Functional Role of Circular NFATC3 in Triple Negative Breast Cancer


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Fatima Al-Dasim1,Thasni Karedath1,Stephanie Aron Ramadan1,Nayra Al-Thani1,Joel Malek1
1Weill Cornell Medicine-Qatar

Abstract

Background

NFATC3 gene is a member of the nuclear factors of activated T cells DNA-binding transcription complex. Studies proved that NFATC3 play a key role in tumor invasion and proliferation besides to normal development and growth, immune and inflammatory responses. In addition to coding NFATC3 genes which is well characterized functionally, many circular isoforms of NFATC3 is also present in the cell system. These circular RNAs (circRNA) that form through non-canonical backsplicing events of pre-mRNA transcripts and functional relevance of circular NFATC3 remains elusive.

Method

Silencing and over-expression circNFATC3 in breast cancer cell lines were done. siRNA mediated silencing of circNFATC3 targeting the circular junctions was adopted. pcDNA3.1(+) CircRNA Mini Vector in triple negative breast cancer cells was used to carry the over-expression. Sanger sequencing was performed to identify circular junctions. Phenotypic functional screening, RT-qPCR and RNA-sequencing were implemented to study the effect of NFATC3 silencing and over-expression in breast cancer cells.

Results

A circular RNA isoform of NFATC3 (circNFATC3) was identified, which is predominantly present in MDA-MB-231 triple negative breast cancer cells and Lymphoblastoid cell lines. CircNFATC3 was characterized through a series of experiments including sanger sequencing to identify the non-canonical splice junctions and full length circNFATC3 by using specific primer pairs. RNaseR treatment was done to confirm the circularization and stability of circNFATC3. siRNA mediated silencing of circNFATC3 revealed the functional relevance of this circRNA in the cell system. CircNFATC3 plays a key role in regulating cell viability, invasion and migration in cancer cells confirmed by using different cell-based assays.

Conclusion

CircNFATC3 is an abundant functionally relevant circular RNA both in cancer cells and immune cells. CircNFATC3 can be a potential therapeutic or prognostic target for the future studies.