Upregulated hsa_circRNA_100269 inhibits the growth and metastasis of gastric cancer through inactivating PI3K/Akt axis

Abstract

The pathogenesis of GC involves the complex networking of multiple signaling pathways; however, the detailed mechanisms of tumorigenesis of GC remains largely unknown. Therefore, it is necessary to explore novel diagnostic/prognostic biomarkers for GC. In this study, the levels of hsa_circRNA_100269 in gastric cancer (GC) samples and cells were examined, and its effects on the biological functions of GC cells were elucidated. The levels of hsa_circRNA_100269 in specimens/cell lines were examined using RT-qPCR. Cell models with hsa_circRNA_100269 overexpression or knockdown were generated using lentiviral vectors. Cell viability was determined by MTT assay; cell migratory/invasive activity was evaluated using wound healing/Transwell assay. Cell cycle arrest and apoptosis were assessed by flow cytometry; expression of associated markers involved in cell apoptosis, EMT and the PI3K/Akt signaling were determined by RT-qPCR/immunoblotting. In vivo study was also performed using hsa_circRNA_100269 knockout mice. Our findings revealed downregulation of hsa_circRNA_100269 in GC tissues compared to non-cancerous control. Additionally, the levels of PI3K were remarkably elevated in GC tissues, where hsa_circRNA_100269 and PI3K was negatively correlated. Moreover, the expression of hsa_circRNA_100269 was associated with histology grade and occurrence of metastasis in GC patients. In addition, hsa_circRNA_100269 was downregulated in GC cells compared to normal gastric epithelial cells. Overexpressed hsa_circRNA_100269 notably inhibited the proliferation, migration, invasion and EMT of GC cells, whereas cell cycle arrest at G0/G1 phase was promoted and cell apoptosis was enhanced. Moreover, the PI3K/Akt signaling was involved in hsa_circRNA_100269-regulated GC cell proliferation, migration, invasion, EMT and apoptosis. Knockdown of hsa_circRNA_100269 also remarkably induced tumor growth in mouse model. In summary, our findings indicated that the levels of hsa_circRNA_100269 were reduced in GC. Furthermore, hsa_circRNA_100269 could suppress the development of GC by inactivating the PI3K/Akt pathway. More importantly, hsa_circRNA_100269/PI3K/Akt axis may be a novel therapeutic candidate for GC treatment.

Fig 1. The expression of hsa_circRNA_100269 is downregulated in GC tissues and cells.

(A) The level of hsa_circRNA_100269 was determined in 56 GC tissues and paired non-tumour samples using reverse transcription-quantitative polymerase chain reaction. (B) Hsa_circRNA_100269 expression was assessed in GC patients with different tumour grades. (C) The level of hsa_circRNA_100269 was evaluated in GC tissues with or without metastasis. (D) Survival analysis of GC patients with low- or high- hsa_circRNA_100269 expression. (E) The expression of hsa_circRNA_100269 was examined in GC cell lines (AGS, NCI-N87 and MKN-45) and one normal human gastric epithelial cell line (GES-1). All the experiments were performed in triplicate. *P<0.05 vs. corresponding control group. GC, gastric cancer.

Fig 2. Upregulated hsa_circRNA_100269 inhibits the proliferation, migration, invasion and EMT of GC cells.

(A) Transfection efficiency of o/e-hsa_circRNA_100269 was confirmed using RT-qPCR. (B and C) The proliferation of GC cells transfected with o/e-hsa_circRNA_100269 or o/e-NC were determined using Cell Counting Kit-8 assay. (D and E) The migration of transfected AGS and MKN-45 cells were evaluated using a wound healing assay (magnificationx100). (F and G) The invasive activity of GC cells transfected with o/e-hsa_circRNA_100269 or o/e-NC were examined (magnificationx200). (H and I) The expression levels of EMT-associated markers were evaluated using RT-qPCR and western blotting. All the experiments were performed in triplicate. *P<0.05 vs. o/e-NC. GC, gastric cancer; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 3. Overexpression of hsa_circRNA_100269 induces cell cycle arrest and apoptosis in GC cells.

