CircSWT1 Promotes NSCLC Progression Via The MiR-370-3p/SNAIL Axis By Inducing Cell Epithelial-mesenchymal Transition

Background and Objective:Globally,lung cancer had the second highest incidence rate among all cancers,with the highest cancer-related mortality rate.It is a serious disease that poses a significant obstacle to increasing human life expectancy.Non-small cell lung cancer(NSCLC)accounts for 80-90% of all lung cancers and is the main pathological type leading to lung cancer incidence and mortality.Despite the widespread use of early screening,improved diagnostic techniques,and thoracoscopic surgery,as well as various treatment options such as radiation and chemotherapy,targeted therapy,and immunotherapy,the overall five-year survival rate for lung cancer patients is only 22%.Therefore,further research into the pathogenesis and treatment options for lung cancer is an urgent and important health problem in today’s society in order to improve patient survival rates.Circular RNA(circ RNA)is widely expressed in eukaryotes and has diverse biological functions.As an oncogene or tumor suppressor,it participates in the occurrence and development of tumors and is a potential biomarker and therapeutic target.There have been extensive studies on the mechanism of circ RNA in NSCLC.SWT1-derived hsa＿circ＿0015677 has been shown to affect the apoptosis of myocardial cells.However,the expression levels and biological functions of SWT1-derived circ RNAs in NSCLC have not been fully investigated.Therefore,we analyzed the expression of SWT1-derived circ RNAs in NSCLC,identified the highly expressed has＿circ＿0004689,and studied its potential biological functions and underlying mechanisms in NSCLC progression.Method:1.Four pairs of NSCLC tissues and matched normal tissues were collected to detect the expression levels of circ RNAs derived from SWT1 by qRT-PCR.2.The expression of circSWT1 in 96 NSCLC tissues was detected by qRT-PCR,and the relationship between circSWT1 expression levels and clinical pathological features and prognosis of NSCLC patients was analyzed.3.NSCLC cell lines with differential expression of circSWT1 were established,and the proliferation,invasion,and migration abilities of cells were detected by CCK-8,clone formation,matrigel Transwell,and wound healing assays.The expression levels of EMT-related biomarkers were detected by qRT-PCR and Western blot.4.RNA pull-down,RIP,and luciferase reporter gene assays were conducted to confirm the interaction between circSWT1 and miR-370-3p,as well as the interaction between miR-370-3p and SNAIL.5.After CRISPR-Cas9 knockout of SNAIL,the effects of differential expression of circSWT1 on the proliferation,invasion,migration,and EMT of NSCLC cells were detected.6.In vivo experiments were conducted to investigate the effects of differential expression of circSWT1 on NSCLC progression.Result:1.Among 41 circ RNAs derived from SWT1,circSWT1(has＿circ＿0004689)has the highest expression level in NSCLC tissue,as detected by qRT-PCR.2.The expression level of circSWT1 is significantly higher in NSCLC tissues(P<0.0001)compared to normal lung tissue.NSCLC patients with tumor diameter >3cm(P<0.01),stage III-IV(P<0.01),or lymph node metastasis(P<0.01)have higher expression levels of circSWT1.NSCLC patients with high circSWT1 expression have poorer OS(P=0.0005)and DFS(P=0.0004).CircSWT1 expression level is an independent risk factor for OS(P=0.015)and DFS(P=0.023).3.Overexpression of circSWT1 promotes proliferation,invasion,and migration of NSCLC cells,reduces E-cadherin expression,and increases N-cadherin and Vimentin expression,promoting EMT process.Knockdown of circSWT1 inhibits proliferation,invasion,and migration of NSCLC cells,increases E-cadherin expression,and reduces N-cadherin and Vimentin expression,suppressing EMT process.4.Bio-circSWT1 pulldown experiment showed significant enrichment of miR-370-3p.Bio-miR-370-3p pulldown experiment showed significant enrichment of circSWT1.RIP experiment with anti-AGO2 antibody showed significant enrichment of both circSWT1 and miR-370-3p.Fluorescent reporter gene assays confirmed that circSWT1 and miR-370-3p,as well as miR-370-3p and SNAIL,can directly bind to each other.5.After knocking out SNAIL,NSCLC cells with differential expression of circSWT1 showed no significant differences in proliferation,invasion,and migration abilities,and the EMT process was no longer regulated by circSWT1.6.The xenograft tumor model in nude mice confirmed that overexpression of circSWT1 promotes the progression of NSCLC,and knockdown of circSWT1 inhibits the growth of NSCLC.Conclusion:1.The expression level of circSWT1 is significantly upregulated in NSCLC tissues.2.Patients with NSCLC who have larger tumor diameter(>3cm),advanced TNM stage(III-IV),or lymph node metastasis tend to have higher expression levels of circSWT1.3.The expression level of circSWT1 is an independent risk factor for overall survival(OS)and disease-free survival(DFS)and could serve as a potential biomarker for predicting the prognosis of NSCLC patients.4.Overexpression of circSWT1 promotes the proliferation,invasion,migration ability,and epithelial-mesenchymal transition(EMT)process of NSCLC cells.5.CircSWT1 acts as a sponge for miR-370-3p,relieves its inhibition of SNAIL, promotes the proliferation,invasion,and migration ability of NSCLC,and enhances the EMT process of NSCLC.