CLDN4 Inhibits Migration And Invasion Of Laryngeal Carcinoma Hep-2 Cells Via Regulating JNK-MMPs Signaling Pathway

At present,the occurrence and development of laryngeal cancer are regulated by various factors,among which the changes of tight junction structure or function play an important role in tumor metastasis and invasion.The changes in the expression of tight junction protein CLDNs affect tumor migration and invasion by regulating related signaling pathways.The PDZ domain at the carboxyl terminus of CLDNs is one of the common peptide binding domains in proteins.This domain is known for its flexibility in ligand selection,the mode of interaction with its ligands,and its association with other protein interactions.Due to the diversity of functions,it can bind various types of signaling proteins,thereby regulating related signaling pathways to affect tumor development,such as binding to the JNK protein in the MAPK family,and the JNK signaling pathway is activated to act on its downstream target genes,Target genes such as MMPs,c-jun,and P53,in particular,affect the expression and activity of related MMPs,which in turn affect cell proliferation,migration,and invasion.However,the specific mechanism of how CLDNs act on their downstream MMPs-related target genes through the JNK signaling pathway to affect cell migration and invasion phenotypes is currently unclear.In the previous work,we found that demethylation treatment can suppress the malignant phenotype of Hep-2 cells by up-regulating CLDN4 expression.However,the specific mechanism of CLDN4 affecting Hep-2 cell migration and invasion is not yet clear.Related studies have shown that some changes in CLDNs expression can mediate the occurrence and development of related tumors by affecting the JNK pathway.MMPs are a very important family of zinc-dependent enzymes.They play a role in promoting tumorigenesis and invasion through various mechanisms such as degrading ECM and disrupting cell adhesion.Many CLDNs can mediate the activation of pro-MMP2 and pro-MMP9,thereby promoting tumor invasion and metastasis.According to current research reports,it is found that JNK-MMPs signaling pathway plays a potential role in the migration and invasion of different types of tumors.However,it is not clear whether the high expression of CLDN4 in laryngeal cancer cells inhibits the migration and invasion of Hep-2 cells related to the JNK-MMPs signaling pathway.Objective:To explore the role of JNK-MMPs signaling pathway in the up-regulation of CLDN4 expression to inhibit the migration and invasion of laryngeal cancer Hep-2cells,and to provide a certain experimental basis for future targeted drug treatment of laryngeal cancer.Method:1.Immunohistochemical staining technique was used to detect the expression of CLDN4,P-JNK,MMP2 and MMP9 in human laryngeal carcinoma tissues.2.RT-PCR and Western-Blot technology to detect CLDN4 expression in Hep-2cells.3.Stably transfect Hep-2 cells with lentivirus carrying CLDN4 to obtain cells(clone group)stably expressing CLDN4,and identify the transfection efficiency of lentivirus by RT-PCR and Western-Blot technology.4.Cell scratches and Transwell cell experiments were used to detect the effect of overexpression of CLDN4 on cell migration and invasion.5.Western-Blot detects the effect of overexpression of CLDN4 on the expression of JNK,P-JNK,MMP2 and MMP9 in the cell;ELISA technology detects the effect of overexpression of CLDN4 on the expression of MMP2 and MMP9 in the cell secreted supernatant.6.Western-Blot technology detects the optimal concentration and time of AM to activate JNK in overexpression of CLDN4 cells.7.Western-Blot technology was used to detect the effect of AM on the expression of JNK,P-JNK,MMP2 and MMP9 in the cell;ELISA technology was used to detect the effect of AM on the expression of MMP2 and MMP9 in the cell supernatant.8.Cell scratches and Transwell chamber experiments were used to detect the effect of AM on the migration and invasion ability of overexpression of CLDN4 cells.Result:1.Immunohistochemical staining showed that CLDN4 expression was significantly down-regulated(X~2=14.519,P<0.05),and P-JNK,MMP2 and MMP9expressions were significantly up-regulated(X~2=8.571,P<0.05;X~2=6.231,P<0.05;X~2=8.248,P<0.05).Spearman correlation analysis showed that CLDN4 expression was negatively correlated with P-JNK,MMP2 and MMP9 expression in human laryngeal carcinoma tissues(r_s=-0.259,P<0.05;r_s=-0.241,P<0.05;r_s=-0.260,P<0.05),and P-JNK expression was positively correlated with the expression of MMP2 and MMP9(r_s=0.395,P<0.05;r_s=0.354,P<0.05).2.RT-PCR and Western-Blot results showed that compared with Hela cells in the control group,CLDN4 expression in Hep-2 cells was lower at both mRNA and protein levels(P<0.05).3.After lentivirus transfection,stable clones overexpressing CLDN4 were obtained;RT-PCR and Western-blot results showed that compared with the empty group,the mRNA and protein levels of CLDN4 in the cloned group cells were significantly increased(P<0.05).4.The results of cell scratches and Transwell cell experiments showed that the migration and invasion ability of cells in the overexpressed CLDN4 group was significantly reduced compared with the empty group(P<0.05).5.Western-Blot results showed that compared with the empty group,the expression of P-JNK,MMP2 and MMP9 in overexpressed CLDN4 cells were significantly down-regulated(P<0.05);ELISA results showed that compared with the empty group,the expressions of MMP2 and MMP9 in the secreted supernatant of overexpressed CLDN4 group cells were significantly down-regulated(P<0.05).6.Western-Blot results showed that the optimal concentration of AM was 2?M,and the optimal time was 24h(P<0.05).7.Western-Blot results showed that the expression of P-JNK,MMP2 and MMP9in cells were significantly up-regulated after AM was applied to CLDN4overexpressing group(P<0.05);ELISA results showed that the expression of MMP2and MMP9 in cell secreted supernatant were significantly up-regulated after AM was applied to CLDN4 overexpressing group(P<0.05).8.The results of cell scratches and Transwell cell experiments showed that after AM was applied to cells in the overexpressed CLDN4 group,the cell migration and invasion ability was significantly enhanced.Conclusion:1.Low expression of CLDN4,high expression of P-JNK,MMP2 and MMP9 in laryngeal carcinoma tissues,and CLDN4 expression is correlated with the expression of P-JNK,MMP2 and MMP9.2.Low expression of CLDN4 in Hep-2 cells,and overexpression of CLDN4obviously inhibits the migration and invasion ability of Hep-2 cells.3.The role of CLDN4 in inhibiting Hep-2 cell migration and invasion is related to the JNK-MMPs pathway.