| Objective:Laryngeal cancer(LCA)is one of the most common malignant tumors in the head and neck.Despite the continuous development of cancer treatment in recent years,the incidence of Laryngeal cancer is increasing year by year,while the mortality rate is not significantly decreased.Therefore,it is important to further reveal the molecular mechanism of LCA occurrence and development for its early diagnosis and targeted prevention and treatment.In this study,the expression and mechanism of mir-139-3p in LCA were investigated,and the mechanism of mir-139-3p in regulating the malignant biological behavior of LCA and the possible signaling pathway were further analyzed by looking for its target gene KDM5 B,so as to provide a basis for finding new therapeutic targets for LCA.Methods:(1)Twenty-five LCA tumor and paracancer tissue specimens were collected,The gene expression levels of KDM5 B,mir-139-3p and SOX2 in LCA tissues and cell lines were detected by RT-q PCR,as well as the gene expression levels of KDM5 B,mir-139-3p and SOX2 under various intervention conditions.(2)The expression levels of KDM5 B and SOX2 proteins in LCA tissues and cell lines were detected by Western Blot and immunohistochemistry.Western Blot was used to detect the protein expression levels of Bcl-2,Bax,Caspase3,Cleaved Caspase3 andβ-catenin in various intervention conditions.(3)Overexpressed and silenced KDM5 B vectors,overexpressed mir-139-3p vectors and silenced SOX2 vectors were constructed to transfect HNO210 and TU177 cell lines,respectively,and the established transfection cell models were verified by RT-q PCR and Western Blot methods.(4)The proliferation of HNO210 and TU177 LCA cells under various intervention conditions was detected by Edu experiment,CCK-8 experiment and clonogenesis experiment.The apoptosis of LCA cells was detected by flow cytometry and Hoechst staining.The migration ability of LCA cells was detected by cell scratch assay.The invasion ability of LCA cells was detected by Transwell chamber assay.(5)The binding site of mir-139-3p and KDM5 B was predicted using Target Scan database,and the targeting relationship between mir-139-3p and KDM5 B was detected by dual luciferase assay.(6)Ch IP assay and RT-q PCR assay were used to detect the binding relationship between KDM5 B and SOX2.(7)The nude mouse transplanted tumor model was established,and the tumor growth was observed under various intervention conditions.The gene expression levels of KDM5 B,mir-139-3p and SOX2 in tumor tissues were detected by RT-q PCR,and the protein expression changes of KDM5 B and SOX2 were detected by Western Blot and immunohistochemistry.The expression of β-catenin protein was detected by Western Blot.(8)Graph Pad Prism 8.4.0 was applied to plot the experimental results and conduct data analysis.The results of the three independent experiments are expressed as mean±standard deviation.T test was used for comparison between the two groups,and P < 0.05 was considered as significant difference.Results:(1)KDM5B was highly expressed in both gene and protein levels in LCA tumor tissues and cells.(2)LCA cell lines HNO210 and TU177 were successfully constructed to silence KDM5 B,which inhibited LCA cell proliferation,migration,invasion,epithelial-mesenchymal transformation(EMT)and promoted LCA cell apoptosis.(3)Mir-139-3p has a targeting relationship with KDM5 B,mir-139-3p inhibits the expression of KDM5 B,and mir-139-3p is underexpressed in LCA tumor tissues and cells.(4)LCA cell lines HNO210 and TU177 with overexpression of mir-139-3p were successfully constructed,and overexpression of mir-139-3p inhibited LCA cell proliferation,migration and invasion,and promoted APOPTOSIS of LCA cells.(5)SOX2 is highly expressed in LCA tumor tissues and cells.KDM5 B promotes SOX2 expression in LCA cells,and SOX2 activates the Wnt/β-catenin pathway.(6)LCA cell lines HNO210 and TU177 were successfully constructed to silence SOX2,which inhibited the proliferation,migration and invasion of LCA cells and promoted the apoptosis of LCA cells.(7)Mir-139-3p targets KDM5 B to inhibit SOX2 and β-catenin expression.(8)Mir-139-3p inhibited LCA cell proliferation,migration and invasion by inhibiting Wnt/β-catenin signaling pathway,and promoted LCA cell apoptosis.(9)In vivo experiments demonstrated that mir-139-3p inhibited the growth of transplanted tumor by targeting KDM5 B and regulating SOX2/Wnt/β-catenin axis.The expression of KDM5 B and SOX2 was inhibited by mir-139-3p in transplanted tumor.Conclusion:(1)In LCA tumor tissues and cells,KDM5 B was highly expressed at both gene and protein levels,mir-139-3p was low expressed,and SOX2 was highly expressed at both gene and protein levels.(2)Silencing KDM5 B inhibited LCA cell proliferation,migration,invasion and EMT,and promoted apoptosis;mir-139-3p inhibits LCA cell proliferation,migration and invasion by targeting KDM5 B and inhibiting its expression,and promotes cell apoptosis by overexpressing mir-139-3p.KDM5 B inhibited LCA cell proliferation,migration,invasion and apoptosis by targeting SOX2 and promoting its expression,and inhibited LCA cell malignant biological behavior by inhibiting Wnt/β-catenin pathway.(3)Mir-139-3p regulates SOX2/Wnt/β-catenin axis by targeting KDM5 B,inhibits LCA cell proliferation,migration and invasion,and promotes cell apoptosis. |
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