| Objective: Esophageal cancer is the fourth most malignant tumor in the world and the sixth most common cause of cancer death,which poses a great threat to human health.Patients with esophageal cancer are often diagnosed in local advanced stage,and with poor effect of surgical treatment.Many patients eventually occur recurrence and metastasis.Methods: CAFs and NFS cells were isolated and cultured from esophageal squamous cell carcinoma and normal tissues to observe the effect of CAFs exosomes on epithelial mesenchymal transformation(EMT)of esophageal squamous cell carcinoma cell line.RT-PCR was used to detect the content of LINC01410 in CAFs and NFS cells.Next,we detected the content of LINC01410 in extracted exosomes and TE-1 cells incubated with exosomes,analyzed the potential target of LINC01410 by bioinformatics analysis technology,and explored the molecular mechanism of LINC01410 in regulating tumor cell migration and inducing epithelial mesenchymal transformation(EMT)of tumor cells.Next,well-differentiated esophageal squamous cell TE-1 cells and low-differentiated esophageal squamous cell carcinoma Eca-109 cells were transfected with LINC01410-OE,mir-122-5p mimic and PKM2-OE.The transfection efficiency of LINC01410,mir-122-5p and PKM2 in TE-1 or Eca-109 cells was detected by q RT-PCR.Transwell test was used to detect the migration and invasion of tumor cells.Western blot was used to detect the expression of EMT related proteins in LINC01410-OE group,LINC01410-OE + mirnamic + PKM2 NC-OE group and LINC01410-OE + mi RNAmic + PKM2-OE group.Remedial experiments were conducted to further clarify the process of CAFs exosomes regulating EMT of tumor cells.Finally,a nude mouse model test was carried out to knock down LINC01410 and inhibit mir-122-5p,so as to verify the mechanism of LINC01410/ pkm2mir-122-5p/PKM2 axis regulating epithelial mesenchymal development of esophageal squamous cell carcinoma cells and regulating tumor progression.Results:1 CAFs exosomes promote the migration,invasion and epithelial mesenchymal of esophageal squamous cell carcinoma1.1 CAF exosomes promote metastasis of esophageal squamous cell carcinoma.We first studied the similarities and differences of fibroblasts in cancer tissues and adjacent normal tissues.The results confirmed that the cells in this study were successfully isolated and purified.The isolated primary cultured cells were human esophageal squamous cell carcinoma associated fibroblasts(CAFs)and normal fibroblasts(NFs).Next,we cocultured esophageal squamous cell carcinoma TE-1 cell line on the above two fibroblast conditioned media,and found that CAF could significantly improve the migration and invasion ability of esophageal squamous cell carcinoma TE-1cell line.The migration and invasion abilities of TE-1/CAF cells were significantly decreased after the addition of exosome inhibitors.The size and morphology of exosomes were observed by electron microscopy,the particle size distribution of exosomes was measured by nanoparticle tracking analysis,and the protein of exosomes was measured by Western blotting.The exosome uptake of esophageal squamous cell carcinoma TE-1 cell line was detected by laser confocal microscope.1.2 CAF exosomes promote epithelial mesenchymal of esophageal squamous cell carcinoma.Compared with TE-1 / NF,the expression of PKM2,snail and vimentin in TE-1 / CAF increased significantly,while the expression of E-cadherin decreased.2 CAFs secrete LINC01410 promotes epithelial mesenchymal transformation of esophageal squamous cell carcinoma cells2.1 The expression of LINC01410 in esophageal squamous cell tissues and fibroblast secretesRT-PCR was used to detect the content of LINC01410 in esophageal squamous cell carcinoma tissues and adjacent tissues.The results showed that the content of LINC01410 in esophageal squamous cell carcinoma tissues increased significantly compared with adjacent tissues.Next,we also detected the content of LINC01410 in cultured NFS and CAFs cells by RT-PCR.The results showed that the expression level of LINC01410 in CAFs was significantly higher than that in NFs.In addition,we detected the content of LINC01410 in exosomes derived from NFs and CAFs and TE-1 cells incubated with exosomes.The results showed that the expression level of LINC01410 in CAFs was significantly higher than that in NFs.2.2 CAFs exosome LINC01410 promotes esophageal squamous cell carcinoma metastasis and epithelial mesenchymalTranswell migration assay showed that overexpression of LINC01410 significantly promoted migration and invasion of TE-1 cells,while knockdown of LINC01410 significantly inhibited migration and invasion of TE-1 cells.Next,we cocultured CAFs exosomes with TE-1 cells.The results showed that the exosomes overexpressing LINC01410 significantly promoted the migration and invasion of TE-1 cells.In addition,it weakened the migration and invasion of TE-1 cells.Western blot showed that the expression of snail,vimentin and PKM2 proteins were up-regulated and the expression of E-cadherin was down regulated in LINC01410 overexpressing cells.Knockdown of LINC01410 increased the expression of E-cadherin and inhibited