Depletion of hsa_circ_0000144 Suppresses Oxaliplatin Resistance of Gastric Cancer Cells by Regulating miR-502-5p/ADAM9 Axis

Abstract

Background: Circular RNAs (circRNAs) have been disclosed to exert important roles in human cancers, including gastric cancer (GC). CircRNA hsa_circ_0000144 was identified as an oncogene in GC development. The aim of our study was to explore the role of hsa_circ_0000144 in oxaliplatin (OXA) resistance of GC.

Methods: Expression levels of hsa_circ_0000144, microRNA-502-5p (miR-502-5p) and A disintegrin and metalloproteinase 9 (ADAM9) were examined by quantitative real-time PCR (RT-qPCR) or Western blot assay. The OXA resistance of GC cells was evaluated by Cell Counting Kit-8 (CCK-8) assay. Colony formation assay was performed to assess the colony formation capacity. Cell apoptosis was determined by flow cytometry and caspase 3 activity. And cell migration and invasion were detected by Transwell assay. Target association between miR-502-5p and hsa_circ_0000144 or ADAM9 was demonstrated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Moreover, role of hsa_circ_0000144 in vivo was analyzed by xenograft tumor assay.

Results: Hsa_circ_0000144 and ADAM9 were highly expressed, while miR-502-5p was downregulated in OXA-resistant GC tissues and cells. Depletion of hsa_circ_0000144 could inhibit OXA resistance, proliferation and metastasis in OXA-resistant GC cells, which was attenuated by miR-502-5p inhibition. Hsa_circ_0000144 sponged miR-502-5p to positively regulate ADAM9 expression. MiR-502-5p suppressed OXA resistance, proliferation and metastasis in OXA-resistant GC cells by targeting ADAM9. Hsa_circ_0000144 knockdown could hamper tumor growth in vivo.

Conclusion: Hsa_circ_0000144 exerted inhibitory effects on OXA resistance, proliferation and metastasis of OXA-resistant GC cells by regulating miR-502-5p/ADAM9 axis, at least in part.

Keywords: ADAM9; GC; OXA resistance; OXA-resistant GC cells; hsa_circ_0000144; miR-502-5p.

Figure 1

Hsa_circ_0000144 expression was upregulated in OXA-resistant GC tissues and cells. (A) Expression of hsa_circ_0000144 in GC tissues and normal tissues showed by GSE78092 database. (B) RT-qPCR assay for the expression of hsa_circ_0000144 in normal tissues, 25 cases of OXA-sensitive and 25 cases of OXA-resistant GC tissues. (C) RT-qPCR assay for the expression of hsa_circ_0000144 in GES-1, AGS, AGS/OXA, MKN45 and MKN45/OXA cells. (D) Nuclear-cytoplasmic fractionation for the location of hsa_circ_0000144 in AGS/OXA and MKN45/OXA cells. (E) RT-qPCR assay for the expression of hsa_circ_0000144 and linear mRNA in RNA isolated from AGS/OXA and MKN45/OXA cells digested with RNase R or not. *P < 0.05.

Figure 2

Hsa_circ_0000144 depletion suppressed OXA resistance, proliferation and metastasis in OXA-resistant GC cells. (A) CCK-8 assay for the survival rate of AGS, AGS/OXA, MKN45 and MKN45/OXA cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (B) RT-qPCR assay for the expression of hsa_circ_0000144 in AGS/OXA and MKN45/OXA cells transfected with si-NC, si-hsa_circ_0000144#1, si-hsa_circ_0000144#2 or si-hsa_circ_0000144#3. (C–H) AGS/OXA and MKN45/OXA cells were transfected with si-NC or si-hsa_circ_0000144#1. (C) CCK-8 assay for the survival rate of transfected cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (D) Colony formation assay for the colony formation ability of transfected cells. (E) Flow cytometry for the cell apoptosis in transfected cells. (F) Caspase 3 activity in transfected cells determined by commercial kit. (G and H) Transwell assay for the cell migration and invasion in transfected cells. *P < 0.05.

Figure 3

MiR-502-5p was a direct target of hsa_circ_0000144. (A) The predicted binding positions between hsa_circ_0000144 and miR-502-5p by Circular RNA Interactome. (B) Dual-luciferase reporter assay for the luciferase activity of AGS/OXA and MKN45/OXA cells co-transfected with hsa_circ_0000144-wt or hsa_circ_0000144-mut and miR-502-5p or NC. (C) RIP and RT-qPCR assays for the binding efficiency of hsa_circ_0000144 and miR-502-5p to Ago2 protein in AGS/OXA and MKN45/OXA cells. (D) RT-qPCR assay for the expression of hsa_circ_0000144 in AGS/OXA and MKN45/OXA cells transfected with circ-NC or hsa_circ_0000144. (E) RT-qPCR assay for the expression of miR-502-5p in AGS/OXA and MKN45/OXA cells transfected with circ-NC or hsa_circ_0000144. (F) RT-qPCR assay for the expression of miR-502-5p in normal tissues, 25 cases of OXA-sensitive and 25 cases of OXA-resistant GC tissues. (G) Pearson’s correlation analysis for the correlation between hsa_circ_0000144 and miR-502-5p in 50 cases of GC tissues. (H) RT-qPCR assay for the expression of miR-502-5p in GES-1, AGS, AGS/OXA, MKN45 and MKN45/OXA cells. *P < 0.05.

