The Mechanism Of CircFOXP1 Inhibiting Lung Cancer Progression Through MiR-558/TNFAIP1 And TPM1

BackgroundLung cancer is a common malignant tumor.Its incidence rate and mortality rate are the first in the world.Lung cancer has various types,difficult treatment and poor prognosis.Therefore,it is urgent to find more effective therapeutic targets and diagnostic markers.At the same time,the research on the specific mechanism of lung cancer will be beneficial to its diagnosis and treatment.In recent years,circular RNA(circRNA)has become a hot topic in biomedical research.It has species conservation,stability and expression specificity,plays an important regulatory role in tumor and other diseases,and is expected to become a new target and marker in clinical diagnosis and treatment.At present,the research of circRNA in lung cancer has made some progress,but more and more in-depth research is needed,hoping to provide more reliable theoretical support for the diagnosis and treatment of lung cancer.Therefore,this study aimed at the low expression of circFOXP1 in lung cancer to carry out specific experiments in vitro and in vivo,to explore the specific molecular mechanism of its effect.MethodsPart 1:The role of circFOXP1 in lung cancer cells.1.Detection of circFOXP1 expression in various lung cancer cell lines by fluorescence quantitative PCR.2.Construction of circFOXPl overexpression vector to transfect lung cancer cells,and detection of its overexpression level by fluorescence quantitative PCR.3.CCK-8 method was used to detect the effect of circFOXP1 on the proliferation activity of lung cancer cells.4.Effect of circFOXP1 on apoptosis of lung cancer cells by flow cytometry.5.Transwell method was used to detect the effect of circFOXP1 on the invasion of lung cancer cells.Part 2:Competitive adsorption of miR-558 by circFOXP1 promotes the expression of TNFAIP1 and TPM1.1.Expression and localization of circFOXP1 by nuclear and cytoplasmic separation.2.Screening miRNAs combined with circFOXP1.3.Detection of the combination of circFOXP1 and miR-558 by dual luciferase reporter gene assay.4.RNA-pull down assay for direct intracellular binding of circFOXP1 and miR-558.5.The binding ability of circFOXP1,miR-558 and AGO2 protein by immunocoprecipitation.6.Detection of miR-558 expression in various lung cancer cell lines by fluorescence quantitative PCR.7.Screening of miR-558 target genes.8.Detection of the binding of miR-558 with target genes TNFAIP1 and TPM1 by dual luciferase reporter gene assay.9.The effects of circFOXP1 and miR-558 on the expression of TNFAIP1 and TPM1.10.Western blot analysis of the effects of circFOXP1 and miR-558 on the expression of TNFAIP1 and TPM1 proteins.Part 3:circFOXP1 inhibits lung cancer progression through miR-558/TNFAIP1 and TPM1 pathway.1.circFOXP1 overexpression vector,miR-558 mimics or inhibitors,TNFAIP1 or TPM1 overexpression vector or siRNA were transfected or co-transfected into lung cancer cells,respectively.2.Detection of proliferative activity of lung cancer cells by CCK-8.3.Flow cytometry was used to detect the apoptosis of lung cancer cells.4.Transwell method for detecting the invasion of lung cancer cells.5.Screening A549 cells with stable and high expression of circFOXP1 or miR-588.6.Construction of human lung cancer xenografts in nude mice.7.Record the growth curve and volume of tumours.8.Expression of circFOXP1,miR-558,TNFAIP1 and TPM1 was detected by fluorescence quantitative PCR.9.Western blot detection of protein expression of TNFAIP1 and TPM1.ResultsPart 1:1.The expression of circFOXP1 was down-regulated in lung cancer cell lines.2.circFOXP1 inhibited the proliferative activity of lung cancer cells.3.circFOXP1 promoted the apoptosis of lung cancer cells.4.circFOXP1 inhibited the invasion of lung cancer cells.Part 2:1.circFOXP1 was mainly expressed in cytoplasm.2.circFOXP1 inhibited the expression of miR-558 in lung cancer cells.3.circFOXP1 directly bound to miR-558 in lung cancer cells.4.miR-558 was high expressed in lung cancer cell lines.5.TNFAIP1 and TPM1 were both target genes of miR-558.6.miR-558 blocked the effect of circFOXP1 on the expression of TNFAIP1 and TPM1.Part 3:1.circFOXP1 inhibited the proliferative activity of lung cancer cells through miR-558/TNFAIP1 and TPM1 pathway.2.circFOXP1 promoted the apoptosis of lung cancer cells through miR-558/TNFAIP1 and TPM1 pathway.3.circFOXP1 reduced the invasion level of lung cancer cells through miR-558/TNFAIP1 and TPM 1 pathway.4.circFOXP1 inhibited the growth and size of lung cancer in nude mice.5.miR-558 terminated the effect of circFOXP1 on tumor growth in nude mice.6.circFOXP1 promoted the expression of TNFAIP1 and TPM1 in tumor tissues of nude mice.Conclusion1.The expression of circFOXP1 is down-regulated in lung cancer cells,while miR-558 is up-regulated.2.circFOXP1 inhibits the expression of miR-558 by competitively binding miR-558,and then promotes the expression of downstream target gene TNFAIP1 and TPM1.3.circFOXP1 inhibits the proliferation and invasion of lung cancer cells,and promotes the apoptosis through miR-558/TNFAIP1 and TPM1 pathway.4.circFOXP1 inhibits the growth of lung cancer in nude mice through miR-558/TNFAIP1 and TPM 1 pathway.