沉默FAM83D基因调控上皮-间质转化对食管鳞癌放射敏感性影响

Effect of FAM83D silencing on radiosensitivity of esophageal squamous cell carcinoma cells by mediating epithelial-mesenchymaltransition

目的探讨沉默FAM83D对X线照射后食管鳞状细胞癌(ESCC)细胞增殖、存活能力及侵袭、迁移能力的影响及机制。方法采用免疫组化法检测69例ESCC患者组织中FAM83D、E-cadherin、vimentin的表达。免疫印迹法检测ECA109、KYSE30细胞FAM83D基因沉默效果。MTS、克隆形成实验和Transwell方法检测ECA109、KYSE30细胞增殖活性、存活能力及侵袭能力。流式细胞术和免疫印迹法检测ECA109、KYSE30细胞凋亡分布、上皮-间质转化(EMT)及凋亡相关蛋白表达。结果ESCC组织中55%(38/69)FAM83D强表达,36%(25/69)E-cadherin强表达,61%(42/69)vimentin强表达;FAM83D强表达与E-cadherin强表达呈负相关(r=-0.350,P<0.01),与vimentin强表达呈正相关(r=0.470,P<0.01)。ECA109、KYSE30细胞沉默FAM83D后降低了FAM83D蛋白表达(P<0.01)。MTS和克隆形成实验数据显示照射后沉默FAM83D显著抑制了ECA109、KYSE30细胞增殖水平(P<0.05)、增加了放射敏感性(P<0.01)。照射后FAM83D shRNA组ECA109、KYSE30细胞侵袭力降低(P<0.01)、E-cadherin表达增加,而N-cadherin、vimentin、Snail、p-Akt、p-GSK-3β表达降低(P<0.01)。照射后FAM83D shRNA组ECA109、KYSE30细胞凋亡率明显增加(P<0.01),同时Bcl-2、Mcl-1表达下调,Cleaved caspase-3的表达上调(P<0.01)。结论FAM83D与ESCC的侵袭发展密切相关。沉默ECA109、KYSE30细胞FAM83D表达联合X线照射降低了其增殖、侵袭和存活能力,诱导了细胞凋亡,增加了放射敏感性,该作用可能通过Snail/Akt/GSK-3β信号途径来调控EMT。

Objective To examine the effect of FAM83D knockdown on proliferation,survival ability and invasion of human esophageal squamous cell carcinoma after X-ray radiation,and explore the mechanism.Methods The expression of FAM83D,E-cadherin and vimentin in tumor tissues was detected in 69 cases of esophageal squamous cell cancer by using immunohistochemical method.The siRNA based on the sequences of the FAM83D mRNA were synthesized to transfect into the cultured ECA109 cells as FAM83D shRNA group.The effect of silencing FAM83D gene was evaluated to determine the protein levels of FAM83D in the human oesophageal squamous cell carcinoma ECA109 and KYSE30 cells using western blotting.MTS,clone formation,and Transwell assay were employed to examine the proliferation,survival ability and invasion of ECA109 and KYSE30 cells in vitro,respectively.We used flow cytometry assay to analyze distribution of cell apoptosis in different groups.Western blotting was used to examine the expression of cell metastasis-related molecules and apoptosis-related protein.Results The strong expression rates of FAM83D,E-cadherin,and vimentin were 55%(38/69),36%(25/69)and 61%(42/69)in the tumor tissues,respectively.FAM83D protein expression was significantly and negatively correlated with the expression of E-cadherin(r=-0.350,P<0.01),and positively with the expression of vimentin(r=0.470,P<0.01).Western blotting results demonstrated that silencing FAM83D gene significantly reduced the FAM83D protein expression(P<0.01).MTS data demonstrated that FAM83D knockdown after irradiation significantly inhibited the proliferation of esophageal squamous cell carcinoma ECA109 and KYSE30 cells(P<0.05).The data from the clone formation assay revealed that the radiosensitivity was increased after downragulation of FAM83D expression(P<0.01).In addition,the invasive abilities of oesophageal carcinoma cells transfected with FAM83D shRNA after irradiation were significantly inhibited compared with those of the NC group(P<0.01),followed by the downregulation of N-cadherin,vimentin,Snail,p-Akt and p-GSK-3βexpression,and the upregulation of E-cadherin expression(P<0.01).The apoptosis rate of tumor cells in FAM83D shRNA group after irradiation was markedly increased(P<0.01),followed by a decrease of Bcl-2 and Mcl-1 expression and an increase of Cleaved caspase-3 expression(P<0.01).Conclusions FAM83D expressions was found to be closely related to the invasion and development of ESCC.Furthermore,siRNA interference technology inhibited the expression of FAM83D gene in oesophageal squamous cell carcinoma cells,reduced the proliferation,invasion of cells,induced cell apoptosis,and increased radiosensitivity,which may be associated with regulating the epithelial-mesenchymaltransition via Snail/Akt/GSK-3βsignaling pathways.