# CircNR3C1 Alleviates Gastric Cancer Development by Inactivating AKT/mTOR

Authors: Luben Wang; Zhen Guo; Baoliang Guo; Fangkai Gao; Xiangdong Liu; Youchao Xu; Yang Wang
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

Differential level and regulatory effect of circNR3C1 in gastric cancer (GC) were determined. The differential levels of circNR3C1 in clinical samples of GC were determined. The association of circNR3C1 level with pathological indicators of GC was analyzed. After intervening circNR3C1 levels in gastric cancer cells, proliferative and migratory changes were investigated. Furthermore, we measured AKT and mTOR protein levels in GC cells intervened by circNR3C1. Finally, the role of AKT/mTOR in GC cell phenotypes regulated by circNR3C1 was explored. circNR3C1 was markedly lowly expressed in GC cells and tissues. A low level of circNR3C1 predicted high incidences of lymphatic or distant metastasis of GC. Knockdown of circNR3C1 enhanced proliferation and migration abilities in BGC-823 cells, whereas overexpression of circNR3C1 yielded the opposite results in AGS cells. circNR3C1 downregulated mTOR and AKT in GC cells. In addition, induction of the AKT activator could reverse the attenuated proliferative and migratory potentials in GC cells overexpressing circNR3C1. On the contrary, induction of the AKT inhibitor reversed the stimulated malignant phenotypes of GC with circNR3C1 knockdown. circNR3C1 inhibits GC to proliferate and migrate by inactivating the AKT/mTOR signaling. It is also closely linked to GC metastasis.


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## Body

## 1. Introduction

In China, the incidence of gastric cancer (GC) is very high, which is the number one diagnosed cancer of the digestive system. Notably, the diagnostic rate of early-stage GC in our country is only about 5–15%, which remains 50% in Japan and South Korea [1–3]. It is estimated that the five-year survival of GC at an early stage is up to 84–99%. Once GC is in the progressive phase, its 5-year survival sharply reduces to 16% even after comprehensive treatment [4, 5]. The etiology and pathogenesis of GC are complicated, involving changes in tumor cell adhesion, detachment from the primary focus, degradation of extracellular matrix, local or vascular infiltration, tumor cell colonization, and neovascularization [6–8]. It is of significance to enhance the diagnostic efficacy of early stage GC [9, 10].circRNAs have been well concerned because of their unique structure and functions [11]. circRNAs have a closed-loop structure, which are stably distributed in complex intracellular or extracellular environments since they are resistant to exonucleases [12, 13]. In addition, they lack 5′ cat and 3′ poly (A), displaying high specificities in different tissues, developmental stages, and diseases [14–16]. Hence, circRNAs are promising regulators involved in pathological progress, including cancer. circNR3C1 contains a functional NBD domain and a C-terminal with a negative charge. Previous studies have reported the vital functions of circRN3C1 in human cancers [16–18]. The present study attempted to investigate the effects of circNR3C1 on the development of GC, aiming to verify an effective molecular target for GC.


## 2. Materials and Methods

### 2.1. GC Samples

Samples were harvested from 40 patients with GC during radical resection. They did not have preoperative chemotherapy or radiotherapy. Tumor node metastasis (TNM) staging and histological classification of GC were determined based on the Union for International Cancer Control (UICC) criteria. The Ethical Committee of The Second Affiliated Hospital of Xuzhou Medical University approved this study. All patients provided informed consents. The study was conducted following the Helsinki Declaration.


### 2.2. Cell Proliferation Assay

SGC-7901, AGS, SNU-16, BGC-823 cell lines, and gastric mucosal epithelial cells (GES-1) purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) were cultured in the plates (96-well). Cell counting kit-8 (CCK-8) assay was performed daily at 1, 2, 3, and 4. After cell culture for 2 h, optical density at 490 nm per sample was measured via a microplate reader, and the cell activity curve was plotted.


