| Objective: Gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer-related deaths.Most patients with gastric cancer are diagnosed at an advanced stage because early gastric cancer is usually asymptomatic,and metastatic recurrence of gastric cancer often occurs even after radical resection.The most common cause of treatment failure after gastric cancer surgery is peritoneal spread,which is mainly caused by the seeding of free cancer cells from the primary gastric cancer.Gastric cancer patients with peritoneal metastatic cancer that are visible to have a poor prognosis.The connection between inflammation and cancer has been well known.At present,it has been found that tumor cells in the abdominal cavity caused by exfoliation will preferentially colonize milk spots,which are mainly composed of immature macrophages.The purpose of our study is to correlate the relationship between inflammation and cancer,and to prove that macrophages can form a TNF-?-rich inflammatory microenvironment when they interact with tumor cells,and pass MZF-1/Cx43 pathway leads to an increase in the proportion of gastric cancer stem cells,thereby promoting the peritoneal metastasis of gastric cancer cells.The aim is to provide new insights into the mechanism of gastric cancer cells undergoing peritoneal metastasis and further provide a theoretical basis for clinical development of anti-gastric cancer peritoneal metastases.Methods: Transwell invasion,migration and scratch tests were used to detect the cell function of Cx43 in gastric cancer cell line HGC-27.To analyze the correlation between the expression of Cx43 and the occurrence of peritoneal metastasis by immunohistochemical staining.After overexpressing Cx43,the relationship between Cx43 and dryness related indicators Sox2,Nanog and Oct4 was detected by Western Blot experiments.Co-culture macrophages and gastric cancer cells to simulate the interaction between intra-abdominal plaques and free tumor cells,and then detect the expression of Cx43 and stem-related indicators in gastric cancer cells by Western Blot experiments.Coculture after knocking down Cx43 to verify the role of Cx43 in between.The TNF-? level in the co-culture medium was detected by Elisa experiment.Detection of TNF-? mRNA levels in macrophages by qRT-PCR experiments.After adding TNF-? to stimulate cells,the changes of Cx43 and dryness related indicators were detected by Western Blot test.After adding TNF-? to stimulate cells,test the spheroidizing ability of tumor stem cells.After knocking down Cx43,add TNF-? stimulation again to verify the role of Cx43 in the process.After knocking down MZF-1,perform Western Blot and qRT-PCR experiments to detect the expression of Cx43.MZF-1 protein expression was detected by Western Blot experiment under TNF-? stimulation.After knocking down MZF-1,coculture or stimulation with TNF-? was added to verify the role of MZF-1 in TNF-? and co-culture regulation of Cx43 and tumor stemness.Results: 1.The results of the Transwell migration,invasion test,and scratch test showed that Cx43 up-regulation can promote the invasion and migration of cancer cells.Immunohistochemical results showed that the expression of Cx43 protein was closely related to the occurrence of peritoneal metastasis in gastric cancer.2.Western Blot results showed that the protein expression of Sox2,Nanog and Oct4 was up-regulated after Cx43 overexpression.3.Western Blot results showed that HGC-27 and MKN-45 co-cultured with macrophages had higher expression levels of Cx43 and dryness indicator proteins,and after knocking down Cx43,co-cultured tumors originally caused by macrophages Increased cell stemness is suppressed.4.The results of the Elisa experiment showed that compared with the culture medium of tumor cells HGC-27,MKN-45 and cultured macrophages alone,the levels of TNF-? in the co-culture medium were increased.qRTPCR results showed that the expression of TNF-? mRNA was increased in co-cultured macrophages.5.Western Blot results showed that the expression of Cx43,Sox2,Oct4,and Nanog proteins in gastric cancer cells gradually increased under the stimulation of TNF-?.The results of tumor stem cell spheroidization experiments showed that the size and number of tumor spheroids increased significantly under the TNF-? stimulation.6.After knocking down MZF-1,Western Blot and qRT-PCR results showed that Cx43 expression was down-regulated.Western Blot results showed that the expression of MZF-1 protein was up-regulated under TNF-? stimulation,and the knockdown of MZF-1 downregulated the expression of Cx43 and gastric cancer stem cells when co-cultured or stimulated with TNF-?.Conclusion: 1.The Cx43 gene may play a role as an oncogene in gastric cancer cells and is related to tumor stemness.It appears to promote the occurrence of peritoneal metastasis in patients.2.When free gastric cancer cells are exposed to milky spot macrophages,macrophages can secrete TNF-?,and TNF-? can enhance the stemness of tumor cells by up-regulating the expression of Cx43.3.TNF-? can regulate Cx43 expression through MZF-1. |
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