Study On The Mechanism Of Baicalin Inhibiting Proliferation And Promoting Apoptosis Of Esophageal Squamous Cell Carcinoma Based On MiR-21 Targeting FOXO3a Pathway

Esophageal cancer is one of the most serious malignancies in the world,and in China,it is the fifth leading cause of cancer-related deaths,claiming nearly 250,000 lives each year.Esophageal squamous cell carcinoma is the main manifestation of esophageal cancer in China.Despite continuous advances in diagnostic techniques and treatments,the overall 5-year survival rate is still far from satisfactory becacuse it is mostly advanced at the time of diagnosis.herefore,it is crucial to identify oncogenes or oncogenes as biomarkers of ESCC to develop more effective treatment strategies for patients with ESCC.As more and more clinical trials related to esophageal cancer have been conducted in recent years,targeted and non-targeted immunotherapies have been gradually developed.These therapies,in combination with surgery,radiotherapy,and chemotherapy,promise to be the future of multimodal treatment for esophageal cancer.However,drug resistance in targeted immunotherapy is a complex issue that cannot be avoided,and TCM plays an important role in immune and targeted therapies,such as synergizing,improving drug sensitivity,and even reversing drug resistance.Among them,the tradtional Chinese medicine Scutellaria baicalensis has been known as the "Chinese antibiotic" and its anti-cancer effects have been widely recognized.Haicalin,the main extract of Scutellaria haicalensis,has been shown to inhibit the malignant progression of various cancers in various studies,but the effect of Scutellaria baicalensis on ESCC and its specific mechanism have not been reported-In this study,we intervened in ESCC cells in vitro by baicalin,and confirmed that baicalin could inhibit the proliferative activity and promote the apoptotic effect of ESCC using cck8,flow cytometry,qPCR,western blot,and TUNNEL.Further,we demonstrated the inhibitory effect of baicalin on the malignant progression of ESCC using xenografted ESCC lotus mice after administration,and it had no toxic effect on It was further demonstrated that baicalin had no toxic effect on other organs.Subsequently,the FOXO pathway was screened by RNA sequencing followed by KEGG analysis and GO analysis,and finally focused on FOXO3a,a transcription factor.As a member of the FOXO family,FOXO3a is involved in regulating a variety of cellular processes,including survival,proliferation,apoptosis,dfferentiation,cell cycle,invasion,metastasis,epithelial mesenchymal transition,DNA damage repair,and autophagy.FOXO3a also mediates cellular stress responses to various deleterious events,such as UV irradiation,DNA damage,hypoxia,heat shock,oxidative stress,and nutritional deficiencies.In addition,FOXO3a plays a fundamental role in neuroprotection,cardiac aging and human longevity.Due to its pleiotropic effects,abnormal regulation of FOXO3a is closely associated with a variety of diseases,including muscle wasting,renal fibrosis,emphysema and cancer,aberrant expression of FOXO3a in many types of cancer tissues and cells has been observed,while its role in ESCC and related mechanisms remain to be explored.We detected an increased expression level of FOXO3a in ESCC cells after baicalein intervention by qPCR and western blot,and confirmed its oncogenic effect in ESCC by knocking down and overexpressing FOXO3a,while knocking down FOXO3a partially alleviated the inhibitory effect of baicalein on ESCC.Subsequently,FOXO3a was detected in tumor tissues and surrounding normal esophageal tissues of esophageal cancer patients by qPCR and western blot,and it was found that FOXO3a was expressed at low levels in ESCC tissues,which was further v-erified by immohistochemical staining to detect FOXO3a levels in tissue microarrays,and combined with follow-up data,it was concluded that low expression of FOXO3a signified a poor prognosis for ESCC patients The conclusion that low expression of FOXO3a signifies poor prognosis of ESCC patients.To gain more insight into how baicalein regulates FOXO3a,bioinformatics tools and literature data combined with qRCR were used to finally target miR-21 to target FOXO3a.miR-21 overexpression in malignant tumors is a unique prognostic biomarker associated with high tumor cell proliferation,tumor growth,and reduced apoptosis.Several studies have reported significantly higher miR-21 expression in ESCC tissues than in normal surrounding squamous epithelium,a result that suggests the involvement of miR-21 in the carcinogenesis of the esophagus.In addtion,miR-21 has been shown to preferentially target tumor suppressors and also tumor suppressor genes.miR-21 was shown to target FOXO3a in lung and breast cancer,but due to the specificity of miRNA targeting in different tumors,we demonstrated by western blot that miR-21 targets FOXO3a in ESCC cells,and used experiments demonstrated that baicalein inhibited ESCC at least partially by mediating miR-21 targeting and regulating FOXO3a.In summary,our stuy elucidates the role and molecular mechanism of baicalin in inhibiting proliferation and promoting apoptosis in ESCC,suggests the possibility of baicalin as a potential novel drug for the treatment of ESCC,and provides a scientific basis for estahlishing FOXO3a and miR-21 as prognostic indicators and therapeutic targets for ESCC.