Figure 4

MiR-502-5p inhibition weakened hsa_circ_0000144 depletion-mediated inhibitory effects on OXA resistance, proliferation and metastasis in OXA-resistant GC cells. (A) RT-qPCR assay for the expression of miR-502-5p in AGS/OXA and MKN45/OXA cells transfected with anti-NC or anti-miR-502-5p. (B–H) AGS/OXA and MKN45/OXA cells were transfected with si-NC, si-hsa_circ_0000144#1 si-hsa_circ_0000144#1+anti-NC or si-hsa_circ_0000144#1+anti-miR-502-5p. (B) RT-qPCR assay for the expression of miR-502-5p in transfected cells. (C) CCK-8 assay for the survival rate of transfected cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (D) Colony formation assay for the colony formation ability of transfected cells. (E) Flow cytometry for the cell apoptosis in transfected cells. (F) Caspase 3 activity in transfected cells determined by commercial kit. (G and H) Transwell assay for the cell migration and invasion in transfected cells. *P < 0.05.

Figure 5

MiR-502-5p could directly target ADAM9. (A) The predicted binding sites between miR-502-5p and ADAM9 3ʹUTR by TargetScan. (B) Dual-luciferase reporter assay for the luciferase activity of AGS/OXA and MKN45/OXA cells co-transfected with ADAM9-wt or ADAM9-mut and miR-502-5p or NC. (C) RIP and RT-qPCR assays for the binding efficiency of miR-502-5p and ADAM9 to Ago2 protein in AGS/OXA and MKN45/OXA cells. (D) Western blot assay for the protein level of ADAM9 in AGS/OXA and MKN45/OXA cells transfected with si-NC, si-hsa_circ_0000144#1, si-hsa_circ_0000144#1+anti-NC or si-hsa_circ_0000144#1+anti-miR-502-5p. (E) RT-qPCR assay for the mRNA expression of ADAM9 in normal tissues, 25 cases of OXA-sensitive and 25 cases of OXA-resistant GC tissues. (F) Western blot assay for the protein level of ADAM9 in normal tissues, OXA-sensitive and OXA-resistant GC tissues. (G) Pearson’s correlation analysis for the correlation between hsa_circ_0000144 and ADAM9 mRNA in 50 cases of GC tissues. (H) Pearson’s correlation analysis for the correlation between miR-502-5p and ADAM9 mRNA in 50 cases of GC tissues. (I) RT-qPCR assay for the mRNA expression of ADAM9 in GES-1, AGS, AGS/OXA, MKN45 and MKN45/OXA cells. *P < 0.05.

Figure 6

Enforced expression of miR-502-5p reduced OXA resistance, proliferation and metastasis in OXA-resistant GC cells by downregulating ADAM9 expression. (A) RT-qPCR assay for the expression of miR-502-5p in AGS/OXA and MKN45/OXA cells transfected with NC or miR-502-5p. (B) Western blot assay for the protein level of ADAM9 in AGS/OXA and MKN45/OXA cells transfected with pcDNA-NC or pcDNA-ADAM9. (C–J) AGS/OXA and MKN45/OXA cells were transfected with NC, miR-502-5p, miR-502-5p+pcDNA-NC or miR-502-5p+pcDNA-ADAM9. (C) Western blot assay for the protein level of ADAM9 in transfected cells. (D and E) CCK-8 assay for the survival rate of transfected cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (F) Colony formation assay for the colony formation ability of transfected cells. (G) Flow cytometry for the cell apoptosis in transfected cells. (H) Caspase 3 activity in transfected cells determined by commercial kit. (I and J) Transwell assay for the cell migration and invasion in transfected cells. *P < 0.05.

Figure 7

Hsa_circ_0000144 depletion inhibited GC tumor growth in vivo. AGS/OXA cells stably expressing sh-hsa_circ_0000144 or sh-NC were injected into nude mice, followed by inoculation with OXA. (A) Tumor volume measured every 5 d, image collected at 30 d post injection. (B) Tumor weight measured at 30 d post injection. (C) RT-qPCR assay for the expression of hsa_circ_0000144 and miR-502-5p in generated tumors. (D) Western blot assay for the protein levels of ADAM9 and PCNA generated tumors. *P < 0.05.