### 2.3. Transwell Migration Assay

The cell suspension was prepared at 2 × 105 cells/mL. A suspension of 200 μL and 700 μL culture medium with 20% fetal bovine serum (FBS) was added and cultured for 48 h. Migrated cells on the bottom of the transwell insert were treated using methanol for 15 min, 0.2% crystal violet for 20 min, and then captured using a microscope.


### 2.4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

TRIzol was used to extract the total RNA. qRT-PCR was performed according to the previously established instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as the internal control. 2−ΔΔCt method was used for the quantitative analysis. Primer sequences were shown as follows: circNR3C1: forward: 5′’-GAAGCCAGTTGAGGGAAGTT-3’, reverse: 5′’-CTTGAATGCACTGAAGTGTTCC-3’; AKT: forward: 5′’-AAGTCATCGTGGCCAAGGAC-3’, reverse: 5′’-ACAGGTGGAAGAACAGCTCG-3’; mTOR: forward: 5′’-AAGCCGCGCGAACCTC-3’, reverse: 5′’-CTGGTTTCCTCATTCCGGCT-3’; GAPDH: forward: 5′’-CCTGGCACCCAGCACAAT-3’, reverse: 5′’-TGCCGTAGGTGTCCCTTTG-3’.


### 2.5. Western Blot

Total protein was extracted from cells and then quantified via the bicinchoninic acid method. Protein samples with the adjusted same concentration were separated and then loaded on polyvinylidene fluoride membranes followed by being blocked with defatted milk (5%) for 2 h and subsequently incubated with primary antibodies at 4°C overnight. Thereafter, secondary antibodies were added for further incubation for 2 h followed by bands being exposed via an electrochemiluminescence (ECL) kit.


### 2.6. Statistical Analysis

Statistical Product and Service Solutions 18.0 (IBM, Armonk, NY, USA) was used for data analysis. Chi-square was employed to analyze the relationship between the level of circNR3C1 and pathological indicators of GC. Statistical significance was set atP<0.05.


## 2.1. GC Samples

Samples were harvested from 40 patients with GC during radical resection. They did not have preoperative chemotherapy or radiotherapy. Tumor node metastasis (TNM) staging and histological classification of GC were determined based on the Union for International Cancer Control (UICC) criteria. The Ethical Committee of The Second Affiliated Hospital of Xuzhou Medical University approved this study. All patients provided informed consents. The study was conducted following the Helsinki Declaration.


## 2.2. Cell Proliferation Assay

SGC-7901, AGS, SNU-16, BGC-823 cell lines, and gastric mucosal epithelial cells (GES-1) purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) were cultured in the plates (96-well). Cell counting kit-8 (CCK-8) assay was performed daily at 1, 2, 3, and 4. After cell culture for 2 h, optical density at 490 nm per sample was measured via a microplate reader, and the cell activity curve was plotted.


## 2.3. Transwell Migration Assay

The cell suspension was prepared at 2 × 105 cells/mL. A suspension of 200 μL and 700 μL culture medium with 20% fetal bovine serum (FBS) was added and cultured for 48 h. Migrated cells on the bottom of the transwell insert were treated using methanol for 15 min, 0.2% crystal violet for 20 min, and then captured using a microscope.


## 2.4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

TRIzol was used to extract the total RNA. qRT-PCR was performed according to the previously established instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as the internal control. 2−ΔΔCt method was used for the quantitative analysis. Primer sequences were shown as follows: circNR3C1: forward: 5′’-GAAGCCAGTTGAGGGAAGTT-3’, reverse: 5′’-CTTGAATGCACTGAAGTGTTCC-3’; AKT: forward: 5′’-AAGTCATCGTGGCCAAGGAC-3’, reverse: 5′’-ACAGGTGGAAGAACAGCTCG-3’; mTOR: forward: 5′’-AAGCCGCGCGAACCTC-3’, reverse: 5′’-CTGGTTTCCTCATTCCGGCT-3’; GAPDH: forward: 5′’-CCTGGCACCCAGCACAAT-3’, reverse: 5′’-TGCCGTAGGTGTCCCTTTG-3’.


