The Study On The Mechanism Of IL-17 On Proliferation, Apoptosis And Migration Of Laryngeal Carcinoma Hep-2 Cells

Objective: Laryngeal squamous cell carcinoma is one of the most common head and neck malignancies,the cause is not very clear,but there are many common predisposing factors,such as smoking,drinking,viral infection,radiation,eating habits,occupational factors.The main clinical treatment of laryngeal cancer is surgical operation with radiotherapy and chemotherapy,although it can extend the survival time of some patients,but the overall treatment is not very satisfactory.There are lots of sequelae complications,such as cough,loss of sound,difficulty swallowing,seriously affecting the quality of life of patients.For further understanding the mechanism of laryngeal cancer development,expecting to find a more effective and optional molecular targets to prevent disease,and carries on the molecular targeted therapy,this is important to improve the diagnosis and treatment of laryngeal cancer and prognosis.In this study,we examined the changes of Hep-2 cell function by transfection of interleukin-17(IL-17)gene,to investigate the effect of IL-17 on the proliferation,apoptosis and migration of human laryngeal carcinoma Hep-2 cells and its possible mechanism.Methods: Build IL-17 recombinant eukaryotic expression vector pEGFP-N1-IL – 17,then transfect it into Hep-2 cells,at the same time set up empty vector group(pEGFP-N1)and normal control group.Fluorescence microscopy was used to observe the transfection of three groups of cells.The expression of IL-17 mRNA and protein was detected before and after transfection by RT-PCR and Western blotting respectively.MTT proliferation assay was used to detect the changes of Hep-2 cells proliferation in the three groups before and after transfection.At the same time,the apoptosis of the three groups was detected by flow cytometry.The expressions of Caspase-3,Caspase-9 and Bcl-2 were detected by RT-PCR.Cell scratch repair experiment and Transwell migration experiment were respectively observed the changes of lateral migration ability and vertical migration ability of three groups of cells.The invasive ability of Hep-2 cells was detected by Transwell matrix test.RT-PCR technique was used to detect mRNA expression of cell invasion related molecular MMP-2 and MMP-9.Western blotting experiment was used to detect the expression of Notch signaling pathway related molecular Notch1,Hey1 and Hes1.Results: There is no green fluorescent protein GFP expression in normal Hep-2 cells,and green fluorescence does not appear even if excited by excitation light.Hep-2 cells transfected with pEGFP-N1 and pEGFP-N1-IL-17 can see green fluorescence by fluorescence microscopy,and the transfection efficiency was gradually increased with the prolongation of transfection time.RT-PCR and Western blotting showed that IL-17 mRNA and protein expression in Hep-2/IL-17 cells transfected with pEGFP-N1-IL-17,normal Hep-2 cells and Hep-2/pEGFP-N1 cells transfected with pEGFP-N1 did not express IL-17.MTT cell proliferation assay and apoptosis experiments showed that the target gene IL-17 could inhibit the proliferation of Hep-2 cells and promote the apoptosis of Hep-2 cells.The expressions of Caspase-3 and Caspase-9 were significantly increased after transfection with pEGFP-N1-IL-17,and the expression of Bcl-2 was significantly decreased.The difference was statistically significant(Caspase-3: F=289.52,P<0.01;Caspase-9: F=20.70,P<0.01;Bcl-2: F= 287.05,P<0.01).Cell migration test and Transwell chamber migration and invasion assay showed that IL-17 could significantly inhibit the migration and invasion of Hep-2 cells.In order to further explore the mechanism of IL-17,the expression of MMP-2 and MMP-9 was detected by RT-PCR.The expression of MMP-2 and MMP-9 in pEGFP-N1-IL-17 group was significantly decreased than that in normal control group(MMP-2: F=223.52,P<0.05;MMP-9: F=722.46,P<0.05).Western blotting was used to detect the expression of Notch1,Hey1 and Hes1 in Notch signaling pathway.The results showed that the expression of Notch1,Hey1 and Hes1 in pEGFP-N1-IL-17 group was significantly higher than that in normal control group(Notch1: F = 176.68,P <0.01;Hey1: F=265.96,P<0.01;Hes1: F=131.11,P<0.01).Conclusion: 1 There was no IL-17 expression in normal Hep-2 cells.Transfection of IL-17 gene can inhibit the proliferation of Hep-2 cells and promote the apoptosis of Hep-2 cells.This may be because that IL-17 can promote the expression of Caspase-3 and Caspase-9 while inhibiting the expression of Bcl-2.2 IL-17 inhibits the lateral migration,longitudinal migration and invasion of Hep-2 cells by inhibiting the expression of MMP-2 and MMP-9.3 The functional effect of IL-17 on human laryngeal carcinoma Hep-2 cells may through the function of the Notch signaling pathway.