The Expression And Significance Of RIPK4 In Non-small Cell Lung Cancer

Objective:To investigate the expression of RIPK4(receptor-interacting protein kinase 4)protein in non-small cell lung cancer(NSCLC)and its relationship with cyclins,and to reveal the relationship between RIPK4 with lung cancer cell proliferation.Methods:111 cases of non-small cell lung cancer tissue paraffin blocks were obtained from the first affiliated hospital of China Medical University from 2013 to 2015.All the paraffin blocks were removed before operation.All the patients had not received any chemical or radiotherapy before surgery.Specimens were fixed with 10% formalin,paraffin embedded.Second,the main reagents and sources,rabbit anti-human RIPK4 polyclonal antibody was purchased from Santa Cruz company;SP immunohistochemistry kit,DAB enzyme substrate color kit were purchased from Fuzhou Mai new biotechnology development company;RIPK4 plasmid and interference Fragments were purchased from Origene.Third,the method of immunohistochemical staining(SP method)detection of 111 cases of non-small cell lung cancer RIPK4 expression and localization.Cell culture In this study,human lung cancer cell lines A549,H1299,HBE,SK,292,H661 and H460 were cultured in DMEM or RPMI1640 containing 10% fresh calf serum in a 5% CO2 saturated incubator.Finally,H1299 of the medium levels of expression was selected to carry out the experiment.The combined use of plasmid transfection technique up-regulated the expression of RIPK4 in lung cancer cell lines H1299,and observed the expression level of cell Cyclins by Western blot and Real-time PCR.Similarly,si RNA interference was used to down-regulate the expression of RIPK4 in lung cancer cell lines H1299,observe the changes of Cyclins and analyze the relationship between RIPK4 expression and cell proliferation.In addition,functional changes were detected by MTS technology and cell colony assay to further analyze and verify the relationship between RIPK4 expression and tumor cell proliferation.Fourth,statistical analysis,using SPSS(PASW)l7.0 statistical analysis software,the relationship between RIPK4 expression and clinicopathological factors by t test to a = 0.05 level,P <0.05 was considered statistically significant.Results:1.Immunohistochemistry was used to detect the expression of RIPK4 protein in 111 non-small cell lung cancer tissues and to prove its localization in the cytoplasm.2.The expression of RIPK4 in lung cancer cell lines such as A549,H1299,HBE,SK,292,H661 and H460 was detected by Western blot.The medium expression of H1299 was selected for the experiment.3.Overexpression of RIPK4,Cyclin D1 and other cyclins in H1299 cell line also increased.Similarly,RIPK4 was disrupted in H1299 cell line behave in the opposite way.4.In vitro functional experiments MTS showed that RIPK4 plasmid transfected,the proliferation of tumor cells increased significantly higher than the control group.Cell colony assay also showed that,compared with the control group transfected RIPK4 plasmid group,tumor cell proliferation stronger than the control group;while,compare with the disrupted RIPK4 group with the control group,tumor cell proliferation lower than the control group.Conclusion:1.RIPK4 is expressed in lung cancer tissues and localized in the cytoplasm.RIPK4 was high expression in adenocarcinoma.2.RIPK4 effects and positively correlates with cyclin.RIPK4 overexpression may promote the proliferation of tumor cells.