The Effect Of Melatonin In IL-1β Mediated Epithelial Mesenchymal Transition In Human Gastric Adenocarcinoma Cells

Objective: To investigate the inhibitory effect of melatonin(MLT)on IL-1β mediated epithelial-mesenchymal transition(EMT)in gastric adenocarcinoma cells and its mechanism.Methods: 1.Human gastric adenocarcinoma cells MGC80-3 and SGC-7901 were used as the research subjects,and a blank control group,IL-1β group and IL-1β+ different concentrations of MLT were established.2.Cell invasion and migration were measured by Transwell assay.3.Detection of EMT biomarker molecules by Real-time fluorescence quantitative PCR,such as epithelial cell markers(β-catenin、E-cadherin)and mesenchymal cell markers(Fibronectin 、 Vimentin 、 Snail)and matrix metalloproteinases(MMP-2 and MMP-9))mRNA expression levels.4.The protein expression levels of MMP-2 and MMP-9 were detected by Western blot.5.The transcription factor NF-κB transcriptional activity was examined by gel migration assay(EMSA).6.The role of NF-κB in IL-1β-induced gastric adenocarcinoma cell line EMT was verified by transfection of NF-κB p65 siRNA,and the potential mechanism of action of NF-κB in MLT inhibition of the above effects.7.Human gastric adenocarcinoma MGC80-3 cells were injected into tail vein to establish gastric adenocarcinoma lung metastasis model in nude mice.After the cancer cells were planted for a week,nude mice were randomly divided into three groups,namely saline control group,IL-1β injection and IL-1β+MLT injection group.The lung metastasis of gastric adenocarcinoma cells was observed by HE staining.The mRNA expression level of EMT biomarker molecules in lung tissue was detected by RT-qPCR.The protein expression levels of MMP-2 and MMP-9 in lung tissue were detected by immunohistochemistry.Results:1.In vitro cell experiment1.1Melatonin inhibits IL-1β-induced epithelial-mesenchymal transition in gastric adenocarcinoma cells:(1)After IL-1β treatment,the cell morphology of human gastric adenocarcinoma cells MGC80-3 and SGC-7901 changed,and the original irregular triangle or tetragonal epithelial cell morphology changed into long fusiform mesenchymal morphology,but these morphological changes could be inhibited by MLT.(2)After treatment with IL-1β,the number of invasion and migration of MGC80-3 and SGC-7901 cells increased significantly,and after MLT treatment,the number of cells invading and migrating was significantly reduced,but there was no concentration-dependent.(3)After RT-qPCR detection,IL-1β treatment significantly down-regulated the mRNA expression levels of epithelial cell markers β-catenin and E-cadherin in gastric adenocarcinoma cells,and significantly up-regulated the mRNA expression levels of mesenchymal cell markers Fibronectin、 Vimentin and Snail.MLT can significantly inhibit the effect of IL-1β on the transcription level of the above genes.1.2 MLT down regulates the expression of MMP-2 and MMP-9 in gastric adenocarcinoma cells: IL-1β significantly up-regulated the mRNA and protein expression levels of MMP-2 and MMP-9 in gastric adenocarcinoma cells MGC80-3 and SGC-7901.MLT significantly inhibited the above effects of IL-1β,but not in a concentration-dependent manner.1.3 MLT inhibits IL-1β-activated NF-κB transcriptional activity: The results of EMSA experiments showed that IL-1β stimulation can enhance the transcriptional activity of NF-κB in gastric adenocarcinoma cells MGC80-3 and SGC-7901,and this effect can be inhibited by MLT.1.4Knockdown of NF-κB expression attenuates IL-1β-induced gastric adenocarcinoma cell EMT:Transfection of NF-κB p65 siRNA significantly inhibited the expression of NF-κB,and the inhibition rate reached more than 75%.Compared with the transfected scramble siRNA group,the migration and invasion abilities of gastric adenocarcinoma cells MGC80-3 and SGC-7901 were significantly increased in the transfected scramble siRNA+IL-1β treatment group,while transfected with NF-κB p65 siRNA+IL-1β treatment group.The migration and invasion ability of gastric adenocarcinoma cells is significantly reduced;This result indicates that NF-κB and IL-1β are closely related to the induction of EMT in gastric adenocarcinoma cells.RT-qPCR and Western Blot results showed that mRNA expression levels of β-catenin and E-cadherin in epithelial cell markers transfected with scramble siRNA gastric adenocarcinoma cells were significantly decreased after IL-1β treatment;andthe mRNA expression levels of mesenchymal cell markers Fibronectin、Vimentin、Snail,as well as the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly enhanced.Transfection of NF-κB p65 siRNA significantly down regulated the mRNA expression of the above-mentioned mesenchymal cell marker genes and the mRNA and protein expression levels of MMP-2 and MMP-9 in gastric adenocarcinoma cells.2.In vivo animal experiment2.1 MLT can effectively inhibit lung metastasis of gastric adenocarcinoma in nude mice:The incidence of lung metastasis of gastric adenocarcinoma was 50% in 6 nude mice of normal saline control group and the average number of lung tumor nodules in nude mice with lung metastasis of gastric adenocarcinoma was 5;The incidence of lung metastasis of gastric adenocarcinoma in nude mice with IL-1β injection was 100%,and the average number of lung tumor nodules in nude mice with lung metastasis of gastric adenocarcinoma was 9;The incidence of lung metastasis of gastric adenocarcinoma was 33.3% in the IL-1β+MLT injection group,and the lung tumor nodules in nude mice with gastric adenocarcinoma lung metastases were 2 on average.The above results indicate that IL-1β can promote lung metastasis of gastric adenocarcinoma,and MLT can inhibit tumor metastasis.2.2 MLT inhibits EMT-related genes and MMP-2 and MMP-9 expression levels in lung tissues of nude mice:IL-1β significantly down-regulated the mRNA expression levels of epithelial cell markers β-catenin and E-cadherin in lung tissues of nude mice,and up-regulated the mRNA expression levels of mesenchymal cell markers fibronectin、Vimentin and Snail in lung tissue,as well as MMP-2 and MMP-9 mRNA and protein levels.The use of MLT inhibits the above effects of IL-1β.The above results further indicate that IL-1β promotes EMT and MLT inhibits EMT from molecular marker levels.Conclusion: NF-κB and IL-1β are closely related to the induction of EMT in gastric adenocarcinoma cells;MLT can significantly inhibit the EMT process of gastric adenocarcinoma cells and reduce the ability of cell invasion and metastasis.The mechanism may be related to the inhibition of NF-κB transcriptional activity and MMP-2 and MMP-9 expression.