ZCCHC4 Interacts with AL133467.2 and gamma H2AX to Promote Chemoresistance in Hepatocellular Carcinoma [UV-CLIP_ZCCHC4]

Purpose: We performed UV-CLIP-seq to investigate the RNAs interacting with ZCCHC4. The goal of this study is to investigate the role of ZCCHC4 in regulating the progression and prognosis of HCC. We have found that ZCCHC4 could inhibit DNA damage agent induced DNA damage and apoptosis of HCC, and promote chemoresistance. We proposed that ZCCHC4 could interact with RNAs to regulate chemoresistance. 2×10^7 HepG2 cells transfected with Flag-tagged ZCCHC4 recombinant vectors were subjected to crosslinking with 0.15J/cm2 of 254nm UV light in a crosslinker HL-2000 (UVP). Cells were harvested and lysed with CLB for 1h at 4℃ to obtain the protein extract. The extract was then treated with RNase T1 (1U/ml) at 22℃ for 15min followed by transferring to ice for 5min. The lysates supplemented with cocktail, PMSF and RNase inhibitor were incubated with anti-Flag M2 Magnetic Beads for 7h at 4℃. The beads were washed with NTEN 900 buffer (1mM EDTA, 0.5%NP40, 20mM Tris-HCl (pH8.0), 900mM NaCl) for once, NTEN 300 buffer (1mM EDTA, 0.5%NP40, 20mM Tris-HCl (pH8.0), 300mM NaCl) for twice. The NTEN buffer mentioned was added with cocktail, PMSF and RNase inhibitor. Then beads were linked with biotin-labeled L3 linker, and separated on a 4–12% NuPAGE gel (Invitrogen). The protein-RNA complexes were transferred to nitrocellulose filter membrane and detected by chemiluminescent kit. The target protein-RNA complexes were cut and digested by Protease K. Finally, RNA was isolated and subjected to high-throughput sequencing using Illumina HiSeq 3000 with SE50 strategy.