TRIM44基因对子宫内膜癌细胞生长及放疗敏感性的影响

Effects of TRIM44 gene on growth and radiosensitivity of endometrial cancer cells

目的探究三结构域蛋白44(TRIM44)对子宫内膜癌(EC)细胞生长和放疗敏感性的调控机制。方法①通过免疫组化检测30例EC患者的癌组织和临近癌旁组织中TRIM44的表达水平。通过qRT-PCR检测癌旁组织以及不同FIGO分期、不同组织学分级、淋巴结转移/未转移的EC患者癌组织中TRIM44 mRNA水平。②将人子宫内膜癌细胞系(HEC-1A)分为对照组、siRNA-NC组、siRNA-TRIM44组、LV-NC组和LV-TRIM44组,根据分组情况,使用Lipofectamine 2000试剂将阴性对照siRNA(siRNA-NC)、靶向TRIM44的siRNA(siRNA-TRIM44)、阴性对照慢病毒(LV-NC)和TRIM44过表达慢病毒(LV-TRIM44)转染到对应分组的HEC-1A细胞中,对照组细胞不进行转染,通过qRT-PCR和Western blot检测各组细胞中TRIM44的mRNA和蛋白表达水平。③将HEC-1A细胞分为siRNA-NC组、siRNA-TRIM44组、LV-NC组、LV-TRIM44组、8 Gy+siRNA-NC组、8 Gy+siRNA-TRIM44组、8 Gy+LV-NC组、8 Gy+LV-TRIM44组。根据分组情况,使用Lipofectamine 2000试剂转染细胞,并采用直线加速器6-MV X射线照射细胞(8 Gy)。通过CCK-8测定细胞增殖,AnnexinⅤ-FITC凋亡检测试剂盒检测细胞凋亡,Transwell检测细胞迁移和侵袭,qRT-PCR和Western blot检测TRIM44、p62、细丝蛋白A(FLNA)和RAD51的表达。结果与癌旁组织相比,癌组织中TRIM44的mRNA表达水平显著升高(t=8.906,P<0.001)。与FIGO分期Ⅰ+Ⅱ期相比,Ⅲ+Ⅳ期癌组织中TRIM44的mRNA表达水平显著升高(t=3.943,P<0.001)。与组织学分级G1级相比,G2+G3级癌组织中TRIM44的mRNA表达水平显著升高(t=4.046,P<0.001)。与淋巴结未转移的患者相比,转移患者癌组织中TRIM44 mRNA表达水平显著升高(t=4.576,P<0.001)。与siRNA-NC组相比,siRNA-TRIM44组相对细胞活力、迁移和侵袭细胞数量均降低,而凋亡率升高(P<0.05)。与siRNA-NC组相比,siRNA-TRIM44组和8 Gy+siRNA-NC组细胞核P62蛋白相对表达量均升高,而细胞核FLNA和RAD51蛋白相对表达量均降低(P<0.05)。与8 Gy+siRNA-NC组相比,8 Gy+siRNA-TRIM44组细胞核P62蛋白相对表达量升高,而细胞核FLNA和RAD51蛋白相对表达量均降低(P<0.05)。结论TRIM44的高表达促进了EC细胞的增殖和转移,TRIM44的高表达抑制了p62的核转位,从而无法降解细胞核内FLNA和RAD51,导致DNA修复增加、癌细胞活性和放疗抗性升高。

Objective To explore the regulatory mechanism of tripartite motif-containing protein 44(TRIM44)on the growth and the radiosensitivity of endometrial cancer(EC)cells.Methods①The expression level of TRIM44 was detected in cancer tissues and adjacent adjacent tissues of 30 EC patients by immunohistochemistry.The level of TRIM44 mRNA was detected in adjacent tissues and cancer tissues of EC patients with different FIGO stages,histological grades,and lymph node metastasis/non-metastasis by qRT-PCR.②Human endometrial cancer cell lines(HEC-1A)were divided into control group,siRNA-NC group,siRNA-TRIM44 group,LV-NC group and LV-TRIM44 group.HEC-1A cells were transfected into negative control siRNA(siRNA-NC),siRNA targeting TRIM44(siRNA-TRIM44),negative control lentivirus(LV-NC)and TRIM44 overexpression lentivirus(LV-TRIM44 using lipofectamine 2000 reagent)in the corresponding groups,and the cells in control group were not transfected.The mRNA and protein expression levels of TRIM44 in each group were detected by qRT-PCR and Western blot.③HEC-1A cells were divided into siRNA-NC group,siRNA-TRIM44 group,LV-NC group,LV-TRIM44 group,8 Gy+siRNA-NC group,8 Gy+siRNA-TRIM44 group,8 Gy+LV-NC group,8 Gy+LV-TRIM44 group.HEC-1A cells were respectively transfected using lipofectamine 2000 reagent and irradiated with linear accelerator 6-MV X-ray(8 Gy).Cell proliferation was detected by CCK-8,apoptosis was detected by AnnexinⅤ-FITC apoptosis detection kit,cell migration and invasion were detected by Transwell,and the expression of TRIM44,p62,filamin A(FLNA)and RAD51 was detected by qRT-PCR and Western blot.Results Compared with adjacent tissues,the mRNA expression level of TRIM44 in cancer tissues was significantly increased(t=8.906,P<0.001).Compared with FIGO stageⅠ+Ⅱ,the mRNA expression level of TRIM44 in stageⅢ+Ⅳcancer tissues was significantly increased(t=3.943,P<0.001).Compared with histological G1 grade,the mRNA expression level of TRIM44 in G2+G3 grade was significantly increased(t=4.046,P<0.001).Compared with non-metastatic patients,the expression level of TRIM44 mRNA in cancer tissues of metastatic patients was significantly increased(t=4.576,P<0.001).Compared with siRNA-NC group,the relative cell viability and the migration and invasion cell numbers were decreased in siRNA-TRIM44 group,while the apoptosis rate was increased(P<0.05).Compared with siRNA-NC group,the relative expression of nuclear P62 protein was increased in siRNA-TRIM44 group and 8 Gy+siRNA-NC group,while the relative expression of nuclear FLNA and RAD51 protein was decreased(P<0.05).Compared with 8 Gy+siRNA-NC group,the relative expression of nuclear P62 protein was increased in 8 Gy+siRNA-TRIM44 group,while the relative expression of nuclear FLNA and RAD51 protein was decreased(P<0.05).Conclusion The high expression of TRIM44 can promote the proliferation and the metastasis of EC cells,and inhibit the nuclear translocation of p62,thereby failing to degrade FLNA and RAD51 in the nucleus,resulting in the enhancement of DNA repair,cancer cell activity,and radioresistance.