The Mechanisms Of GATA6 Regulates Shh Expression And Inhibits The Proliferation And Migration Of Lung Squamous Cell Cancer Cells

Background:Lung cancer is still a major health problem for human owing to the high incidence rate and mortality rate ? Lung Squamous Cell Carcinoma(SCC)is one of the predominant subtypes of Non-Small Cell Lung Cancers(NSCLCs),which usually shows a poor prognosis,mainly because of the occult onset of lung squamous cell carcinoma,and the lack of effective clinical diagnostic molecular markers in treatment,so the current use of conventional chemotherapy drugs treatment,often poor prognosis.Therefore,in-depth study of the pathogenesis of lung squamous cell carcinoma and the search for the treatment of lung squamous cell carcinoma of the key targets,has a very important translational significance.Abnormal activation of Hedgehog(Hh)signaling pathways is often associated with tumors.The Hh signaling pathway is abnormally activated in lung squamous cell carcinoma,and Shh ligand is the most common ligand in the Hh signaling pathway.GATA6 is a transcription factor involved in cell lineage differentiation and organ formation in a variety of tissues.GATA6 is associated with tumorigenesis and involved in transcriptional regulation of lung respiratory epithelial genes,which is very important for lung development and branching morphogenesis.Studies have shown that GATA6 can inhibit the metastasis of lung adenocarcinoma.However,the role of GATA6 in lung squamous cell carcinoma is still unclear,and whether GATA6 can regulate the expression of Shh in lung squamous cell carcinoma remains to be clarified.The expression of GATA6 can be regulated in a variety of ways,and MicroRNAs(miRNAs)have been proved to regulate the expression of GATA6.On the other hand,epigenetic modification is related to the expression of GATA6,and it remains to be determined whether DNA methylation and histone epigenetic modification in GATA6 promoter region regulate the expression of GATA6 in lung squamous cell carcinoma.Objectives: 1.Detect the expression of GATA6 in lung squamous cell carcinoma cells.2.Verified GATA6 affects cell proliferation and migration by inhibiting the expression of Shh.3.Explore the regulatory effect of epigenetic modification on GATA6 expression.Methods: 1.The pathological samples and frozen tissue samples of lung squamous cell carcinoma obtained after clinical lung squamous cell carcinoma resection were used to detect the expression levels of GATA6 in the lung squamous cell carcinoma tissues and control tissues in the samples by immunohistochemistry and real-time PCR.2.In terms of cell function,EDU and Cell Migration were used to verify the effect of overexpression of GATA6 in lung squamous cell carcinoma cells on the proliferation and migration of tumor cells.3.Bioinformatics software was used to analyze the relationship between GATA6 and Shh promoter,Dual-Luciferase reporter assay and chromatin immunoprecipitation were conducted to explore the mechanism of GATA6 regulating Shh expression.4.The medium containing N-Shh was used to culture the lung squamous cell cells overexpressing GATA6,and the EDU and Cell Migration were conducted again to detect whether the proliferation and migration ability of the cells were changed.5.Correlation analysis was used to compare the correlation between the expression levels of GATA6 and DNMT1 and HDACs in tumors,and to detect whether DNA methylation and histone acetylation affect the regulation of GATA6 expression.Results: 1.Immunohistochemistry and real-time PCR of clinical tissue samples showed that GATA6 expression in lung squamous cell carcinoma decreased compared with normal lung tissues.2.EDU and cell migration experiments demonstrated that overexpression of GATA6 in lung squamous cell carcinoma resulted in decreased proliferation and migration of lung squamous cell carcinoma cells.3.Dual-Luciferase reporter assay and chromatin immunoprecipitation assay confirmed GATA6 transcriptional inhibition of Shh expression.4.After the use of the medium containing N-Shh,the inhibition of cell proliferation and migration caused by overexpression of GATA6 was reversed by shh.5.Both DNA methylation and histone acetylation can regulate the expression of GATA6,and HDAC6 can regulate histone acetylation in GATA6 promoter region,thereby reducing the expression of GATA6.Conclusion:GATA6 expression was significantly decreased in lung squamous cell carcinoma compared with control normal tissue.The overexpression of GATA6 inhibited the proliferation and migration of lung squamous cell carcinoma cells.Dual-Luciferase reporter assay and chromatin immunoprecipitation assay demonstrated that GATA6 negatively regulated Shh expression by directly binding its promoter region.In addition,N-Shh saved the inhibition of proliferation and migration of lung squamous cell cells caused by overexpression of GATA6.The regulation of GATA6 is affected by epigenetic modification.HDAC6 regulates the acetylation of histones in GATA6 promoter region,thereby reducing the expression of GATA6.