-206. As displayed in Figure 7a-c, PTPN14 expression at mRNA and protein levels were notably decreased via knockdown of circ_AKT3, which were restored by inhibition of miR-206, indicating that circ_AKT3 regulated PTPN14 by associating with miR-206.

circ_AKT3 knockdown decreases xenograft tumor growth
The study continued to explore the effect of circ_AKT3 in vivo. As shown in Figure 8a and b, the volume and weight of tumors were obviously declined in sh-circ_AKT3 group in comparison to sh-NC group. In addition, circ_AKT3, PTPN14 and P-gp levels were reduced and miR-206 abundance was increased in tumor samples from sh-circ_AKT3 group in comparison to sh-NC group (Figure 8c-e). The above data ascertained that circ_AKT3 knockdown inhibited MKN-7/DDP cell malignancy in vivo.

Discussion
Previous studies demonstrated that circRNAs could act as miRNA sponges or competing endogenous RNAs (ceRNAs) to further modulate drug resistance in GC [20,21]. Moreover, it was reported that circ_AKT3 contributed to cisplatin resistance in GC [9]. Nevertheless, more insights in the mechanism of circ_AKT3 regulating cisplatin resistance in GC are needed. In the current research, circ_AKT3 was highly expressed in GC tissues and cells with cisplatin resistance. Knockdown of circ_AKT3 decreased cisplatin resistance by downregulating P-gp expression. For the first time, we demonstrated a ceRNA network of circ_AKT3/miR-206/PTPN14 that was associated with cisplatin resistance.
In this study, we found high level of circ_AKT3 might be associated with cisplatin resistance in GC, indicating the positive role of circ_AKT3 in GC, which was opposite to that in other cancers [7,8]. We hypothesized that the exact roles of circRNAs in cancers might be induced due to the alteration of tumor microenvironment. By using MKN-7/DDP and HGC-27/DDP cells, we found that silencing circ_AKT3 decreased cisplatin resistance as reduction in IC 50 of cisplatin and increase in apoptosis after circ_AKT3 knockdown, which was consistent with the findings of previous study [9]. P-gp is one of the key proteins associated with drug resistance and can trigger multidrug resistance in GC [22,23]. This study found that circ_AKT3 knockdown inhibited P-gp protein expression in GC, suggesting the inimical role of circ_AKT3 in chemotherapy. The ceRNA regulatory network, including cir-cRNA, miRNA and mRNA, is one of the main mechanisms associated with tumorigenesis and pathogenesis of GC [24]. To explore whether circ_AKT3 regulated cisplatin resistance by functioning as a ceRNA, its potential target miRNAs were searched. As a result, we confirmed circ_AKT3 acted as a sponge for miR-206 in GC cells.
miR-206 has been reported to negatively modulate drug resistance in human cancers [11][12][13][14]. In the current work, low level of miR-206 was involved in cisplatin resistance and its overexpression attenuated the cisplatin resistance by regulating P-gp. This was also consistent with previous studies [14,15]. Moreover, the effect of circ_AKT3 silence on cisplatin resistance was weakened by miR-206 exhaustion, indicating that circ_AKT3 regulated drug resistance by sponging miR-206 in GC. To further explore the ceRNA crosstalk of circ_AKT3, the target mRNA of miR-206 was searched. Previous studies focusing on the role of miR-206 in human cancers have showed some targets, such as cyclinD2, CXC chemokine receptor 4 and mitogen activating protein kinase 3 (MAPK3) [15,25,26]. This research validated PTPN14 as a functional target of miR-206 using luciferase reporter analysis.
Previous studies suggested PTPN14 served as an oncogene in GC by regulating proliferation, migration and epithelial-to-mesenchymal transition [16,27,28]. Moreover, PTPN14 was implicated in paclitaxel and doxorubicin resistance in GC [17]. Our study showed that PTPN14 expression was elevated in cisplatin-resistant GC, indicating that PTPN14 might also contribute to cisplatin resistance. Furthermore, we found that PTPN14 weakened the effect of miR-206 on cisplatin resistance, and it was regulated by circ_AKT3 through miR-206. Collectively, cir-c_AKT3 induced PTPN14 to participate in the regulation of cisplatin resistance by sponging miR-206 in GC cells. To better understand the underlying mechanism, in vivo experiments were performed. And our data confirmed the suppressive effect of circ_AKT3 knockdown on growth of cisplatin-resistant GC cells in vivo.

Conclusion
In conclusion, our findings described the sensitization role of circ_AKT3 knockdown in GC, uncovered by decrease in IC 50 of cisplatin and P-gp expression and increase in apoptosis. The inner mechanism was that circ_AKT3 regulated miR-206/PTPN14 axis in a ceRNA-based manner. This work first confirmed the ceRNA network of circ_AKT3/miR-206/PTPN14, providing a new idea for ameliorating cisplatin sensitivity in GC treatment.