| Objective ESCC(Esophageal squamous cell carcinoma)is a primary malignant tumour of the oesophagus that has been experiencing high morbidity and mortality in recent years.The tumour microenvironment is closely related to ESCC progression.CAFs(Cancer Associated Fibroblasts)are one of the most important cellular components of the ESCC tumour microenvironment and are associated with ESCC progression and prognosis.However,the effects of CAFs on ESCC and the mechanisms of action are not fully understood.Hedgehog pathways are widely activated in ESCC and ESCC-associated fibroblasts and are highly associated with tumour cell proliferation,migration and stemness.In this study,We use CAFs as the target,Hedgehog pathway as the entry point,and ESCC cells as the experimental object,aiming to investigate the interaction between CAFs in an activated state and ESCC cells,and the possible function of CAFs on ESCC progression,to provide new directions for targeted therapeutic measures using CAFs in the tumour microenvironment.Methods Five primary human esophageal squamous carcinoma CAFs cells were isolated and identified using a tissue block crawl-out primary cell culture technique.Then the consistency of the response to the Hedgehog pathway between NIH/3T3 cells and primary CAFs cells was verified,as well as the effect on ESCC cells.Then,Hh-SMO knockdown NIH/3T3 cells(called NIH/3T3-SMO-KO cells,control cells called NIH/3T3-SMO-NC cells)were established using CRISPR Cas9 technology.The CM(conditioned medium)was collected from NIH3T3-SMO-NC and NIH3T3-SMO-KO cells for culturing ESCC cells to detect the phenotypic changes such as proliferation and migration of ESCC cells,and then transcriptome analysis of NIH3T3-SMONC and NIH3T3-SMO-KO cells was performed by transcriptome sequencing to identify possible downstream targets and mechanisms of action.Real-time PCR and Western Blot were used to validate the results.Results 1.Western Blot and Real-time PCR showed that both primary ESCC-associated CAFs and NIH/3T3 cells expressed α-SMA,a marker of CAFs,and Vimentin,a mesenchymal indicator,and that Hedgehog pathway activity was increased in the cells.2.Compared to culture alone,The proliferative capacity of KYSE140 cells was significantly enhanced when co-cultured with NIH/3T3 cells or No.5 CAFs.In contrast,the proliferation ability was reduced after treatment with Hedgehog pathway inhibitors.3.CM from NIH/3T3-SMO-NC cells significantly promoted the proliferation of ESCC cells compared with NIH/3T3-SMO-KO cells.4.Compared with cells in the control(serum-free DMEM medium)group,CM from the NIH/3T3-SMO-NC group CM significantly promoted the migration and invasion of ESCC cells compared to the control(serum-free DMEM medium)group of cells,while CM of NIH/3T3-SMO-KO cells with SMO knockdown had significantly weaker migration and invasion of ESCC cells compared to the non-knockdown group(P<0.05).5.Transcriptome sequencing results were analyzed to screen out four extracellular matrix genes,the expression of which was decreased in NIH3T3-SMO-KO cells.Real-time PCR results showed that the m RNA expression of the above four genes was significantly decreased in NIH/3T3-SMO-KO cells compared with NIH/3T3-SMO-NC cells,with 16.67,1.72,2.13 and7.14-fold decreases,respectively.And KEGG pathway analysis demonstrated that Tenascin-W and COL6A2 were enriched in the PI3K-AKT pathway.6.The expression of P-AKT and Pm TOR in the NIH/3T3-SMO-KO group was reduced after CM treatment of ESCC cells with NIH/3T3-SMO-NC/KO cells compared to the NIH/3T3-SMO-NC cell group.Conclusion The experimental results showed that in ESCC,tumour cells secreted the Hedgehog ligands SHH and IHH,and both primary ESCC-associated fibroblasts and NIH/3T3 cells could respond to this ligand signal and promote the proliferation of ESCC cells,and NIH/3T3-SMO-NC cells affected the tumour cells by secreting factors such as COL6A2 PI3K-AKT signalling,promoting migration and invasion of ESCC cells. |
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