DLL3 Reduces Chemosensitivity And Promotes The Proliferation And Invasion Of Tumor Cells Of Advanced Small Cell Lung Carcinoma

Part one Expression and significance of DLL3 in advanced SCLC tumor tissues Objective: To explore the relationship between the expression of DLL3 in advanced in small cell lung cancer(SCLC)tumor tissues and chemosensitivity to platinum / etoposide therapyandprognosis of patients.Methods: 1.Selected patients with SCLC who met the enrollment criteria and collected their general clinical information.2.Immunohistochemistry was used to detect the expression of DLL3 in SCLC samples.All patients were assigned into DLL3-high group DLL3-low group according to the expression of DLL3.3.?2 test was applied for analyze the relationship between DLL3 expressionandclinicopathological features and platinum-based chemotherapy response.Kaplan-Meieranalysis for exploring the effects of DLL3 on progression-free survival(PFS)and overall survival(OS)in patients with advanced SCLC.Results: 1.DLL3 was mainly located in the cell membrane.The positive expression rate was 79.17%(57/72)in advanced SCLC tumor tissues,and there was no statistical correlation between the expression level and the patient's age,gender,smoking history,TNM stage,and tumor diameters(P>0.05);2.Compared to DLL3-low group,the chemotherapy response in DLL3-high group was decreased(34.8% vs 63.3%,p = 0.041),the difference was statistically significant(P <0.05).The median PFS in DLL3-low group was 6.8 months,and the median PFS in DLL3-high group was 4.9 months.The difference between the two groups was statistically significant(P<0.05).The median OS between the two groups had no statisticallydifference(P=0.866).Conclusions: The expression of DLL3 in tumor tissues was negatively correlated with chemotherapy response rate and PFS in patients with advanced SCLC.DLL3 may be a biomarker for predicting chemotherapy efficancy in patients with advanced SCLC.Part two The effect of DLL3 on the proliferation and invasion of SCLC H69 and H20 cell lines Objective: To explore the role of DLL3 in the proliferation and invasion of SCLC cells.Methods: 1.q RT-PCR method to detect the relative expression of DLL3 m RNA in five SCLC cell lines(H2227,H446,H524,H69 and H20)and normal lung epithelial cells(16HBE);2.Down-regulated the DLL3 m RNA in SCLC cells by transfected with sh-RNA;3.CCK8 and Edu proliferation assays were used to detect the proliferation of SCLC cell lines H69 and H20 after silencing DLL3 m RNA;4.Transwell chamber detects changes in the invasion and metastasis ability of SCLC cell lines H69 and H20 after down-regulating DLL3 m RNA;5.Western blot and immunofluorescence were used to detect the expression of NCadherin and E-Cadherin in H69 and H20 cell lines after silencing DLL3 m RNA.Results:1.qRT-PCR showed that the expression of DLL3 m RNA in five SCLC lineswas higher than that in bronchial epithelial cells.H69 and H20 cells,which exhibit the highest expression level of DLL3;2.After transfected with sh-DLL3#1,sh-DLL3#2,sh-DLL3#3,DLL3 m RNA expression in H69 and H20 cells were significantly decreased.Sh-DLL3#2 was used for all subsequent experiments due to its optimal interference efficiency;3.CCK-8 and Ed Ushowed that the proliferation of SCLC cell lines H69 and H20 were inhibited after silencing DLL3 m RNA(P<0.001);4.Transwell chamber results showed that the invasive ability of H69 and H20 cell lines decreased after DLL3 gene down-regulated;5.Western blot and immunofluorescence showed that the expression of E-cadherin was enhanced but the expression of N-cadherin was decreased after silencing DLL3 gene which indicating that EMT process was inhibited.Conclusions: DLL3 promotes the proliferation and invasion of SCLC cells.Part three Mechanism of DLL3 in malignant behavior of small cell lung cancer Objective: To explore the mechanism associated with the upregulation of DLL3 in SCLC.method: 1.The relevance between DLL3 and LIN28 B in lung samples were obtained from Starbase v.3.0(http://starbase.sysu.edu.cn/).In addition,binding sites of mi R-518d-5p and DLL3 or LIN28 B were predicted from Starbase.2.Co-transfection of mi R-518d-5p and DLL3 m RNA 3'UTR binding target vector(DLL3-WT),mutant(DLL3-MUT)and LIN28 B m RNA 3'UTR binding target vector(LIN28B-WT)and mutation(LIN28B-MUT),and then detected the luciferase activity using dual luciferase reporter gene detection system.Ago2-RIP was used verification mi R-518d-5p and DLL3 and LIN28B3'UTR have direct binding sites;3.q RT-PCR and Western blot were used to detect the expression of DLL3 m RNA,LIN28 B m RNA and protein after transfected with mi R-518d-5p mimics.4.The sh-DLL3 and LIN28 B overexpression vectors were co-transfected to detect the proliferation,metastasis and invasion of small cell lung cancer H69 and H20 cell lines and the changes of N-Cadherin and E-Cadherin protein levels.5.Transfected with mi R-518d-5p inhibitor or LIN28 B vector,then detecting the proliferation,metastasis and invasion of SCLC H69,H20 cell lines and the expression of NCadherin and E-Cadherin protein.Result: 1.The Starbase database shows that LIN28 B is an RNA binding protein that binds to DLL3.RIP assays confirmed that LIN28 B interacts with DLL3.Further studies have revealed that down-regulation of LIN28 B leads to the decay of DLL3 m RNA.2.Bioinformatics predicts that mi R-518d-5p has potential binding sites with LIN28 B m RNA 3'UTR,DLL3 m RNA 3'UTR.3.Ago2-RIP shows that mi R-518d-5p,LIN28 B and DLL3 can interact with AGO2 protein.Further double luciferase reporter assay results showed that luciferase activity was significantly decreased after co-transfection of mi R-518d-5p mimics and DLL3 3'UTR or LIN 28 B 3'UTR(P < 0.001);In contrast,no significant suppressive effect on luciferase activity was observed in cells transfected with mutant DLL3 3'UTR or LIN 28 B 3'UTR.4.After transfected mi R-518d-5p inhibitor or LIN28 B overexpression vector,the proliferation,metastasis and invasion ability of H69 and H20 cell lines were enhanced,ECadherin protein expression was decreased whease N-Cadherin protein expression was increased.Down-regulated mi R-518d-5p or overexpressed LIN28 B Partially recover the proliferation and invasion of sh-DLL3 transfected cells(P <0.05).Conclusion: Mi R-518d-5p negatively regulate the malignant biological behavior of SCLC by targeting DLL3 and LIN28 B.