the expression of snail,vimentin and PKM2 protein.2.3 LINC01410 adsorbs mir-122-5p and promotes the expression of PKM2Bioinformatics analysis of the potential target of LINC01410 using Diana database showed that LINC01410 contains the potential site combined with mir-122-5p.Verification with double luciferase reporter gene showed that mir-122-5p significantly reduced the luciferase activity of LINC01410-WT group,but had no significant effect on the luciferase activity of LINC0-1410-MUT group.In addition,RIP results showed that LINC01410 and mir-122-5p were enriched in Ago2 of TE-1 cells.For downstream molecules involved in tumor cell migration and epithelial mesenchymal,PKM2 and mir-122-5p have potential binding targets.Similarly,through the double luciferase reporter gene,we showed that mir-122-5p significantly reduced the luciferase activity of PKM2-WT group,but had no significant effect on the luciferase activity of PKM2-MUT group.In addition,RNA pull-down results showed that the binding of mir-122-5p to PKM2 was significantly increased compared with the control.3 CAFs exosomes LINC01410 promote esophageal squamous cell carcinoma cell migration and epithelial mesenchymal through mir-122-5p / PKM2 axis3.1 CAFs exosomes promote esophageal squamous cell carcinoma migration through LINC01410 / mir-122-5p / PKM2.LINC01410-OE,mir-122-5p mic and PKM2-OE were transfected into well-differentiated esophageal squamous cell carcinoma cell line TE-1 and low-differentiated esophageal squamous cell carcinoma cell line Eca-109 for remedial experiments.q RT-PCR was used to detect the transfection efficiency and relationship of LINC01410,mir-122-5p and PKM2 in TE-1 or Eca-109 cells.The results showed that the expression of LINC01410,mir-122-5p and PKM2 increased in the transfected cells,and overexpression of LINC01410 could play the role of molecular sponge,adsorb mir-122-5p and reduce the expression of mir-122-5p.Transwell experiment was used to detect the migration and invasion of TE-1 and Eca-109 cells after remedial expression of LINC01410,mir-122-5p and PKM2.The results showed that the migration and invasion of cells in remedial expression of LINC01410 group were significantly increased,while remedial expression of mir-122-5p reversed the migration and invasion of cells.The above results show that LINC01410 in CAFs exosomes can promote the migration and invasion of tumor cells induced by PKM2 by adsorbing mir-122-5p.3.2 CAFs exosomes promote epithelial mesenchymal of esophageal squamous cell carcinoma through LINC01410 / mir-122-5p / PKM2.The contents of snail,vimentin,E-cadherin and PKM2 protein,the markers of epithelial mesenchymal of tumor cells were detected.The results showed that the expression of PKM2,snail and vimentin protein increased significantly and the content of E-cadherin protein decreased significantly in the group expressing LINC01410.After remedial expression of PKM2,the expression of PKM2,snail and vimentin protein increased significantly,and the content of E-cadherin protein decreased significantly.The same results were verified in the low-differentiated Eca-109 cell line.3.3 CAFs exosomes promote the migration and epithelial mesenchymal development of esophageal squamous cell carcinoma in mice through LINC01410 / mir-122-5p / PKM2Nude mice constructed the mouse model of esophageal squamous cell carcinoma by subcutaneous injection of stably transfected CAFs(NC-KD and LINC01410-KD)and TE-1(antigomir NC group and antigomir-mir-122)cells.After 40 days of subcutaneous transplantation,the average tumor volume of LINC01410-KD+ antigomir NC group was significantly smaller than that of NC-KD + antigomir NC group,while the average tumor volume of NC-KD +mir-122-5p inhibition group increased significantly.The tumor volume of LINC01410-KD + mir-122-5p inhibition group was between the above two groups.The weight of the tumor was consistent with its volume.q RT-PCR and Western blot were used to detect LINC01410,mir-122-5p and epithelial mesenchymal related molecules in tumor tissues.The results showed that the expression of LINC01410 was decreased and mir-122-5p was enhanced in LINC01410-KD + antigomir NC and LINC01410-KD + mir-122-5p inhibition groups.At the same time,Western blot showed that the expression levels of PKM2,snail and vimentin were inhibited in LINC01410-KD group and increased in mir-122-5p inhibition group.Conclusions:1.CAFs promote the epithelial mesenchymal function of esophageal squamous cell carcinoma cells through exosomes.2.CAFs exosomes may be involved in the regulation of epithelial mesenchymal transformation of tumor cells through LINC01410/mir-122-5p/PKM2 axis.3.1.The secretion of CAFs can participate in the regulation of EMT in esophageal squamous cell carcinoma cells through the expression of LINC01410/mir-122-5p/PKM2 axis,so as to regulate tumor migration and invasion.These findings support that LINC01410 plays a carcinogenic role in esophageal squamous cell carcinoma and may be used as a potential diagnostic biomarker and a new target for the treatment of esophageal squamous cell carcinoma. |
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