Biochanin A Reversed Epithelial-mesenchymal Transformation And PD-L1 Overexpression To Inhibit Colorectal Cancer Progression By Targeting ZEB1

ObjectivesColorectal cancer is one of the most common digestive cancers,and ranks third in morbidity and mortality of all malignancies worldwide.Despite advances in the treatment of colorectal cancer,the long-term prognosis of patients with advanced colorectal cancer remains poor.Therefore,exploring the molecular mechanism of colorectal cancer,identifying novel therapeutic targets,and improving its prognosis have always been the goals of research in the field of colorectal cancer.An increasing number of studies have attempted to reveal the molecular mechanisms of the oncogenesis,development,invasion,and metastasis of colorectal cancer,but so far they have not been fully elucidated.Epithelialmesenchymal transition(EMT)refers to the biological process in which epithelial cells are transformed into cells with a mesenchymal phenotype through specific programs,which plays an important role in embryonic development,chronic inflammation,tissue remodeling,tissue fibrosis as well as malignant tumor progression.More importantly,the EMT process plays an important role in the oncogenesis and development of tumors,and the EMT process can promote immune evasion.Over the past few decades,more and more traditional Chinese medicines have been found to have anti-inflammatory and anti-tumor abilities,which may open up new prospects for the treatment of malignant tumors.A lot of mature Chinese medicine monomers have gradually entered preclinical and clinical applications.Existing studies have shown that Biochanin A has various pharmacological effects,such as anti-tumor,anti-inflammatory,antibacterial,hypoglycemic,antioxidant,and neuroprotective.Although there are a lot of studies on the regulation of the EMT process by traditional Chinese medicine monomers,there is no relevant research on whether Biochanin A can be used as an inhibitor of the EMT process in colorectal cancer.This study aimed to investigate whether Biochanin A could affect the immune state and malignant biological processes of colorectal cancer by regulating ZEB1,the key transcription factor of EMT.Methods(1)Bioinformatics analysis:The RNA-seq data and relative clinical information of colorectal cancer in the TCGA dataset were downloaded from the Xena Browser;singlesample gene set enrichment analysis(ssGSEA)was used to calculate the EMT score for each colorectal cancer sample in the TCGA database;the ESTIMATE score,immune regulatory genes,immune checkpoints,immune cell markers,tumor-infiltrating immune cell abundance,tumor immune cycle activities were used to describe immune microenvironment characteristics from multiple perspectives;the relationships between the EMT score and features of the cancer-immune microenvironment were subsequently assessed.(2)Clinical sample collection and immunohistochemistry analysis:colorectal cancer tissue microarray HColA160Bc01 was purchased from BioTech Co.,Ltd(Shanghai,China).The tissue microarray contains 80 colorectal cancer tissues and paired para-cancerous tissues;immunohistochemistry staining was performed on the HColA160Bc01 microarray;the stained tissue microarray was scanned by Aperio digital pathological slide scanner;the immuno-reactivity score standard was used to evaluate immunohistochemistry results.The correlation between ZEB1 and PD-L1 expression in colorectal cancer was subsequently evaluated.(3)In vitro cell experiments:HCT116 and SW620 colorectal cancer cells were cultured and used for subsequent in vitro experiments;siRNA and overexpression plasmids against ZEB1 were transfected,after then,the expressions of ZEB1 and PD-L1 were detected;Biochanin A was added to the cell culture medium,after then,ZEB1 expression,EMT marker expression,and PD-L1 expression were detected;CCK8 assay,wound healing assay,and Transwell assay were used to detect cell proliferation,migration,and invasion abilities.(4)Animal experiments:mouse colon cancer MC38 cells and C57BL/6 mice were used to establish animal models,and mice bearing MC38 cells were divided into control group and Biochanin A treatment group,to observe the effects of Biochanin A on the growth of MC38 cells in vivo and the regulation of the expression of ZEB1 and PD-L1.ResultsPart 1:in the TCGA dataset,the EMT score were highly correlated with ESTIMATE scores,immune regulatory genes,immune checkpoints,immune cell markers,tumorinfiltrating immune cell abundance,and tumor immune cycle activities.ZEB1 and PD-L1 were significantly highly expressed in colorectal cancer tissues.ZEB1 was significantly associated with N stage,M stage and TNM clinical stage,and PD-L1 was significantly associated with pathological grade.In addition,there was a significant positive correlation between ZEB1 and PD-L1 expression;in HCT116 and SW620 colorectal cancer cells,knockdown of ZEB1 led to the downregulation while overexpression of ZEB1 led to upregulation of PD-L1 at both the transcriptional and protein levels.Part 2:Biochanin A inhibited ZEB1 expression and nuclear translocation in HCT116 and SW620 cells;Biochanin A inhibited the EMT process of colorectal cancer cells in vitro;Biochanin A inhibited cancer cell proliferation,migration,and invasion in vitro;Biochanin A inhibited the growth of colorectal cancer in vivo but did not affect the body weight of C57BL/6 mice.Part 3:Biochanin A downregulated ZEB1-mediated PD-L1 expression in vitro;Biochanin A inhibits ZEB1 and PD-L1 expressions in MC38 subcutaneous tumor of C57BL mice.ConclusionsZEB1,a key transcription factor in the EMT process,positively regulates the expression of PD-L1.Biochanin A,a Chinese medicine monomer,inhibits the malignant progression and PD-L1 expression of colorectal cancer by down-regulating the expression of ZEB1.In conclusion,this study reveals that the Chinese medicine monomer Biochanin A can be used as a candidate drug for the treatment of colorectal cancer,and its molecular mechanism is preliminarily explained in the current research.