RNA-Seq Explores the Mechanism of Oxygen-Boosted Sonodynamic Therapy Based on All-in-One Nanobubbles to Enhance Ferroptosis for the Treatment of HCC

Background: The combination of sonodynamic therapy and oxygenation strategy is widely used in cancer treatment. However, due to the complexity, heterogeneity and irreversible hypoxic environment produced by hepatocellular carcinoma (HCC) tissues, oxygen enhancing sonodynamic therapy (SDT) has failed to achieve the desired results. With the emergence of ferroptosis with reactive oxygen species (ROS) cytotoxicity, this novel cell death method has attracted widespread attention. Methods: In this study, nanobubbles (NBs) were connected with the sonosensitizer Indocyanine green (ICG) to construct a 2-in-1 nanoplatform loaded with RAS-selective lethal (RSL3, ferroptosis promoter) (RSL3@O2-ICG NBs), combined with oxygen enhanced SDT and potent ferroptosis. In addition, nanobubbles (NBs) combined with low frequency ultrasound (LFUS) are called ultrasound-targeted nanobubble destruction (UTND) to ensure specific drug release and improve safety. Results: MDA/GSH and other related experimental results show that RSL3@O2-ICG NBs can enhance SDT and ferroptosis. Through RNA sequencing (RNA-seq), the differential expression of LncRNA and mRNA before and after synergistic treatment was identified, and then GO and KEGG pathways were used to enrich and analyze target genes and pathways related ferroptosis sensitivity. We found that they were significantly enriched in the ferroptosis-related pathway MAPK cascade and cell proliferation. Then, we searched for the expression of differentially expressed genes in the TCGA Hepatocellular carcinoma cohort. At the same time, we evaluated the proportion of immune cell infiltration and the identification of co-expression network modules and related prognostic analysis. We found that it was significantly related to the tumor micro environment of hepatocellular carcinoma. The prognostic risk genes "SLC37A2" and "ITGB7" may represent new hepatocellular carcinoma ferroptosis-inducing markers and have guiding significance for treating hepatocellular carcinoma. Conclusion: The therapeutic effect of the in vitro synergistic treatment has been proven to be significant, revealing the prospect of 2-in-1 nanobubbles combined with SDT and ferroptosis in treating HCC.