Mechanism Of Fisetin Inhibiting Oral Squamous Cell Carcinoma Progression Through MUC1

Objective: Oral squamous cell carcinoma(OSCC)is mainly originated from oral mucosal epithelial cells.Its occurrence is a complex multi-step process.Combined surgery and chemoradiotherapy is the first-line effective treatment.Current antibody drugs against OSCC are limited and unspecific,and the application of these emerging agents although greatly improving the survival time and quality of life of patients with OSCC,the treatment against OSCC still lacks precise biological targets and effective small molecule compounds,and the search for new therapeutic targets and sensitive small molecule targeted agents to prevent the occurrence and development of OSCC is an urgent problem.Fisetin(3,3’,4’,7-tetrahydroxyflavone),also known as 3,7,3’,4’-tetrahydroxyflavone,is a bioactive flavonol commonly found in a variety of plants,fruits and vegetables in nature.Numerous studies have shown that Fisetin has the effects of inhibiting malignant transformation,proliferation,angiogenesis,invasion and metastasis,and is able to induce cell cycle arrest,promote tumor cell apoptosis and necrosis,reverse multiple drug resistance,and so on.Fisetin has been reported to have anticancer effect in various tumor tissues,however,its functional mechanism in OSCC is poorly defined.This research aims to provide new diagnostic indicators and therapeutic targets for the diagnosis and treatment of OSCC by exploring the role of Fisetin in OSCC and its mechanisms.Methods:1、According to the pre-experimental optimal treatment time and concentration of Fisetin for oral squamous cell carcinoma Tca8113 cell line,a control group(DMSO)and an experimental group(Fisetin)were set up,and RNA was extracted for nanoporebased sequencing analysis to find out the differential genes related to OSCC induction by Fisetin.2、5-10 differentially expressed genes were selected for which Real-time qPCR primers were designed to detect gene expression in OSCC cell lines treated with Fisetin,the target gene MUC1,whose changing trend was consistent with sequencing result,was selected to validate its protein expression level in OSCC cell lines using Western blot with corresponding protein antibodies.3、For pathological sections of patient tumor tissues,the expression level and distribution of target gene MUC1 were determined by immunohistochemistry,and for fresh pathological samples,total protein and RNA were extracted from these samples.4、Lentiviral vectors for overexpression and knockdown of target gene MUC1 were constructed to infect OSCC cells,respectively,and cell lines with stable overexpression and knockdown of MUC1 were obtained.Then changes in the proliferation,migration and invasion abilities of the stable cell lines were tested to verify the functions of MUC1,meanwhile,the collected OSCC clinicopathological tissue samples were subjected to immunohistochemical techniques,and the MUC1 expression levels and localization were statistically analyzed.5、MUC1-overexpressed OSCC cell lines were treated with Fisetin,cell proliferation was assessed by colony formation and CCK-8 assays,apoptosis was detected by Annexin V / PI double staining,and migration and invasion were evaluated by scratch assay and Transwell assay.6、To construct OSCC nude mice model,human OSCC cells overexpressing MUC1 and empty group were injected into the armpits of nude mice,respectively.After the tumorigenesis was observed,Fisetin was used for gavage treatment,and placebo was used for the control group.After 35 days,mice were sacrificed and dissected,tumor size was assessed,and a portion of tumor tissue was harvested for protein and IHC detection of expression changes of Fisetin regulated MUC1.Results:1.Fisetin inhibited proliferation and migration,and promotes apoptosis in human OSCC cells.2.Both pathological sections and fresh tumor tissue samples from patients with OSCC detected higher levels of MUC1 gene and protein in tumor tissue compared with para-cancerous(para-cancerous tissue n=10,tumor tissue n=16,P < 0.0001).3.In OSCC cell lines treated with Fisetin,both gene and protein levels of MUC1 were reduced as detected by Western blot and Real-time qPCR.4.Using NC group as control,when MUC1 was silenced in Cal27 cells,,the proliferation capacity decreased,the apoptosis rate increased,and the migration ability declined.5.After overexpressing in Cal27 cells,the proliferation ability of Cal27 cells became stronger,the apoptosis rate decreased,and the migration and invasion rate increased.However,when Fisetin was added to the culture medium,a series of intracellular changes due to overexpression of MUC1 were significantly suppressed.6.In animal experiments,the tumor of nude mice injected subcutaneously with MUC1-overexpressed cells was larger than that of control group,and high expression of MUC1 was detected by protein,whereas the tumor of nude mice receiving treatment with Fisetin by gavage were significantly smaller,and the intensity of the detected MUC1 protein signal was also markedly reduced.Conclusion:MUC1 is a highly expressed oncogene in human oral squamous cell carcinoma.Overexpression of MUC1 in human OSCC cells significantly promotes cell proliferation,migration and invasion.As an inhibitor of MUC1,Fisetin can inhibit the proliferation,migration and invasion,while promote cell apoptosis of human OSCC cell lines by downregulating MUC1 expression.Animal experiments also confirmed that Fisetin inhibits the growth of OSCC tumor by down-regulating the expression of MUC1 in vivo.