PRMT5 attenuates regorafenib-induced DNA damage in hepatocellular carcinoma cells through symmetric dimethylation of RPL14.

Abstract

Background: Regorafenib has been approved for second-line treatment of hepatocellular carcinoma (HCC) following sorafenib failure, but resistance to targeted therapy remains a major challenge. Enhancing the therapeutic sensitivity of HCC cells to regorafenib is crucial for improving treatment outcomes. This study aims to elucidate the role of PRMT5 in HCC and its impact on regorafenib sensitivity. Specifically, it focuses on the regulatory relationship between PRMT5 and RPL14, investigating their influence on DNA damage repair and drug resistance mechanisms in HCC.

Methods: A stable PRMT5-overexpressing HCC cell line was constructed via lentiviral infection. Immunoprecipitation was employed to examine whether PRMT5 catalyzes the symmetric dimethylation of RPL14 at arginine residues. Western blot (WB) was used to assess changes in DNA damage markers (γ-H2AX) and DNA repair markers (RAD51) after RPL14 knockdown. Huh7 cells with PRMT5 overexpression, RPL14 knockdown, and combined PRMT5 overexpression and RPL14 knockdown were treated with regorafenib. DNA damage repair-related factors were analyzed using WB and immunofluorescence.

Results: Mass spectrometry and immunoprecipitation confirmed the interaction between PRMT5 and RPL14, with PRMT5 catalyzing symmetric dimethylation of RPL14. RPL14 knockdown inhibited HCC cell proliferation, increased sensitivity to regorafenib, and disrupted DNA damage repair, while overexpression had the opposite effect. Regorafenib-treated PRMT5-overexpressing cells showed reduced γ-H2AX expression and improved survival, whereas RPL14 knockdown enhanced γ-H2AX levels and decreased survival. Notably, simultaneous PRMT5 overexpression and RPL14 knockdown significantly elevated γ-H2AX expression compared to PRMT5 overexpression alone, leading to reduced cell viability. These results suggest that PRMT5 modulates DNA damage repair through RPL14, influencing the sensitivity of HCC cells to regorafenib.

Conclusions: PRMT5-mediated symmetric dimethylation of RPL14 stabilizes the protein, promoting DNA damage repair and contributing to regorafenib resistance in HCC. RPL14 plays a key role in PRMT5-driven enhancement of DNA damage repair and reduced drug sensitivity, identifying RPL14 as a potential therapeutic target to overcome regorafenib resistance in HCC.