## 2.5. Western Blot

Total protein was extracted from cells and then quantified via the bicinchoninic acid method. Protein samples with the adjusted same concentration were separated and then loaded on polyvinylidene fluoride membranes followed by being blocked with defatted milk (5%) for 2 h and subsequently incubated with primary antibodies at 4°C overnight. Thereafter, secondary antibodies were added for further incubation for 2 h followed by bands being exposed via an electrochemiluminescence (ECL) kit.


## 2.6. Statistical Analysis

Statistical Product and Service Solutions 18.0 (IBM, Armonk, NY, USA) was used for data analysis. Chi-square was employed to analyze the relationship between the level of circNR3C1 and pathological indicators of GC. Statistical significance was set atP<0.05.


## 3. Results

### 3.1. circNR3C1 Was Highly Expressed in GC

Firstly, circNR3C1 was markedly downregulated in GC tissues in comparison to normal ones (Figures1(a) and 1(b)). In addition, circNR3C1 was lowly expressed in GC cells (Figure 1(c)). Among the four tested GC cells, BGC-823 and AGS cells expressed the most differential level of circNR3C1, which were used to generate circNR3C1 overexpression and knockdown model by transfection of pcDNA-circNR3C1 and anti-circNR3C1, respectively (Figure 2(a)).Figure 1
circNR3C1 was overexpressed in GC. (a, b) circNR3C1 was detected in GC and normal tissues. (c) circNR3C1 levels in GC cell lines.∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001.
(a)(b)(c)Figure 2
circNR3C1 attenuated proliferation and migration potentials in GC. (a) Transfection efficacy of pcDNA-circNR3C1 and anti-circNR3C1 in GC cells, respectively. (b) Viability in GC cell lines regulated by circNR3C1. (c) GC cell migration regulated by circNR3C1.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)Moreover, clinical manifestations of recruited GC patients were retrospectively analyzed according to the median circNR3C1 level in cancer tissues. It is shown that circNR3C1 was significantly associated with incidences of lymphatic metastasis and distant metastasis in GC. As a result, we believed that circNR3C1 may be a novel biomarker for GC.


### 3.2. circNR3C1 Inhibited Proliferative and Migratory Potentials in GC

In AGS cells overexpressing circNR3C1, viability and migratory cell numbers were reduced (Figures2(b)-2(c)). On the contrary, knockdown of circNR3C1 enhanced proliferative and migratory rates in BGC-823 cells (Figures 2(b) and 2(c), right).


### 3.3. circNR3C1 Inactivated the AKT/mTOR Signaling

Interestingly, AKT and mTOR were remarkably downregulated in AGS cells overexpressing circNR3C1 (Figure3(a)). Conversely, upregulated AKT and mTOR were detected after the knockdown of circNR3C1 in BGC-823 cells (Figure 3(b)). It is suggested that circNR3C1 was able to inactivate the AKT/mTOR pathway in GC.Figure 3
circNR3C1 inactivated the AKT/mTOR signaling. (a) Western blotting detected AKT and mTOR protein levels in AGS with circNR3C1 overexpression. (b) Western blotting measured of AKT and mTOR in BGC-823 with circNR3C1 knockdown.∗∗P<0.01.
(a)(b)


