ROCK1 Promotes Migration And Invasion Of Non-small-cell Lung Cancer Cells Through The PTEN/PI3K/FAK Pathway

Lung cancer is one of the malignant tumors which seriously threaten human health.The morbidity and mortality of lung cancer are increasing year by year,and it is the biggest cancer in China.Traditional radiotherapy and chemotherapy-based lung cancer treatment methods have been used clinically for many years.However,these treatments have brought great pain to patients due to the selectivity lackness.Targets and targeted dr ugs have achieved great clinical success,but with their widespread application,the occurrence of target mutations and drug resistance,as well as fewer targeted drugs to choose in the clinic,have become the bottleneck of molecular targeted therapy for lung cancer.Therefore,looking for new molecular targets for lung cancer will be of great significance for the diagnosis,treatment,and prognosis of lung cancer.Rho-associated coiled-coil protein kinase(Rho-associated coiled-coil protein kinase 1,ROCK1)is a member of the Ras family of proteins with serine/threonine-protein kinase activity.It has a molecular weight of 160 KD and the gene is located on chromosome18(18q11.1),which is a trimeric small molecule GTP binding protein,mainly composed of three parts,including N-terminal amino kinase domain,central frizzled protein RBD(Rho-binding domain)domain and the C-terminal of the cysteine-rich PH(pleckstrin homology)domain.Previous studies have reported that ROCK1 plays a very important role in the regulation of tumor cell proliferation,invasion,metastasis,and apoptosis.ROCK1 is highly expressed in a variety of human malignant tumors,such as prostate cancer,liver cancer,gastric cancer,ovarian cancer,and osteosarcoma,compared with the corr esponding normal tissues,and can promote the proliferation and metastasis of cancer cells.ROCK1 knockdown can greatly improve the anti-malignant glioma efficacy of Nimustine.As reports in lung cancer,the expression of ROCK1 in NSCLC tissues was significantly higher than that in normal lung tissues adjacent to cancer.We have found that the expression of ROCK1 in SK-MES-1(Human lung squamous cell carcinoma cells),A549(Human lung adenocarcinoma cells),NCI-H1299(Human lung adenocarcinoma cells),and NCI-H226(Human lung squamous carcinoma cells)were all higher than it in human embryonic lung fibroblast WI-38 cells,indicating that ROCK1 may play a key role in lung cancer,but the mechanism is not clear.In this study,we investigated the effect of ROCK1 on the migration and invasion of NSCLC cells and preliminarily explored its possible mechanism,suggesting that ROCK1 may become a new target for the diagnosis,treatment,and prognosis of lung cancer.Methods:1.The expression of ROCK1 in lung cancer tissues and corresponding normal tissues were analyzed by the bioinformatics method.2.Western blot was used to detect the expression of ROCK1 in human embryonic lung fibroblasts cells(WI-38)and various lung cancer cell lines(SK-MES-1,A549,NCI-H1299,NCI-H226).3.Correlation between ROCK1 and survival time of lung cancer patients was analyzed by Kaplan-Meier survival analysis database.4.The knockdown sh RNA of ROCK1 was transfected into A549 and NCI-1299 cells to knock down the expression of ROCK1 in cells and verify the effect of ROCK1 on cell migration and proliferation.5.Cell scratching assay and Transwell assay were used to verify the migration and invasion of NSCLC cells.6.Cell adhesion assay was used to detect the adhesion ability of A549 and NCI-H1299 cells.7.Immunofluorescence was used to detect the expression of FAK on the cell membrane and the co-localization with Actin.8.The PI3K specific inhibitor LY294002 was used to treat cells to inhibit the PI3K signaling pathway.9.The knockdown sh RNA of PTEN was transfected into A549 and NCI-1299 cells to knock down the expression of PTEN and verify the effect of PTEN on cell migration and proliferation.Results:1.ROCK1 is highly expressed in non-small cell lung cancer,and patients with high expression of ROCK1 have poor survival.The analysis of the oncomine database showed that ROCK1 was more highly expressed in lung cancer tissues than the expression in normal tissues.The Kaplan-Meier plotter analysis showed that the high expression of ROCK1 was associated with poor survival,GSE19188(P=0.016),GSE30219(P=0.033);2.ROCK1 was significantly reduced in A549 and NCI-H1299 NSCLC cells by sh RNA knockdown;3.Knocking down ROCK1 significantly reduced the migration and invasion ability of NSCLC cells;4.Knocking down ROCK1 reduced the expression of FAK in the local phosphorylation part of the cell membrane and decreased the co-localization with Actin,reducing the formation of lamellipodia,and inhibited the ability of cell adhesion.5.The PI3K specific inhibitor LY294002 was added to inhibit the phosphorylation of FAK and reduce cell adhesion.6.Knocking down PTEN increased the expression of phosphorylated PI3K and phosphorylated FAK,and prevented the down-regulation of phosphorylated PI3K and phosphorylated FAK mediated by ROCK1 knockdown.Conclusion:In this study,it shows that ROCK1 can induce cell migration and invasion.ROCK1 can promote the phosphorylation and activation of FAK,improve cell adhesion and accelerate cell migration and invasion by regulating the PTEN/ PI3K/FAK signaling pathway,thus affecting the occurrence and development of NSCLC.It is suggested that ROCK1 may be a new target for the diagnosis and treatment of NSCLC.