CLDN4 Inhibits Proliferation Of Laryngeal Carcinoma Hep-2cells Via JAK2/STAT3 Signaling Pathway

The development of laryngeal carcinoma is regulated by many factors,among which the tight junction proteins(CLDNs)family plays an important role.CLDNs are the main components of tight junctions(TJs).Early studies have shown that CLDNs have cell barrier and palisade functions.Abnormal expression of CLDNs affects the structure or function of TJs.In recent years,studies have found that CLDNs also play an important role in cell signal transduction.CLDNs contain a carboxyl terminal which have PDZ binding domain and potential phosphorylation sites,which can bind to corresponding signal proteins and regulate cell biological activity.For example,that can bind to proteins containing PDZ structure,such as ZO-1;Tyrosine on the carboxyl terminal of tight junction protein family members can be phosphorylated binding sites.JAKs/STATs(Janus kinase/activator of signal transduction)are involved in cell proliferation,differentiation and apoptosis.When the receptors on the cell surface bind to their corresponding ligands,JAKs are activated,and then the downstream protein stats are stimulated to phosphorylate,forming dimer structure and transferring into the nucleus,changing the transcription of corresponding genes.Studies have found that JAK2/STAT3 signaling pathway is abnormal in a variety of tumor cells,and affect its malignant phenotype.In our previous experiments,we found that CLDN4 was low expressed in laryngeal carcinoma.Overexpression of CLDN4 can inhibit the migration and invasion of Hep-2 cells,but its effect on cell proliferation and its mechanism still need to be studied.Many studies have found that CLDNs regulate JAKs/STATs signaling pathway affect the malignant phenotype of tumor cells.For example,CLDN6 inhibits the migration and invasion of breast cancer cells through JAK2 / STAT3 signaling pathway.CLDN9 promotes the proliferation of hepatoma cells through TYK2 / STAT3 signaling pathway.CLDN 12 promotes the metastasis of lung cancer cells through TYK2 / STAT1 signaling pathway.Therefore,we speculate that CLDN4 may affect the proliferation of laryngeal cancer Hep-2 cells,and may be related to JAK2/STAT3 signaling pathway,but its specific mechanism is not clear.Objective:To explore the effect of CLDN4 on the Hep-2 cells and its relationship with JAK2/STAT3 signaling pathway,which provides a new idea for clinical targeted therapy of laryngeal cancer.Method:1.Immunohistochemical staining experiment to detect the expression of CLDN4,p-JAK2 and p-STAT3 in 90 cases of laryngeal carcinoma and 70 cases of adjacent tissues;2.Western-Blot technology identifies the expression of CLDN4 in Hep-2 cells in the CLDN4 overexpression group;3.CCK8,Ed U and two-dimensional plate clone formation experiments were used to detect the changes in the proliferation ability of Hep-2 cells in the CLDN4 overexpression group;4.Western-Blot technology detects the expression of JAK2,p-JAK2,STAT3,and p-STAT3 in Hep-2 cells in the CLDN4 overexpression group;5.Western-Blot technology detects the optimal concentration and time of AG490 on Hep-2 cells in the CLDN4 overexpression group;6.Western-Blot technology to detect the effect of AG490 on the expression of pJAK2,JAK2,STAT3 and p-STAT3 in Hep-2 cells in the CLDN4 overexpression group;7.CCK8 experiment,Ed U experiment and two-dimensional plate clone formation experiment to detect the effect of AG490 on the proliferation of Hep-2 cells in the CLDN4 overexpression group.Result:1.Immunohistochemical staining experiments showed that CLDN4,p-JAK2 and p-STAT3 were lower expressed in laryngeal cancer tissues compared with adjacent tissues;Spearman correlation analysis showed that CLDN4 is positively related to pJAK2 and p-STAT3 in human laryngeal cancer tissues;2.Western-Blot results confirmed that CLDN4 was highly expressed in Hep-2cells in the CLDN4 overexpression group compared with the no-load group;3.CCK8,Ed U and two-dimensional plate clone formation experiments showed that the proliferation of Hep-2 cells in the CLDN4 overexpression group was inhibited compared with the empty group;4.Western-Blot results showed that compared with the no-load group,the expression of p-JAK2 and p-STAT3 in Hep-2 cells with CLDN4 overexpress was significantly increased,while the expression of JAK2 and STAT3 did not change significantly;5.Western-Blot results showed that the best concentration of AG490 on Hep-2cells with CLDN4 overexpress was 50μM,and the best time of action was 24 hours;6.Western-Blot experiments showed that compared with the control group,the expressions of p-JAK2 and p-STAT3 in the AG490 treatment group decreased significantly,while the expressions of JAK2 and STAT3 did not change significantly;7.The results of CCK8,Ed U and two-dimensional plate clone formation experiments showed that compared with the control group,the cell proliferation ability of the AG490 treatment group was enhanced.Conclusion:1.CLDN4,p-JAK2 and p-STAT3 are low expressed in laryngeal carcinoma tissues,and CLDN4 is positively correlated with the expression of p-JAK2 and p-STAT3.2.CLDN4 inhibits Hep-2 cell proliferation and is related to JAK2/STAT3 signaling pathway.