摘要
目的:探讨环状RNA酮戊二酸脱氢酶(circular oxoglutarate dehydrogenase,Circ⁃OGDH)调节miR⁃127⁃3p/蛋白酶体β5亚基(proteasome subunit beta type⁃5,PSMB5)轴对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)增殖、迁移和侵袭的影响。方法:qRT⁃PCR与Western Blotting测定人口腔上皮细胞及人OSCC细胞系CAL⁃27、HN6、Bca885中Circ⁃OGDH、miR⁃127⁃3p及PSMB5表达。体外培养CAL⁃27细胞并分组转染后测定细胞Circ⁃OG⁃DH、miR⁃127⁃3p及PSMB5表达;Ki67免疫荧光及TUNEL染色检测细胞增殖、凋亡,细胞划痕及Transwell侵袭实验检测细胞迁移、侵袭;Western Blotting检测细胞凋亡(Bcl⁃2、Bax)及上皮间充质转化相关蛋白(Vimentin、E⁃cadherin)表达。于裸鼠皮下接种分组转染后的CAL⁃27细胞构建其移植瘤模型,测定裸鼠肿瘤体积与质量,瘤组织中Ki67阳性表达,TUNEL染色检测细胞凋亡情况。双荧光素酶报告基因实验验证CAL⁃27细胞Circ⁃OGDH对miR⁃127⁃3p、miR⁃127⁃3p对PSMB5的靶向调控作用。结果:与人口腔上皮细胞相比,CAL⁃27、HN6、Bca885细胞Circ⁃OGDH、PSMB5 mRNA及蛋白表达均升高,miR⁃127⁃3p表达均降低(P<0.05)。与对照组相比,Circ⁃OGDH敲低组、miR⁃127⁃3p过表达组细胞和瘤组织中PSMB5 mRNA及蛋白表达、细胞增殖率、迁移率、侵袭数目、瘤体积及质量、Ki67阳性表达、细胞和瘤组织中Bcl⁃2与Vimentin蛋白表达降低,miR⁃127⁃3p表达、凋亡率、Bax与E⁃cadherin蛋白表达升高(P<0.05);与Circ⁃OGDH敲低组相比,Circ⁃OGDH敲低+miR⁃127⁃3p敲低组细胞和瘤组织中PSMB5 mRNA及蛋白表达、细胞增殖率、迁移率、侵袭数目、瘤体积及质量、Ki67阳性表达、细胞和瘤组织中Bcl⁃2与Vimentin蛋白表达升高,miR⁃127⁃3p表达、凋亡率、Bax与E⁃cadherin蛋白表达降低(P<0.05)。Circ⁃OGDH可靶向下调CAL⁃27细胞miR⁃127⁃3p表达,miR⁃127⁃3p可靶向下调其PSMB5表达。结论:敲低Circ⁃OGDH
Objective:To explore the effect of circular oxoglutarate dehydrogenase(Circ⁃OGDH)on the proliferation,migration and invasion of oral squamous cell carcinoma(OSCC)by regulating the miR⁃127⁃3p/proteasome subunit beta type⁃5(PSMB5)axis.Methods:qRT⁃PCR and Western Blotting were used to determine the expression of Circ⁃OGDH,miR⁃127⁃3p,and PSMB5 in human oral epithelial cells and human OSCC cell lines CAL⁃27,HN6,and Bca885.CAL⁃27 cells were cul⁃tured in vitro and after grouping and transfection,to determine the expression of Circ⁃OGDH,miR⁃127⁃3p,and PSMB5 of cells in each group.Ki67 immunofluorescence and TUNEL staining were performed to detect cell proliferation and apoptosis,cell scratch and Transwell invasion test were applied to detect cell migration and invasion.Western Blotting were applied to detect the expression of cell apoptosis(Bcl⁃2,Bax)and epithelial mesenchymal transition related proteins(Vimentin,E⁃cad⁃herin).The transplanted tumor model was constructed by subcutaneous inoculation of the transfected CAL⁃27 cells in each group of nude mice.The tumor volume and mass of nude mice were measured,the positive expression of Ki67 in tumor tissues was detected by immunohistochemistry,the apoptosis was detected by TUNEL staining.The double luciferase Reporter gene experiment was applied to verify the targeting regulation of Circ⁃OGDH on miR⁃127⁃3p,and targeting regulation of miR⁃127⁃3p on PSMB5 in CAL⁃27 cells.Results:Compared with human oral epithelial cells,the expression of Circ⁃OGDH,PSMB5 mRNA and protein in CAL⁃27,HN6,and Bca885 cells increased,the expression of miR⁃127⁃3p decreased(P<0.05).Com⁃pared with the control group,the expression of PSMB5 mRNA and protein in cells and tumor tissues,cell proliferation,migra⁃tion rate,number of invasions,tumor volume and mass,positive expression of Ki67,and expression of Bcl⁃2 and Vimentin pro⁃teins in cells and tumor tissues in the Circ⁃OGDH knockdown group and miR⁃127⁃3p overexpression group were de
作者
赵利芬
彭麟微
刘晓璐
夏斌
ZHAO Li-fen;PENG Lin-wei;LIU Xiao-lu;XIA Bin(Department of Oral Medicine,Haiyuan College,Kunming Medical University,Yunnan Kunming 650106,China;Deparment of Oral and Maxillo-facial Surgery,Affiliated Stomatological Hospital of Kunming Medical University,Yunnan Kunming 650106,China)
出处
《临床口腔医学杂志》
2023年第9期525-532,共8页
Journal of Clinical Stomatology
基金
云南省医疗卫生单位内设研究机构科研项目(2018NS0210)。
