Inhibition of the U12-type splicing factor ZCRB1 mediates retention of the USP21 minor intron to suppress malignant progression in hepatocellular carcinoma.

[BACKGROUND] RNA splicing dysregulation plays an important role in hepatocellular carcinoma (HCC). However, the critical functions of zinc finger CCHC type and RNA binding motif 1 (ZCRB1), one of the key components of the minor spliceosome, in HCC remains unclear.

[METHODS] The mRNA and protein expression of ZCRB1 were evaluated by bioinformatics analysis, western blotting and immunohistochemistry. The role of ZCRB1 on biological functions of HCC were measured in vitro and in vivo. rMATS software analyzes RNA-seq data to identify ZCRB1-related alternative splicing (AS) events. Therapeutic targeting of ZCRB1 using GalNAc-conjugated small-interfering RNA was investigated in the HCC mouse model.

[RESULTS] We discovered that ZCRB1 was dramatically upregulated in HCC tissues, and high ZCRB1 expression was associated with poor prognosis in HCC patients. Knockdown of ZCRB1 significantly reduced HCC cell proliferation and promoted cell apoptosis. In addition, high enrichment levels of H3K27ac in the promoter region activated ZCRB1 expression. Mechanistically, AS event analysis revealed that knockdown ZCRB1 resulted in retention of intron 11 of USP21 thereby reducing USP21 expression. USP21 overexpression partially rescued the attenuation of malignant behavior of HCC cells caused by inhibition of ZCRB1, and inhibition of USP21 reduced proliferation in HCC cells. Furthermore, therapeutic targeting of ZCRB1 using GalNAc-conjugated small-interfering RNA significantly reduced tumor burden and increased the infiltration level of CD8+ T cells in the HCC mouse model.

[CONCLUSIONS] This study identified ZCRB1 as an oncogenic U12-type splicing factor that supports malignant growth through blocking intron 11 retention of USP21 in HCC cells. Thus, ZCRB1 could serve as a new target for HCC therapy and a potential biomarker for HCC prognosis.