### 3.4. AKT/mTOR Signaling Participated in circNR3C1-Regulated GC Progression

To further elucidate the involvement of the AKT/mTOR in GC progression, we applied SC79 (AKT activator) and MK-2206 (AKT inhibitor) in GC cells. SC79 application markedly upregulated circNR3C1 in AGS cells overexpressing circNR3C1. In addition, MK-2206 application downregulated circNR3C1 level in BGC-823 cells with circNR3C1 knockdown (Figure4(a)). Moreover, SC79 induced increases in viability and migratory cell number in AGS cells overexpressing circNR3C1 (Figures 4(b) and 4(c), left). However, MK-2206 caused declines in proliferation and migration of BGC-823 cells with circNR3C1 knockdown (Figures 4(b) and 4(c), right). Collectively, circNR3C1 alleviated GC progression through inactivating the AKT/mTOR signaling.Figure 4
AKT/mTOR signaling was involved in circNR3C1-regulated GC progression. (a) CircNR3C1 levels in 2 GC cell lines induced with SC79 and MK-2206, respectively. (b) Cell viability in 2 GC cell lines induced using SC79 and MK-2206, respectively; (c) Cell migration in 2 GC cell lines induced by SC79 and MK-2206.∗P<0.05, ∗∗P<0.01.
(a)(b)(c)


## 3.1. circNR3C1 Was Highly Expressed in GC

Firstly, circNR3C1 was markedly downregulated in GC tissues in comparison to normal ones (Figures1(a) and 1(b)). In addition, circNR3C1 was lowly expressed in GC cells (Figure 1(c)). Among the four tested GC cells, BGC-823 and AGS cells expressed the most differential level of circNR3C1, which were used to generate circNR3C1 overexpression and knockdown model by transfection of pcDNA-circNR3C1 and anti-circNR3C1, respectively (Figure 2(a)).Figure 1
circNR3C1 was overexpressed in GC. (a, b) circNR3C1 was detected in GC and normal tissues. (c) circNR3C1 levels in GC cell lines.∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001.
(a)(b)(c)Figure 2
circNR3C1 attenuated proliferation and migration potentials in GC. (a) Transfection efficacy of pcDNA-circNR3C1 and anti-circNR3C1 in GC cells, respectively. (b) Viability in GC cell lines regulated by circNR3C1. (c) GC cell migration regulated by circNR3C1.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)Moreover, clinical manifestations of recruited GC patients were retrospectively analyzed according to the median circNR3C1 level in cancer tissues. It is shown that circNR3C1 was significantly associated with incidences of lymphatic metastasis and distant metastasis in GC. As a result, we believed that circNR3C1 may be a novel biomarker for GC.


## 3.2. circNR3C1 Inhibited Proliferative and Migratory Potentials in GC

In AGS cells overexpressing circNR3C1, viability and migratory cell numbers were reduced (Figures2(b)-2(c)). On the contrary, knockdown of circNR3C1 enhanced proliferative and migratory rates in BGC-823 cells (Figures 2(b) and 2(c), right).


## 3.3. circNR3C1 Inactivated the AKT/mTOR Signaling

Interestingly, AKT and mTOR were remarkably downregulated in AGS cells overexpressing circNR3C1 (Figure3(a)). Conversely, upregulated AKT and mTOR were detected after the knockdown of circNR3C1 in BGC-823 cells (Figure 3(b)). It is suggested that circNR3C1 was able to inactivate the AKT/mTOR pathway in GC.Figure 3
circNR3C1 inactivated the AKT/mTOR signaling. (a) Western blotting detected AKT and mTOR protein levels in AGS with circNR3C1 overexpression. (b) Western blotting measured of AKT and mTOR in BGC-823 with circNR3C1 knockdown.∗∗P<0.01.
(a)(b)


