The Molecular Mechanism Of Silencing ROR1 By Small Interfering RNA To Inhibit Metastasis And Invasion Of Lung Adenocarcinoma Based On AKT/mTOR Pathway

Background and purpose: Lung cancer is one of the most common cancer in the world.Smoking is the main cause of lung cancer,followed by air pollution,occupational carcinogens,ionizing radiation,activation of certain oncogenes,and loss of anti-cancer genes.Lung cancer is mainly divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),and non-small cell lung cancer accounts for about 85% of lung cancer.Lung adenocarcinoma is one of the non-small cell lung cancers,accounting for 40% of all lung cancer types.Due to tumor invasion and metastasis,the 5-year survival rate of lung adenocarcinoma is poor,and the therapeutic effect of traditional treatment methods is not obvious.The receptor tyrosine kinase-like orphan receptor 1(ROR1)is a type I surface transmembrane protein that contributes to the development and migration of fetal organs during embryonic development.ROR1 has been shown to inhibit apoptosis,enhance EGFR signaling,and induce proliferation,and is therefore closely related to tumor cell progression.This study was designed to investigate the role of ROR1 in the metastasis and invasion of lung adenocarcinoma,and to provide a potential target for the treatment of lung adenocarcinoma.Experimental approach:(1)Small interfering RNA(si RNA)was used to blocking the expression of ROR1 in lung adenocarcinoma cell lines PC9,PC9 erlo,NCI-H1975 and NCI-H358.(2)Detect the migration of lung adenocarcinoma cell lines PC9 and PC9 erlo cells after ROR1 via would healing assay.(3)Detect the invasion level of lung adenocarcinoma cell lines PC9,PC9 erlo and NCI-H1975 by cell invasion assay.(4)Detect the expression level of epithelial-mesenchymal transition marker proteins in PC9,PC9 erlo and NCI-H1975 cell line by immunofluorescence assay.(5)Detect the expression level of key markers and transcription factors in epithelial-mesenchymal transition and the activity of AKT signaling pathway in PC9,PC9 erlo,NCI-H1975 and NCI-H358 cell lines after interference with ROR1 by Western blotting assay.was used to detect the expression changes of key proteins and epithelial-mesenchymal transition key transcription factors in the AKT signaling pathway of PC9,PC9 erlo,NCI-H1975 and NCI-H358 cell lines after interference with ROR1.Results:(1)The expression level of ROR1 in si ROR1 group was significantly lower than that in si NC group after interference with si RNA in four different lung adenocarcinoma cell lines PC9,PC9 erlo,NCI-H1975 and NCI-H358.(2)The migration of cells in the si ROR1 group was significantly inhibited compared with the si NC group after 48 hours of interference with si RNA in two different lung adenocarcinoma cell lines,PC9 and PC9 erlo.(3)The invasion of cells in the si ROR1 group was significantly inhibited compared with that in si NC group after 48 hours of interference with si RNA in three different lung adenocarcinoma cell lines PC9,PC9 erlo and NCI-H1975.(4)The Vimentin fluorescence level in the si ROR1 group was lower than that in the si NC group,while the E-cadherin fluorescence level in the si ROR1 group was higher than that in the si NC group in three different lung adenocarcinoma cell lines PC9,PC9 erlo and NCI-H1975.(5)The expression of Snail,Slug and Vimentin in the si ROR1 group was lower than that in the si NC group,while the expression of E-cadherin was higher than that in the si NC group.The key proteins in the AKT/m TOR signaling pathway are then detected.The expression of p-m TOR,p-AKT,p-p70S6 K and p-Raptor in the si ROR1 group was decreased compared with the si NC group in the NCI-H1975 and NCI-H358 cell lines;The expression of p-m TOR,p-AKT and p-p70S6 K in the si ROR1 group was decreased compared to the si NC group in the PC9 and PC9 erlo cell lines.Conclusions and Implications: Our data confirmed that ROR1 has a potential effect on the metastasis and invasion of human lung adenocarcinoma,and via upregulating the activity of critical protein in AKT/m TOR signaling pathway.This study provides a potential target for the treatment of lung adenocarcinoma.