Molecular Mechanism Of Tumor Cells Promoting Cancer Associated Fibroblast Transformation By Regulating GLI2-TGFβ1 Signaling Axis Through MiR-30d

Background and Purpose:Lung cancer is the malignant tumor with the highest morbidity and mortality,and its 5-year survival rate is less than 20%.Cancer-associated fibroblast(CAFs)are an important part of the tumor microenvironment.Most of the CAFs are transformed from fibroblasts induced by tumor cells and play a key role in the occurrence and development of cancer.The development of targeted therapy strategies based on the remodeling mechanism of tumor cells on the microenvironment will effectively improve the survival time of patients.In this study,we explored the role and molecular mechanism of abnormal expression of miRNAs in tumor cells in promoting fibroblast remodeling from the perspectives of bioinformatics,molecular biology and cell biology.Methods:1.The key miRNAs related to CAFs were screened by bioinformatics research methods,and the expression of miR-30d-5p in tumor cells and bronchial epithelial cells was verified by real-time quantitative PCR(q PCR);2.Use q PCR,Western blotting(WB)and immunofluorescence(IF)techniques to verify the relationship between the abnormal expression of miR-30d-5p and the transformation of normal-associated fibroblasts into CAFs induced by tumor cells;3.Explore the regulation of miR-30d-5p on TGFβ pathway in tumor cells by bioinformatics,RNA-seq,q PCR and WB;4.Using RNA-seq,Double luciferase reporter gene assay,q PCR,WB and IF to study the molecular mechanism of miR-30d-5p activating Hedgehog-GLI2 pathway through non-canonical pathway;5.Binding site prediction and Double luciferase reporter gene assay were used to explore the regulatory relationship between transcription factor GLI2 and TGFβ1.To study GLI2 regulates the expression of TGFβ1 in lung adenocarcinoma cells,thereby affecting the transformation of fibroblasts.Result:1.miR-30d-5p has low expression in CAFs-enriched tumors and high expression in normal cells,and is associated with prognosis;2.Inhibition of miR-30 d in normal lung epithelial cells resulted in tumor cellinduced conversion of NAFs to CAFs,whereas overexpressed miR-30 d in LUAD cell lines could inhibit the conversion of NAFs to CAFs;3.Deletion of miR-30d-5p promoted the upregulation of TGFβ1,in addition,miR-30d-5p promoted the transformation of CAFs through TGFβ1;4.miR-30d-5p directly inhibits GLI2 and regulates TGFβ1 expression through non-canonical pathways,and miR-30d-5p affects fibroblast transformation through GLI2;5.TGFβ1 is the downstream target gene of transcription factor GLI2,which mediates the transformation effect of GLI2 on fibroblasts.Conclusion:Low expression of miR-30d-5p in lung adenocarcinoma cells non-canonically activates GLI2 to regulate TGFβ1 secretion and promote the conversion of NAFs to CAFs.