| Objective:The invasion and metastasis of tumors are not only related to the abnormalities of tumor cells,but also the microenvironment in which they are located.The fibroblasts adjacent to tumor cells called cancer-associated fibroblasts(CAFs)are the most important stromal cells in the tumor microenvironment.The interaction between CAFs and tumor cells plays an important role in tumor progression.We plan to analyze the role of CAFs with high expression of MLCK or secreted excessive IL-6 in the promotion of gastric cancer,and try to illuminate the cross-talk between cancer cells and their microenvironment from the perspective of tumor microenvironment.Methods:1)Isolation and culture the tumor-associated fibroblast(CAFs)from gastric cancer and normal fibroblasts(NFs)from adjacent normal tissues,which were the basis for subsequent studies.2)Protein levels of IL-6 in gastric cancer tissues and cells were tested by ELISA.The localization in tissue was detected by immunofluorescence.3)Gastric cancer cells were stimulated with CAFs and exogenous IL-6.The changes of metastatic ability were measured by Transwell assay.Western blot was used to detect the protein level of EMT markers.4)IL-6 secreted by CAFs was blocked by the neutralizing antibody.Then,the effects of CAFs on gastric cancer cell metastasis and EMT,and the activation of downstream JAK2/STAT3 signaling pathway were detected.5)The JAK2 inhibitor AG490was used to inhibit the signal pathway activated by IL-6,and the effect of CAFs on gastric cancer cell metastasis and EMT were determined.6)After down-regulation of IL-6expression in CAFs by IL-6 siRNA or inhibition CAFs-secreted IL-6-activated JAK2/STAT3 signaling pathway by AG490,the number of peritoneal metastatic nodules generated by gastric cancer cells in nude mice was determined.7)The expression levels of MLCK in gastric cancer tissues and cells was detected by IHC and Western blot,and the localization of MLCK in CAFs and NFs was analyzed by IF.8)The expression levels of MLCK in CAFs were down regulated by siRNA,and then CAFs were co-cultured with gastric cancer cells and the effects on invasion,metastasis ability and EMT of gastric cancer cells were observed.9)After co-cultured with CAFs down-regulated expression of MLCK,the translocation and accumulation of?-catenin in gastric cancer cells were measured.11)The growth of subcutaneous tumors and the formation of peritoneal metastatic nodules in nude mice were determined after inoculation CAFs and gastric cancer cells in the subcutaneous and peritoneal cavity of mice.Results:1)We isolated and cultured gastric cancer-associated fibroblast CAFs and adjacent normal control fibroblast NFs,and confirmed that the expression of?-SMA,FAP and vimentin in CAFs was significantly higher than that in NFs.2)The level of IL-6 expression was significantly higher in gastric cancer tissues than that in adjacent normal tissues.Immunofluorescence staining showed that IL-6 expression was mainly co-localized with?-SMA~+stromal cells in gastric cancer tissues.Furthermore,the expression level of IL-6 in CAFs was significantly higher than NFs or other gastric cancer cell lines.3)Stimulated gastric cancer cells with CAFs or exogenous IL-6,the metastasis ability of gastric cancer cells was increased,and the expression of EMT markers such as?-catenin,?-catenin,N-cadherin,SLUG,SNAIL and ZEB2 was elevated,while the expression levels of E-cadherin was inhibited.4)Adding neutralizing IL-6 antibody into the co-culture system led to significantly decreased migration of gastric cancer cells,and the expression of EMT markers such as N-cadherin and ZEB2 was suppressed,while the decrease of E-cadherin triggered by CAFs was restrained.In addition,the expression of p-STAT3 and p-JAK2 was significantly decreased in the presence of IL-6 neutralizing antibodies.5)The expression of p-STAT3 and p-JAK2 were significantly decreased when adding the inhibitor AG490 into the co-culture system.The effect of CAFs on the migration and EMT of gastric cancer cells was inhibited.6)The expression of IL-6 in CAFs was down-regulated by siRNA.CAFs-siIL-6 was co-inoculated into the peritoneal cavity of mice with gastric cancer cells.The number of peritoneal metastatic nodules in the mice in CAFs-siIL-6 group was significantly lower than that in the control group.AG490 significantly inhibited the formation of peritoneal metastatic nodules in mice compared with the control group injected with DMSO.7)The expression of MLCK in CAFs was significantly higher than that in gastric cancer cells.The expression levels of MLCK protein and mRNA in CAFs were higher than gastric cancer cells and NFs.8)The expression of MLCK in CAFs was knocked down by siRNA,and then CAFs-siMLCK was co-cultured with gastric cancer cells.In CAFs-siMLCK group,the number of invasive and migrated gastric cancer cells was significantly reduced,and the expression of N-cadherin and MMP2 was inhibited,while the expression of E-cadherin was significantly increased,compared with control group CAFs-siNC.9)Gastric cancer cells was stimulated with the CAFs-siMLCK supernatant,and expression level of?-catenin in gastric cancer cell nucleus was significantly lower than that in CAFs-siNC group.10)Co-inoculating gastric cancer cells and CAFs-siMLCK into the subcutaneous of mice,the growth rate and tumor formation size of gastric cancer cells was significantly lower than that of CAFs-siNC group.Moreover,the CAFs-siMLCK were co-inoculated with the gastric cancer cells into the peritoneal cavity of mice,and the number of peritoneal metastatic nodules was significantly lower than the control group.Conclusion:CAFs can promote EMT and metastasis of gastric cancer cells by secreting a large amount of IL-6,which mainly achieved through JAK2/STAT3 signaling pathway.In addition,CAFs with high expression of MLCK can promote the activation of Wnt/?-catenin signaling pathway in gastric cancer cells,and down-regulation of MLCK expression in CAFs inhibits the invasion and metastasis of gastric cancer cells induced by CAFs. |
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