Encoding gene RAB3B exists in linear chromosomal and circular extrachromosomal DNA and contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma via inducing autophagy

Abstract

Cisplatin (DDP) resistance is an important factor that decreases the effect of chemotherapy, thus leading to local recurrence and lymph node metastasis of hypopharyngeal squamous cell carcinoma (HSCC). We aimed to explore the role and mechanism of extrachromosomal circular DNA (eccDNA) in the DDP resistance of HSCC. In our research, the HSCC cell line FaDu and the DDP-resistant cell line FaDu/DDP were used as subjects. eccDNA sequencing and whole transcriptome sequencing were conducted, followed by a combined analysis of the two sequencing profiles. Outward PCR, inward PCR and Sanger sequencing were used to verify sequences of the eccDNAs. Bioinformatics analysis based on TCGA/GEO was performed in addition to plasmid transfection, RNA interference, qRT-PCR and Western blot experiments to verify the expression level of RAB3B amplified from eccDNA. mRFP-GFP-LC3 adenoviral particle transfection and transmission electron microscopy were used to detect autophagic flux. Finally, we evaluated the role of RAB3B in FaDu/DDP cells and patient-derived organoids. Our results showed that we purified and sequenced more than 10 thousand eccDNAs from the two cell lines, and the size of the eccDNAs was distributed from 0.01 kb to 1000 kb. The combined analysis between eccDNA and transcript sequencing indicated that there were some highly expressed genes that were completely or partially transcribed from related sequences of eccDNAs and not from genome linear DNA. We further screened and verified the encoding gene RAB3B using full-length sequences that might be amplified from eccDNA [chr1circle 46219-52682 kb]. Finally, we confirmed that RAB3B could promote DDP resistance in HSCC by inducing autophagy. The eccDNA might play significant roles in DDP resistance in HSCC by amplifying related functional genes. Further study is needed to explore the novel mechanisms of eccDNA in the drug resistance of HSCC.