MicroRNA-333-5p Inhibits The Cell Proliferation And Metastasis In Non-small Cell Lung Cancer By Targeting CPNE 1

Objectives:This study investigated the expression of CPNE 1 in non-small cell lung cancer tissues and its correlation with clinical pathological parameters.MicroRNA-335-5p inhibits proliferation and metastasis of non-small cell lung cancer cells by targeting CPNE 1 from in vitro cell level and in vivo animal level,respectively,and further studies on related machinery.Methods:1.The expression of CPNE 1 in 60 cases of non-small cell lung cancer and matched paracancerous tissues was detected by immunohistochemistry(IHC)and real-time fluorescent quantitative PCR.The correlation between the expression of CPNE 1 and clinical data was analyzed;2.CCK-8 assay,colony formation assay,flow cytometry,scratch and Transwell assay were used to detect the effects of interference and overexpression of CPNE 1 on proliferation and metastasis of non-small cell lung cancer cells.Western Blot assay was used to detect downstream proliferation of CPNE 1,transfer signal protein levels change;3.Bioinformatics was used to predict potential microRNAs upstream of CPNE 1 and microRNA-3 3 5-5p was screened by tissue microarray results;dual luciferase assay verified the targeting regulation of microRNA-335-5p and CPNE 1;CCK-8 method,colony formation assay,flow cytometry,scratch and Transwell assay were used to verify the effect of microRNA-335-5p on proliferation and metastasis of non-small cell lung cancer cells.Western Blot method was used to detect microRNA-335-5p downstream.Proliferation,transfer signal protein levels change;4.The effect of overexpression of CPNE 1 on the tumorigenic ability of non-small cell lung cancer was observed by constructing a subcutaneous tumor formation model in nude mice.Results:1.The expression of CPNE 1 in non-small cell lung cancer tissues was higher than that in adjacent tissues(P<0.05).The expression of CPNE 1 was found in stratified analysis.The gender,age,smoking history,pathological type and clinical stage of CPNE 1 were analyzed.There was no significant correlation(P>0.05),but the expression in patients with lymph node metastasis was higher than that in non-lymph node metastasis(P<0.05);2.After interference and overexpression of CPNE 1 in A549 and H1299 lung cancer cell lines,the proliferation and metastasis ability of lung cancer cells were weakened or enhanced,respectively.Flow cytometry results indicated that CPNE 1 mainly affected cell cycle,but had no significant effect on cell apoptosis.Western Blot results suggest that p-EGFR and downstream p-FAK,p-SRC,p-AKT,p-ERK,Cyclin D1 and Snail,N-cadherin,MMP9 and CPNE 1 protein levels are consistent,interfering with CPNE 1 expression Decreased EGF-induced up-regulation of p-EGFR,p-FAK,p-SRC,p-AKT,and p-ERK proteins;Co-IP results suggest that CPNE 1 can bind to EGFR,thereby activating EGFR and its downstream signaling pathways.Leading lung cancer cell proliferation and metastasis,there is co-expression between the two;3.The expression of microRNA-335-5p mRNA in 60 non-small cell lung cancer tissues was lower than that in adjacent tissues(P<0.01).The expression of microRNA-335-5p mRNA was related to the age,sex and smoking history of patients.There was no significant relationship between pathological type and clinical stage(P>0.05),However,patients with lymph node metastasis have lower expression than non-lymph node metastasis,and the difference was statistically significant(P<0.05).Dual luciferase assay confirmed that microRNA-335-5p can be targeted and regulated.The expression of CPNE 1 and the expression of microRNA-335-5p mRNA were significantly up-regulated after transfection of microRNA-335-5p mimics in A549 and H1299 lung cancer cell lines,and the expression of CPNE 1 mRNA and protein were down-regulated,further confirming the results of dual luciferase;Compared with the miR-NC group,transfection up-regulated microRNA-335-5p mRNA can inhibit cell proliferation and metastasis,while p-EGFR and its downstream p-FAK,p-SRC,p-AKT,p-ERK Cyclin D1 and Snail,N-cadherin,and MMP9 protein expression decreased;4.By observing the tumor of nude mice,it was found that tumor growth was observed on the 22nd day after CPNE 1 group inoculation,while the PLVX group showed macroscopic tumors on the 26th day after inoculation;the growth of transplanted tumors in CPNE 1 overexpression group was significantly accelerated,while the PLVX group was significantly accelerated.The transplanted tumor grew slowly and the volume was significantly smaller than that of the control group.The nude mice were sacrificed to a tumor volume of 1000 cm3.The tumors were completely removed and photographed and weighed.The average weights were:0.494 g in CPNE 1 group and 0.293 g in PLVX group.The weight and gross volume were significantly higher than those in the PLVX group(P<0.05).The immunohistochemistry results showed that the expression level of CPNE 1 protein in CPNE 1 group was significantly higher than that in the control group.Conclution:1.CPNE 1 is highly expressed in non-small cell lung cancer tissues and is higher in patients with lymph node metastasis than in patients with non-lymph node metastasis;2.CPNE 1 promotes proliferation and metastasis of non-small cell lung cancer cells via EGFR and its downstream signaling pathway;3.microRNA-335-5p inhibits the proliferation and metastasis of non-small cell lung cancer cells by targeting CPNE 1,which is related to EGFR and its downstream signaling pathway;4.CPNE 1 can promote the proliferation of non-small cell lung cancer in vivo.