Roles Of ARID1A In Liver Tumorigenesis And Its Underlying Mechanisms

BackgroundLiver cancer is the second leading cause of cancer death worldwide. China alone accounts for about 50% of the total patients, which threats people’s life greatly. The most frequent liver cancer is hepatocellular carcinoma (HCC). The most prominent factors associated with HCC include chronic hepatitis B and C viral infection, chronic alcohol consumption and aflatoxin-B1-contaminated food. The development and progression of HCC is considered as a multistage process with the deregulation of several oncogenes and tumor-suppressor genes. Further research is still needed to explore the mechanisms of liver tumorigenesis, which may provide new ideas for the prophylaxis and treatment of HCC.Genomic analyses have defined the extensive landscape of altered genes and pathways in HCC. In 2012, three papers in Nature Genetics have identified some mutated genes implicated in HCC, including CTNNB1, AXIN1, P53 and ARID1A. The roles of CTNNB1, AXIN1, P53 in liver tumorigenesis have been be elaborated somewhere else. However, the mechanisms by which mutations in ARID1A drive tumorigenesis are unclear. And now, it becomes a focus of attention for researchers.AimsThis research aimed to study the roles of ARID1A inactivation in liver tumorigenesis and its underlying mechanisms. We looked at whether it can drive tumor initiation by knocking out Arid1a in hepatocytes and whether it can accelerate tumor initiation by knocking out Arid1a in liver cancer models. Based on this, we further investigated the biological function of ARID1A in the HCC cell line and elucidated the mechanisms of ARID1A in liver tumorigenesis.Methods1. CRISPR-Cas9 system was built for site-specific genome editing in eukaryotic cells.2. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly targeted Arid1a in wild-type mice. We further assessed the phenotype following sgArid1a treatment.3. We co-injected sgArid1a, sgPten and sgP53 into wild-type mice and compared the phenotype with sgPten and sgP53 treatment.4. We used hydrodynamic injection to deliver sgAridla to the liver of the DEN model. We compared the phenotype between sgArid1a and DEN treatment and DEN treatment only.5. We knocked out ARID1A in Bel7404 cell line using CRISPR-Cas9 system. We analyzed differential gene expression on transcription level using RNAseq.6. We compared the ability of cell proliferation between ARID1A KO and wild-type Bel7404 cell line using CCK-8 and tumor formation analysis in nude mice.7. We verified the expression of ARID1A in samples of clinical HCC tumors using IHC analysis and then investigated the correlation between prognosis and the level of ARID1A.8. We observed the mitosis of ARID1A KO cells using confocal microscopy.9. We blocked cell cycle using MG132 and Nocodazole and analyzed the level of ARID1A at different stages of cell cycle.10. We compared the differences of cell cycle between ARID1A KO and wild-type Bel7404 cell line using flow cytometry after cell synchronization treatment.11. DNA damage was induced in ARID1A KO and wild-type Bel7404 cell line using IR, UV and cisplatin, and r-H2A.X was measured as a marker of DNA damage repair by Western blot.12. Apoptosis was induced using IR in vivo and in vitro, Cleaved-PARP was measured by Western blot and Cleaved-Caspase-3 was detected by IHC.Results1. It is feasible to edit targeted genome using CRISPR-Cas9 system in vivo and in vitro.2. The mice did not exhibit liver tumors at ten months post-injection with sgAridla only. But more hepatocytes were positive by IHC staining of P-Histone-H3 and r-H2A.X, compared with that of the control group.3. The mice co-injected with sgAridla, sgPten and sgP53 developed liver tumors more quickly than the control group. At 3 months post-injection, abnormal nodules of liver can be found in those mouse.4. The DEN treated mice injected with sgArid1a developed liver tumors more quickly than the control group. At 6 months post-natal, tumors can be found in those mouse.5. We successfully generated ARID1A KO Bel7404 cell line using CRISPR-Cas9 system, which was identified by Western blot, sequencing, immunofluorescence stain and IHC.6. The proliferation ability of ARID1A KO cells and wild-type Bel7404 cells was of no significant difference in vivo and in vitro, which was supported by the data of RNAseq.7. Immunohistochemistry indicated that 12.17%(14 of 115) of patients with HCC had lost ARID1A in tumors. ARID1A negative and positive cases did not show any statistically significant difference regarding overall survival and disease-free survival.8. We found that deletion of ARID1A in Bel7404 cells led to "anaphase bridge" formation and a G2/M-phase block characteristic. ARIDIA accumulated in G2 and was completely eliminated during mitosis. The ARIDIA KO cells re-entered the cell cycle more quickly than the wild-type Bel7404 cells after double thymidine treatment.9. Deletion of ARIDIA resulted in inefficient H2A.X phosphorylation (r-H2AX) and nuclear focus formation after DNA damage induced by IR, UV and cisplatin. ARID1A interacts with r-H2AX by co-immunoprecipitation.10. Deletion of ARIDIA in Bel7404 cells resulted in resistance to apoptosis induced by IR in vivo and in vitro.Conclusions1. Deletion of Aridl a alone is insufficient for tumor initiation in mouse liver, but can accelerate tumor initiation in different tumor models.2. Deletion of ARID1A has no significant effects in proliferation of Bel7404. Loss of ARID1A in HCC tumors is not significantly associated with survival of patients.3. The normal process of chromosome separation at mitosis requires ARIDIA and it suggests this activity contributes to the role of ARID1A as a tumor suppressor.4. ARIDIA has important roles in H2A.X phosphorylation during the DNA damage repair, which is associated with tumorigenesis.5. ARIDIA can function as a positive regulator of apoptosis which can contribute to tumor suppression.