Abstract LB-307: Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis
Authors: Liang Gehao , Ling Yun , Liu Zihao , Mehrpour Maryam , Hamai Ahmed , Tian Zhenluan , Tan Luyuan , Zhong Wenjing , Lin Wanyi , Codogno Patrice , Song Erwei , Gong Chang 
Published: August 2019

Abstract
Introduction: Autophagy plays a critical role in cancer initiation and progression. CircRNAs were recently identified as new molecules in regulation of cancer cell biology. However, it remains unclear whether circRNAs are involved in the process of autophagy and regulate cancer biology via autophagy.
Methods: We performed circRNAs microarray to screen autophagy-associated circRNAs in hypoxia-induced autophagy model and qRT-PCR was used to verify the expression level of candidate circRNAs after autophagy induction in both hypoxia and starvation induced autophagy models. RNase R digestion assay and Poly A detective experiment were used to verify the circular characteristic of circRNA candidates. The proliferative effects of knocking down and overexpressing circRNA in breast cancer cells were tested by colony formation and cell counting assays. Western Blot was used to test the protein level of LC3 and ATG7 in cancer cells. Ago2 RIP assay was used to test the miRNA sponge potential of circRNA candidates. CHiRP assays, miRNA pull down assays and dual-luciferase reporter system assays were used to verify the miRNA sponge of circRNA candidate. The location and expression levels of AUACA1 and ATG7 in human breast tissue was detected by ISH and IHC respectively. Survival rates and curves in a breast cancer cohort of 109 patients were determined by the Kaplan-Meier method and the comparison of survival differences was evaluated by using the log-rank test.
Results:
In our study, we found that breast cancer cells after autophagy induction displayed specific circRNA profiles. Among the altered circRNAs, upregulation of AUACA1 (named Autophagy Associated CircRNA 1) was most obvious in both hypoxia and starvation-induced autophagy. Moreover, AUACA1 upregulation was parallel to the degree of autophagy in breast cancer cells, while inhibition of autophagy did not affect the level of the AUACA1. Rnase R digestion and Poly A detective experiment showed that character of AUACA1 was circular rather than linear. Nuclear plasma separation assay and in-situ hybridization showed that AUACA1 mostly located in the cytoplasm. Knocking down of AUACA1 significantly inhibited the proliferation of breast cancer cells, but did not affect cell apoptosis and migration. Knocking down AUACA1 decreased autophagic level by detecting LC3-II. Proliferative assay demonstrated that AUACA1 promoted proliferation via autophagy in MDA-MB-231. Ago2 RIP assay significantly enriched AUACA1, indicating AUACA1 may worked as miRNA sponge. CHiRP and miRNAs microarray proved that AUACA1 worked as miR-1275 sponge. MiRNA pull down assay and dual-luciferase reporter system assays also verified that AUACA1 worked as miR-1275 sponge. MiR-1275 affected ATG7 expression at both RNA and protein level after transfecting miR-1275 mimics in MDA-MB-231. In addition, clinical data indicated that AUACA1 expression level was positively correlated with ATG7 and Ki-67 index. High expression of AUACA1 was correlated with high tumour burden and poor disease-free survival in 109 breast cancer patients.
Conclusion: Our findings indicate that AUACA1 is an autophagy–associated circRNA and plays a critical role in promoting the proliferation of breast cancer cell via miR-
1275/ATG7/autophagic axis. AUACA1 may be a potential prognostic biomarker for breast cancer.
Citation Format: Gehao Liang, Yun Ling, Zihao Liu, Maryam Mehrpour, Ahmed Hamai, Zhenluan Tian, Luyuan Tan, Wenjing Zhong, Wanyi Lin, Patrice Codogno, Erwei Song, Chang Gong. Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-307.