Acetylation-dependent regulation of core spliceosome modulates hepatocellular carcinoma cassette exons and sensitivity to PARP inhibitors

Despite the importance of spliceosome core components in cellular processes, their roles in cancer development, including hepatocellular carcinoma (HCC), remain poorly understood. In this study, we uncover a critical role for SmD2, a core component of the spliceosome machinery, in modulating DNA damage in HCC through its impact on BRCA1/FANC cassette exons and expression. Our findings reveal that SmD2 depletion sensitizes HCC cells to PARP inhibitors, expanding the potential therapeutic targets. We also demonstrate that SmD2 acetylation by p300 leads to its degradation, while HDAC2-mediated deacetylation stabilizes SmD2. Importantly, we show that the combination of Romidepsin and Olaparib exhibits significant therapeutic potential in multiple HCC models, highlighting the promise of targeting SmD2 acetylation and HDAC2 inhibition alongside PARP inhibitors for HCC treatment.

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Data analysis
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Lianxin Liu Mar 28, 2024
The RNA-seq libraries were sequenced using an an Illumina sequencing platform, specifically the HiSeq™ 2500 or or HiSeq X Ten systems, producing 125 bp bp or or 150 bp bp paired-end reads.The raw data (raw reads) were subsequently processed with Trimmomatic.MS/MS data were processed using MaxQuant search engine (v.1.6.15.0),searching tandem mass spectra against the human SwissProt database concatenated with the reverse decoy database.Constitutive exons were obtained from HEXEvent, while cassette exons were identified using RNA-seq data.Reference alternative exons, which are not regulated by by SmD2, were determined by by excluding exons affected by by SmD2 based on on data from HEXEvent.

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All studies must disclose on these points even when the disclosure is negative.This study did not apply to just one gender, and we did not take gender into account in our study.We also did not apply the term gender in our article.
This study did not involve socially relevant categorical variables.
HCC patient study participants were matched for age (range 37-79 years old), sex, and ethnicity in the discovery.
Label-free quantitative proteome analysis and Sm proteins expression analysis were performed on specimens from HCC patients undergoing surgery at the First Affiliated Hospital of USTC between June and December 2021.PDX models were established using the specimen procured from one HCC patient scheduled for surgical treatment in September 2022.
Informed consent was acquired from all participants, and the study received approval from the Institutional Review Board.Two HCC tissue microarrays (TMAs) were used, with TMA1 containing 48 formalin-fixed, paraffin-embedded HCC tissues and TMA2 consisting of 58 HCC tumor and adjacent normal tissues.These samples were collected from patients who underwent curative resection at the Department of Hepatobiliary Surgery, First Affiliated Hospital of USTC.
Informed consent was obtained from the patients who provide HCC specimens, and the research was approved by the Ethics Committee of the First Affiliated Hospital of USTC, the approval number is 2022-KY293.This statement has been included in the Methods section of our manuscript.
Our HCC sample selection originated from the hepatocellular carcinoma sample library at the First Affiliated Hospital of the University of Science and Technology of China.These samples were randomly selected without specific inclusion criteria to minimize selection bias and better represent a broad patient demographic.This approach ensures that our findings can be generalized to a wider HCC patient population.
Initially, Sm protein was identified as the primary research target from a preliminary screening of samples from 6 patients.Subsequently, we analyzed the expression of SmD2 in 105 pairs of HCC samples.The decision on this sample size was influenced by the availability of samples from the library and the intention to achieve a statistically meaningful analysis of SmD2 expression variability.Although no pre-defined sample size calculation was performed, the chosen sample size is deemed sufficient based on prior research experiences and the capacity of our sample library, allowing for significant statistical analysis and conclusions.
For in vitro experiments, we did not conduct a predetermined sample size calculation but opted for independent experiments with n=3, incorporating at least 3 biological replicates and, where possible, technical replicates for each experiment.This protocol is based on laboratory norms and preliminary experiment outcomes, considered adequate for ensuring the reproducibility and reliability of our results.
In our animal studies, we ensured a minimum of 5 mice per group.This decision was guided by preliminary data and standard practices in the In summary, our sample size choices were informed by prior research experience, the availability of samples, and the requirements of our experimental design.Despite the absence of rigorous pre-study sample size calculations, we believe our approach, combined with careful experimental design and data analysis, supports the validity of our research objectives and conclusions.
No data were excluded from analysis.
Each experiment was conducted a minimum of three times to ensure reproducibility.Additionally, to account for biological variability, we used multiple independent samples for our assays.Where applicable, experiments were also independently replicated by different team members to confirm the consistency of the results.
All samples were randomly assigned to various groups.
In our study, blinding was not applicable as it is exploratory in nature and does not involve any elements that could be influenced by bias from the subject or observer.
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