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Background Liver cancer is the fifth leading cause of cancer death worldwide, but early diagnosis and treatment of liver cancer remains a clinical challenge. How to screen and diagnose liver cancer early and prolong the survival rate is still the focus of researchers. Methods Cell experiments were used to detect the effect of WZ35 on the colony formation ability and proliferation activity of hepatoma cells, nude mouse experiment to observe the in vivo anticancer activity and toxic side effects of WZ35; metabolomics analysis, glucose metabolism experiment and Seahorse analysis of liver cancer cells treated with WZ35; cell experiments combined with bioinformatics analysis to explore the mechanism of WZ35-mediated metabolic reprogramming to exert anticancer activity; tissue microarray and case analysis to evaluate the clinical significance of biomarkers for early diagnosis, treatment and prognosis evaluation of liver cancer. Results WZ35 inhibited the proliferation activity of various cell lines of liver cancer, and showed good therapeutic effect in nude mice model of hepatocellular carcinoma without obvious toxic and side effects; WZ35 inhibited the absorption of glucose in hepatoma cells, and the drug effect glycolysis, phosphorylation and purine metabolism are relatively seriously damaged; WZ35 mainly inhibits YAP from entering the nucleus as a transcription factor activator by activating oxidative stress in liver cancer cells, reducing the transcription of GLUT1, and finally reducing its GLUT1. Tissue microarray and case analysis showed that GLUT1 and YAP were highly expressed and correlated in liver cancer patients, and were associated with poor patient prognosis. The GLUT1-YAP risk model had a high score in predicting prognosis. Conclusion The study confirms that WZ35 is a small molecule glycolysis inhibitor, and through its properties, it mediates metabolic reprogramming dominated by impaired glycolysis, oxidative phosphorylation and purine metabolism to inhibit the proliferation activity of liver cancer cells. Our findings present novel insights into the pathology of liver cancer and potential targets for new therapeutic strategies. GLUT1-YAP has important reference significance for predicting the stages of disease progression in liver cancer patients and have the potential to serve as novel biomarkers for the diagnosis and treatment of liver cancer.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3′- splicing site on intron 2 and the alternative 3′- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.

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Background Aberrant splice variants play different roles in the formation of tumors. We observed the splice isoform of Protein arginine methyltransferase 5 (PRMT5-ISO5) increases in HCC patients undergoing stereotactic body radiotherapy, which is associated with improvement of poor prognosis. However, the mechanism of alternative splicing of PRMT5-ISO5 induced by ionizing radiation (IR) is still unclear. Methods The transcriptional changes of PRMT5-ISO5 induced by IR were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Bioinformatic analyses were performed to identify potential splicing factors involved in regulating PRMT5 splicing. Small interferring RNA and overexpressing plasmids for SRSF3 and HNRNPH1 were introduced into HCC cell lines, followed by in vitro functional experiments in regulating PRMT5 splicing by RT-qPCR, western blot and RNA-immunoprecipitation assay in vitro. The roles of IR-induced PRMT5-ISO5 and hepatocyte-specific Prmt5 knockout on HCC progression were evaluated in vivo. Results we indicated IR could induce PRMT5-ISO5 transcript in HCC cells by virtue of splicing factors SRSF3 and HNRNPH1. Mechanistically, HNRNPH1 silencing resulted in the decrease of PRMT5-ISO5 while SRSF3 silencing led to the increase of PRMT5-ISO5. In addition, both SRSF3 and HNRNPH1 bound to PRMT5 precursor mRNA on the region around 3' splicing site of intron 2 and alternative 3’ splicing site on exon 4, leading to their opposite functions on regulating PRMT5 splicing. In vivo, the increase of PRMT5-ISO5 induced by IR led to tumor regression, and liver-specific Prmt5 depletion decelerated the progression of Akt/N-Ras-derived spontaneous HCC. Conclusion Our study not only provides mechanistic views that IR-induced SRSF3 downregulation leads to the imbalance of SRSF3 and HNRNPH1 in regulating PRMT5-ISO5 transcript, but also indicates a potential radiotherapeutic of PRMT5-ISO5 in HCC formation since liver-specific Prmt5 knockout inhibits spontaneous HCC tumorigenesis.