(A and B) The distribution of cell cycle and apoptosis in GC cells overexpressing hsa_circRNA_100269 were examined. (C and D) The apoptosis of AGS and MKN-45 cells transfected with o/e-hsa_circRNA_100269 were also determined using flow cytometry. (E and F) The expression levels of apoptosis-associated markers were evaluated in transfected GC cells compared with the control. All the experiments were performed in triplicate. *P<0.05 vs. o/e-NC. GC, gastric cancer.

Fig 4. Knockdown of hsa_circRNA_100269 promotes the proliferation, migration, invasion and EMT of GC cells.

(A) Transfection efficiency of sh-hsa_circRNA_100269 was evaluated by RT-qPCR. (B and C) The viabilities of GC cells transfected with sh-hsa_circRNA_100269 or sh-NC were examined using Cell Counting Kit-8 assay. (D-G) The migration and invasion of transfected AGS and MKN-45 cells were determined using wound healing (magnificationx100) and Transwell assay (magnificationx200). (H and I) The levels of EMT-related molecules were determined using RT-qPCR and western blotting. All the experiments were performed in triplicate. *P<0.05 vs. non-transfected cells. GC, gastric cancer; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 5. Knockdown of hsa_circRNA_100269 inhibits cell cycle arrest and apoptosis of GC cells.

(A and B) The distribution of cell cycle and apoptosis in GC cells transfected with sh-hsa_circRNA_100269 were determined. (C-F) The apoptosis of AGS and MKN-45 cells with hsa_circRNA_100269 knockdown were also examined compared with the controls. All the experiments were performed in triplicate. *P<0.05 vs. non-transfected cells. GC, gastric cancer.

Fig 6. The PI3K/Akt signaling is a promising target of hsa_circRNA_100269 in GC cells.

(A) The levels of molecules involved in the PI3K/Akt signaling were assessed in AGS and MKN-45 cells with overexpression/knockdown of hsa_circRNA_100269. (B) The expression of PI3K was examined in GC and matched non-tumour tissues. (C) PI3K expression was evaluated in GC patients with various tumour grades. (D) The expression level of PI3K was determined in GC tissues with metastasis compared to the controls. (E) Spearman’s correlation analysis indicated the inverse correlation between hsa_circRNA_100269 and PI3K in GC samples (r = -0.3291; P = 0.00938). (F) The level of PI3K was also assessed in GC cells compared with normal human gastric epithelial cells. All the experiments were performed in triplicate. *P<0.05 vs. corresponding control group. GC, colorectal cancer.

Fig 7. Inactivation of the PI3K/Akt signaling reverses the effects of hsa_circRNA_100269 knockdown in GC cells.

(A and B) The proliferation of GC cells transfected with sh-NC, sh-hsa_circRNA_100269 or co-treated with LY294002 was assessed. (C-F) The migration and invasion of treated AGS and MKN-45 cells were determined. (G and H) EMT and apoptosis of transfected GC cells were evaluated. All the experiments were performed in triplicate. *P<0.05 vs. sh-NC. GC, gastric cancer; NC, negative control.

Fig 8. Doxycycline-induced expression of hsa_circRNA_100269 suppress the growth of GC in vivo.

(A) The tumours in mice were harvested six weeks post-injection. (B) The average tumor volumes were examined in dox-induced group compared with the control. (C) Orthotopic tumor weights at day 42 post-injection were determined. (D) The numbers of nodules in mice were also calculated six weeks post-injection. (E) Protein levels of EMT, apoptosis and PI3K/Akt-associated molecules in xenograft tumors were evaluated by western blot analysis. A total of five mice were included in each experimental group. All the experiments were performed in triplicate. *P<0.05 vs. corresponding control group. n = 5 per each group. GC, gastric cancer.