ZCCHC14 Regulates Proliferation And Invasion Of NSCLC Through The MAPK-P38 Signalling Pathway

The incidence of lung cancer have always been in the forefront of global malignant tumors.Non-small cell lung cancer(NSCLC)accounted for 85% of the total number of lung cancers.Patients with advanced stage lung cancer or brain metastases have a poor prognosis.In this study,we mainly involved samples from patients with NSCLC,and in addition,we introduced samples from patients with brain metastases.Using Meta analysis associated with whole genome analysis,researchers screened the loss of ZCCHC14 may lead to nicotine dependence(ND)susceptibility and the reason of alcohol addiction and analysis to identify new genetic mutations associated with stroke,found ZCCHC14 may exist correlation with small vessel stroke,so do not rule out ZCCHC14 play a role in the process of development of lung cancer the and cerebrovascular formation and management.However,the function of ZCCHC14 in cancer tissues have not been studied.In this study,we mainly studied the role and significance of ZCCHC14 expression in human lung cancer tissues and brain metastases tissues.There are few studies on CCHC-type protein in tumors.Only a few studies have shown that CCHC-type zinc finger protein plays a biological role in regulating MAPK signalling pathway in tumor tissues.ZCCHC14 also belongs to CCHC-type zinc finger protein containing zinc finger motif,and its possible participation in signalling pathway has not been studied.Therefore,in the following study,we will confirm whether ZCCHC14 regulates MAPK signalling pathway and which subfamily regulation mechanism,this is also the focus of the next experiment.In the verification process,the inhibitor Doramapimod(BIRB796),which entered the national clinical phase III trial,was introduced to further verify the molecular mechanism of ZCCHC14 in the occurrence of NSCLC.Objective: To investigate the expression and significance of ZCCHC14 in NSCLC and brain metastasis,as well as its function and mechanism in NSCLC cell proliferation and invasion.Methods: Immunohistochemistry was specifically used to detect the expression of ZCCHC14 in NSCLC cancer tissues and brain metastases tissues.SPSS software was mainly utilized to analyze the correlation between ZCCHC14 and clinical factors of lung cancer.The relationship between ZCCHC14 and prognosis of lung cancer patients was analyzed by combining lung cancer patient sample data and KM Plotter database.SPSS software was to analyze the correlation between ZCCHC14 and NSCLC cancer with brain metastases.Promoter methylation levels of ZCCHC14 in lung squamous cell carcinoma and lung adenocarcinoma,common types of NSCLC,were measured in the UALCAN database.The localization of ZCCHC14 was detected by immunofluorescence assay.Western blot detected the protein expression of ZCCHC14 in lung cancer cells.By transfecting ZCCHC14 or ZCCHC14-KO plasmids in cells,the expression of each subfamily and downstream related molecular proteins in MAPK signalling pathway was detected by western blot assay.The proliferation and invasion of NSCLC cells transfected with ZCCHC14 or ZCCHC14-KO plasmids were investigated by transwell and colony formation assay.Transwell and colony formation assay were applied to investigate the effects of ZCCHC14 expression on proliferation and invasion ability of NSCLC cells transfected with or without ZCCHC14-KO plasmids.After adding inhibitors of MAPK-P38 signalling pathway into non-small cell lung cancer cells transfected with and without ZCCHC14-KO plasmids,we detected the effects of ZCCHC14 on the regulation of MAPK-P38 signalling pathway and the MMP7 and Cyclin D1 expression by western blot,to further explore the mechanism of ZCCHC14.The effect of ZCCHC14 on tumorigenicity of mice was detected by tumor formation experiment.Results : 1.ZCCHC14 is expressed in the nuclei and cytoplasm of tumor adjacent tissues,but its expression is weakened or absent in NSCLC patients.2.The abnormal expression of ZCCHC14 in cancer tissues was significantly correlated with the degree of differentiation,TNM stage and gender(P<0.05).3.The survival curve revealed that the survival time of patients with low ZCCHC14 expression in cancer tissue was significantly shorter than that of patients with high ZCCHC14 expression(P = 0.023).KM Plotter database revealed that the survival rate of NSCLC patients with high ZCCHC14 m RNA expression was higher than that of NSCLC patients with low ZCCHC14 m RNA expression(P<0.001).4.The rate of low expression of ZCCHC14 in NSCLC patients with brain metastases was statistically higher than that in tissues without brain metastases,the difference was statistically significant(P<0.05).5.Promoter methylation of ZCCHC14 was significantly enhanced in NSCLC.6.ZCCHC14 was expressed in both HBE and lung cancer cells,and was decreased in LK2,H460 and H292 cells to varying degrees.Immunofluorescence staining showed that ZCCHC14 was expressed in both the nucleus and cytoplasm of the cells.7.The results of transwell assay and colony formation assay indicated that down-regulation of ZCCHC14 significantly improved the invasion ability of cancer cells.8.After transfection of ZCCHC14-KO plasmids in A549 and H1299 cells,p-P38 and P38 molecular downstream signalling molecules including Cyclin D1 and MMP7 were significantly increased.9.After transfection of ZCCHC14 plasmids in LK2 cells,p-P38 were significantly decreased,the invasion and proliferation ability of cancer cells were reduced.10.The results of colony formation assay and transwell assay indicated that the addition of P38 signaling pathway inhibitor BIRB796 in cells could eliminate the effects of adding ZCCHC14-KO plasmids on cell proliferation and invasion.11.Western blot analysis showed that transfection of ZCCHC14-KO plasmids promoted the change of P38 pathway and downstream related molecular proteins,which was abolished by the inhibitor BIRB796.12.ZCCHC14 could regulate p-PI3 K protein expression.13.Vivo experiments showed that ZCCHC14 could induce changes in tumorigenicity.Conclusion: 1.The expression of ZCCHC14 is weakened or absent in NSCLC.2.The expression of ZCCHC14 in lung cancer is significantly correlated with gender,differentiation,TNM stage,prognosis and brain metastases.3.ZCCHC14 could inhibit the proliferation and invasion of lung cancer cells.4.ZCCHC14 can regulate the proliferation and invasion of lung cancer cells through MAPK-P38 signalling pathway.5.ZCCHC14 affects the P38 signalling pathway by regulating phosphorylation of PI3 K.6.ZCCHC14 can regulate the tumorigenicity of mice.