Promoting effect of breast cancer anti-estrogen resistance 1 on proliferation and growth of lung adenocarcinoma cells and potential networks of its interacting proteins

Abstract

Objective To explore the biological functions of breast cancer anti-estrogen resistance 1 (BCAR1, also known as p130cas) in the incidence and development of lung cancer, and investigate the potential networks of its interacting proteins. Methods ① Western blotting was performed to study the expression of BCAR1 in 15 pairs of lung cancer tissues and para-cancerous normal tissues, and immunohistochemical (IHC) assay was carried out in paraffin-embedded tissues from 54 patients with early stage lung cancer in order to determine the relationship between BCAR1 level and prognosis. TCGA analysis was also performed as further validation. ②BCAR1 knockout (KO) cell lines were established in H1975 and H1299 lung cancer cells, respectively, using CRISPR/Cas9. And A549 cells with overexpression (OE) of BCAR1 were constructed by transfection of BCAR1 cDNA. MTT analysis and plate colony formation assay were used to detect the proliferation and viability of the cells, respectively. ③Immunoprecipitation mass spectrometry (IP-MS) along with bioinformatics analysis was employed to identify the key protein of BCAR1 interacting networks in 293T tool cells. ④The expression variations of the key node gene were confirmed in BCAR1-KO and BCAR1-OE cells using Western blotting, and the correlation between the key node gene and BCAR1 was verified in the lung cancer tissue microarray. Results ① The expression of BCAR1 was significantly higher in the lung cancer tissues than para-cancerous tissues (P < 0.05). Higher expression of BCAR1 was associated with worse prognosis in the patients with early-stage lung cancer (HR=5.026, P= 0.001) and TCGA cohort study (HR=1.5, P=0.008 7). ②BCAR1-KO and BCAR1-OE cells were successfully constructed, and the cell proliferation and colony formation were decreased with BCAR1-KO and increased following BCAR1-OE respectively. ③Mammalian lethal with SEC13 protein 8 (GβL, mLST8) was revealed to be a key protein of BCAR1 interacting network. ④MLST8 was significantly expressed at a higher level in the lung cancer tissues than the para-cancerous tissues (P < 0.05), and its expression was positively correlated with the expression of BCAR1 (R=0.422 1, P=0.001 5). Furthermore, the expression of MLST8 was decreased in BCAR1-KO cells and increased in BCAR1-OE cells. Conclusion BCAR1 plays a critical carcinogenetic role in lung cancer. MLST8 expression is significantly correlated with BCAR1, and may be a key downstream regulatory protein of BCAR1.

Keywords

• bcar1
• mammalian lethal with sec13 protein 8
• cell proliferation
• cell colony formation
• immunoprecipitation mass spectrometry
• lung cancer