Mechanism of ARID1A Knockout Promoting Invasion and Metastasis of Lung Adenocarcinoma by Regulating the EMT Pathway

Objective To explore the effect and molecular mechanism of ARID1A on the invasion and metastasis of lung adenocarcinoma PC9 cells, aiming to clarify the role and regulatory pathway of ARID1A in the progression of lung adenocarcinoma, and to provide experimental basis for the targeted therapy of lung adenocarcinoma.

Methods The PC9-ARID1A knockout (KO) cell line was constructed by CRISPR/Cas9 technology. The efficiency of gene knockout and protein silencing was verified by Sanger sequencing and Western blot (WB). Cell migration and invasion assays were used to detect the effect of ARID1A knockout on the migration and invasion ability of PC9 cells. WB was used to detect the expression changes of key EMT pathway markers (E-cadherin, N-cadherin, Vimentin). RNA-SEQ sequencing and bioinformatics analysis (GO, KEGG, GSEA) were used to clarify the differentially expressed genes and EMT pathway enrichment characteristics after ARID1A knockout. Nude mouse subcutaneous tumorigenesis and ventricular injection metastasis experiments were used to verify the effect of ARID1A knockout on the in vivo growth and metastasis of lung adenocarcinoma.

Results  TCGA database analysis showed that low ARID1A expression was closely associated with poor prognosis of lung adenocarcinoma patients n=2 661, median overall survival (OS): 60 months vs. 79 months, hazard ratio (HR)=0.74, 95% confidence interval (CI) 0.66~0.84, P<0.001. The PC9-ARID1A KO cell line was successfully constructed with a gene knockout efficiency of 100% and a protein silencing efficiency of 90.2% ± 0.5% (P=0.029). After ARID1A knockout, the migration and invasion abilities of PC9 cells were significantly enhanced (number of migrating cells: 433.00±86.02 vs. 1 140.00±84.02, P=5.07×10⁻⁴; number of invading cells: 640.67±45.17 vs. 2 037.00±182.01, P=8.23×10⁻⁴ ). WB results showed that the expression of E-cadherin was downregulated, and the expressions of N-cadherin and Vimentin were upregulated in PC9-ARID1A KO cells. RNA-SEQ analysis detected 2 284 significantly differentially expressed genes, which were significantly enriched in EMT-related pathways, and 18 key EMT-related differentially expressed genes were screened out. In vivo experiments showed that the volume of subcutaneous tumors in nude mice of the ARID1A knockout group was significantly larger than that of the control group（47.19±36.97 mm³ vs. 8.43±7.17 mm³, P<0.05）, the whole-body fluorescence signal intensity was significantly higher than that of the control group (P=8.76×10⁻⁶ ), and the number of lung tumor metastases increased.

Conclusions ARID1A knockout can significantly enhance the migration, invasion ability and in vivo growth and metastasis ability of PC9 lung adenocarcinoma cells by activating the EMT pathway. ARID1A is expected to be a potential target for prognosis judgment and targeted therapy of lung adenocarcinoma, providing experimental support for clarifying the molecular mechanism of lung adenocarcinoma invasion and metastasis.