ASB16-AS1 up-regulated and phosphorylated TRIM37 to activate NF-κB pathway and promote proliferation, stemness, and cisplatin resistance of gastric cancer

Abstract

Background

Long non-coding RNA (lncRNA) ASB16 antisense RNA 1 (ASB16-AS1) is recognized as an oncogene in several cancer types, but its relation to GC is unknown. Tripartite motif containing 37 (TRIM37) has been proven to accelerate the development of gastric cancer (GC), whereas the molecular mechanism assisted ASB16-AS1 and TRIM37 in regulating GC progression remains unclear.

Methods

Differentially expressed lncRNAs in GC samples were analyzed based on Gene Expression Omnibus (GEO) data. CCK-8 and colony formation assays were applied to determine the proliferative ability of GC cells. Stem cell-like phenotype of GC cells was assessed by sphere formation assay and flow cytometry analysis. Luciferase reporter assay, RNA immunoprecipitation (RIP), pulldown, and co-immunoprecipitation (Co-IP) were performed to verify the interplay of RNA molecules.

Results

ASB16-AS1 was upregulated in GC samples according to GEO data and qRT-PCR analysis. ASB16-AS1 strengthened the proliferative ability and stem cell-like characteristics in GC cells. More importantly, ASB16-AS1 encouraged GC cell growth in vivo. Mechanistically, ASB16-AS1 strengthened TRIM37 expression by sequestering miR-3918 and miR-4676-3p. ASB16-AS1 activated NF-kappa B (NF-κB) pathway by cooperating with ATM serine/threonine kinase (ATM) to induce TRIM37 phosphorylation.

Conclusion

In summary, ASB16-AS1 exerted oncogenic functions in GC through modulating TRIM37 expression at both mRNA and protein levels.

Graphic abstract

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Acknowledgements

We sincerely appreciate all lab members.

Funding

This study was supported by Science Foundation of Peking University Cancer Hospital 2020-12.

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Correspondence to Zhaode Bu or Jiafu Ji.

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With the informed consents signed by all patients, this study had received the approval of the Ethics Committee of Peking University Cancer Hospital and Institute.

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Figure S1. A. qRT-PCR detected the knockdown efficiency of ASB16-AS1, RP11-475B2.1 and AC010518.3. B-C. qRT-PCR and western blot detected TRIM37 expression with RP11-475B2.1 or AC010518.3 silence. D. qRT-PCR of ASB16-AS1 overexpression under pcDNA3.1-ASB16-AS1 transfections. E. qRT-PCR and western blot of TRIM37 level under ASB6-AS1 overexpression in GC cells and normal cells. **P < 0.01, n.s. suggested no significance. (TIFF 2443 kb)

Figure S2 A. CCK-8 data of GC cell viability under ASB16-AS1 overexpression. B. Pictures of colonies from GC cells with ASB16-AS1 overexpression were taken and colony number was counted. C. Spheres of GC cells were pictured and sphere-forming efficiency was determined. Scale bar: 100 μm. D. CD133+ cell ratio of GC cells with ASB16-AS1 overexpression was measured by flow cytometry analysis. E. CCK-8 data of GC cells transfected with pcDNA3.1 or pcDNA3.1-ASB16-AS1 under the treatment of cisplatin at 2, 4, and 8 μM relative to 0 μM. F. Apoptosis ratio of GC cells with ASB16-AS1 overexpression was detected by flow cytometry under cisplatin treatment. **P < 0.01. (TIFF 1071 kb)

Figure S3 A. CCK-8 data of normal GES-1 cell viability under ASB16-AS1 overexpression. B. Pictures of colonies from normal GES-1 cell with ASB16-AS1 overexpression were taken and colony number was counted. C. Spheres of normal GES-1 cell were pictured and sphere-forming efficiency was calculated. Scale bar: 100 μm. D. CD133+ cell ratio of normal GES-1 cell with ASB16-AS1 overexpression was measured by flow cytometry analysis. E. CCK-8 data of normal GES-1 cell transfected with pcDNA3.1 or pcDNA3.1-ASB16-AS1 under the treatment of cisplatin at 2, 4, and 8 μM relative to 0 μM. F. Apoptosis ratio of normal GES-1 cell with ASB16-AS1 overexpression was detected by flow cytometry under cisplatin treatment. **P < 0.01, n.s. suggested no significance. (TIFF 831 kb)

Figure S4 A. Luciferase reporter assay detected the luciferase activity of reporters containing TRIM37 promoter. B. RNA pull-down assay detected the enrichment of TRIM37 mRNA pulled down by ASB16-AS1. C. qRT-PCR examination revealed the expression of 12 candidate miRNAs in GC tissues versus adjacent normal ones. D. qRT-PCR examined miR-3918 and miR-4676-3p expressions in GC cells versus GES-1 cells. E. Correlation between ASB16-AS1 and miR-3918/miR-4676-3p was analyzed based on TCGA data and determined by Pearson correlation analysis in 76 GC samples. F. qRT-PCR verified overexpression efficiency of miR-3918 and miR-4676-3p. *P < 0.05, **P < 0.01, n.s. suggested no significance. (TIFF 738 kb)

Figure S5 A. qRT-PCR measured TRIM37 mRNA stability after ASB16-AS1 inhibition in GC cells treated with ActD. B. Western blot analysis of TRIM37 protein stability after ASB16-AS1 inhibition in GC cells with CHX treatment. C. p-TRIM37 level in GC cells and GES-1 cells was detected by western blot under ASB16-AS1 overexpression. D. qRT-PCR verified depletion efficiency of ATM. **P < 0.01, n.s. suggested no significance. (TIFF 404 kb)

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Fu, T., Ji, K., Jin, L. et al. ASB16-AS1 up-regulated and phosphorylated TRIM37 to activate NF-κB pathway and promote proliferation, stemness, and cisplatin resistance of gastric cancer. Gastric Cancer (2020). https://doi.org/10.1007/s10120-020-01096-y

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Keywords

  • TRIM37
  • ASB16-AS1
  • Gastric cancer
  • ATM