## 3.4. AKT/mTOR Signaling Participated in circNR3C1-Regulated GC Progression

To further elucidate the involvement of the AKT/mTOR in GC progression, we applied SC79 (AKT activator) and MK-2206 (AKT inhibitor) in GC cells. SC79 application markedly upregulated circNR3C1 in AGS cells overexpressing circNR3C1. In addition, MK-2206 application downregulated circNR3C1 level in BGC-823 cells with circNR3C1 knockdown (Figure4(a)). Moreover, SC79 induced increases in viability and migratory cell number in AGS cells overexpressing circNR3C1 (Figures 4(b) and 4(c), left). However, MK-2206 caused declines in proliferation and migration of BGC-823 cells with circNR3C1 knockdown (Figures 4(b) and 4(c), right). Collectively, circNR3C1 alleviated GC progression through inactivating the AKT/mTOR signaling.Figure 4
AKT/mTOR signaling was involved in circNR3C1-regulated GC progression. (a) CircNR3C1 levels in 2 GC cell lines induced with SC79 and MK-2206, respectively. (b) Cell viability in 2 GC cell lines induced using SC79 and MK-2206, respectively; (c) Cell migration in 2 GC cell lines induced by SC79 and MK-2206.∗P<0.05, ∗∗P<0.01.
(a)(b)(c)


## 4. Discussion

Gastric cancer (GC) is insidious and can occur in any part of the stomach, most often in the antrum, and its clinical manifestations lack specificity [1–3]. At present, surgery is the main treatment for GC, which is divided into radical surgery and palliative surgery. With the innovation of medical technologies, great advances have been made on improving efficacies of radiotherapy, chemotherapy, biological therapy, interventional therapy, molecular targeted therapy, etc. [4–8]. Popularized screening of GC should be applied to detect GC as early as possible, therefore prolonging the survival of affected people [9, 10].A growing number of circRNAs have been demonstrated to have a close relation to lung carcinoma and esophageal carcinoma [11–15]. Recently, plentiful circRNAs were reported to be aberrantly expressed and were regarded as potential biomarkers in the carcinogenesis and progression of GC [19]. circNR3C1 was previously reported mostly in bladder cancer [16–18]. Xie et al. demonstrate that circNR3C1 inhibited the progression of bladder cancer via acting as endogenous blocker of BRD4/C-myc complex [16]. There is no evidence showing the role of circNR3C1 in the progression of gastric cancer. The novelty of our present study was that it was the first attempt to study the biofunctions of circNR3C1 in GC and to explore the underlying molecular mechanism. The current study demonstrated that circNR3C1 was significantly upregulated in GC cell lines and tissues. By retrospective analysis of recruited GC patients, circNR3C1 was closely related to the incidences of lymphatic metastasis and distant metastasis. Later, we generated circNR3C1 overexpression and knockdown model in GC cells, respectively. circNR3C1 overexpression remarkably decreased proliferative and migratory rates in AGS cells, while knockdown of circNR3C1 enhanced them in BGC-823 cells. It is concluded that circNR3C1 inhibited GC to proliferate and migrate as a tumor-suppressor gene.It was demonstrated that circNR3C1 negatively regulated AKT/mTOR in GC cells. The AKT/mTOR signaling is widely regulated by extracellular signal-regulated kinases, nuclear factors, matrix metalloproteinases, and growth factors [20, 21]. More and more clinical data supported the effectiveness of oral AKT/mTOR-targeted drugs in specific types of malignant tumors [20]. By applying SC79, the AKT activator, proliferative and migratory potentials were enhanced in AGS cells overexpressing circNR3C1. Moreover, the application of MK-3206, the AKT inhibitor, weakened proliferative and migratory potentials in BGC-823 cells with circNR3C1 knockdown. It is concluded that circNR3C1 alleviated the malignant development of GC via inactivating the AKT/mTOR pathway. There are some evident limitations in this study. Lack of in vivo validations in animal models weakened the confidence level of our findings. Our future study will focus on the validation of circNR3C1 expression in a larger cohort of GC patients. More GC samples should be collected and more patients need to be enrolled for the longer follow-up. Also, we should further complete the in vivo assays in mice models of GC to explore the role of circNR3C1 in the growth of GC.


## 5. Conclusions

circNR3C1 inhibits GC to proliferate and migrate by inactivating the AKT/mTOR signaling. It is also closely linked to GC metastasis. In summary, circNR3C1/AKT/mTOR may be the key mechanism and a promising therapeutic target in GC in the years to come.



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*Source: 8402732-2022-03-18.xml*