摘要
目的探究双链RNA结合蛋白核因子(NF45)在喉鳞状细胞癌(LSCC)中的表达,及其对LSCC细胞辐射敏感性的影响及机制。方法实时反转录PCR(RT-qPCR)和免疫组化染色检测LSCC及癌旁组织内NF45表达。将NF45-shRNA慢病毒转染至Hep-2细胞,RT-qPCR和蛋白质印迹法(WB)测定细胞转染效果。将Hep-2细胞分为对照组、2 Gy组、sh-NC+2 Gy组和sh-NF45+2 Gy组,进行慢病毒感染和2 Gy X射线照射处理,CCK-8法检测细胞增殖活性,流式细胞术测定细胞凋亡率。采用mCherry-EGFP-LC3B处理各组Hep-2细胞,免疫荧光染色检测细胞自噬水平,WB测定细胞内自噬相关蛋白微管相关蛋白轻链3(LC3)-Ⅱ/LC3-Ⅰ比值及Beclin-1、p62蛋白表达水平。结果NF45在LSCC组织中的表达水平显著高于癌旁组织(P<0.01)。感染NF45-shRNA的Hep-2细胞中NF45 mRNA相对表达量和蛋白相对表达量均显著低于对照组和sh-NC组(P值均<0.05)。与对照组比较,2 Gy组、sh-NC+2 Gy组及sh-NF45+2 Gy组细胞增殖活性均降低,细胞凋亡率增加,细胞内自噬溶酶体增多,LC3-Ⅱ/LC3-Ⅰ比值升高,Beclin-1蛋白相对表达量增加,p62蛋白相对表达量减少(P值均<0.05)。与2 Gy组比较,sh-NF45+2 Gy组细胞增殖活性降低且细胞凋亡率增加,同时,细胞内自噬溶酶体增多,LC3-Ⅱ/LC3-Ⅰ比值升高,Beclin-1蛋白相对表达量增加,p62蛋白相对表达量减少(P值均<0.05)。结论NF45在LSCC组织中表达升高,靶向下调NF45表达能够抑制LSCC细胞增殖活性,促进细胞凋亡,提高肿瘤细胞的辐射敏感性,该机制可能与调控细胞自噬水平有关。
Objective To investigate the expression of double-stranded RNA-binding protein nuclear factor 45(NF45)in laryngeal squamous cell carcinoma(LSCC),and the effect of NF45 on the radiation sensitivity of LSCC cells and its mechanism.Methods NF45 expression in LSCC and adjacent tissues was detected by real-time reverse transcription PCR(qRT-PCR)and immunohistochemical staining.The NF45-ShRNA lentivirus was transfected into Hep-2 cells,and cell transfection efficiency was determined by qRT-PCR and Western blot.Hep-2 cells were randomly divided into the control group,2 Gy group,sh-NC+2 Gy group and sh-NF45+2 Gy group.Lentivirus infection and 2Gy X-ray irradiation treatment were carried out.Cell proliferation activity was assessed by CCK-8 assay.Apoptosis rate was determined by flow cytometry.Hep-2 cells in each group were treated with mCherry-EGFP-LC3B.The levels of autophagy were detected by immunofluorescence staining.The ratio of autophagy-related protein microtubule-associated protein 1 light chain 3(LC3)-Ⅱ/LC3-Ⅰand the expression levels of Beclin-1 and p62 proteins were determined by Western blot.Results The expression level of NF45 in LSCC tissues was significantly higher than that in adjacent tissues(P<0.01).The relative expression levels of NF45 mRNA and protein in Hep-2 cells infected with NF45-shRNA were significantly lower than those in the control and sh-NC groups(all P<0.05).Compared with the control group,the cell proliferation activity was decreased,the apoptosis rate was increased,the intracellular autophagy-lysosome were increased,the ratio of LC3-Ⅱ/LC3-Ⅰwas increased,the relative expression levels of Beclin-1 protein were up-regulated,and the relative expression levels of p62 protein were down-regulated in the 2 Gy,sh-NC+2 Gy and sh-NF45+2 Gy groups(all P<0.05).Compared with the 2 Gy group,the cell proliferation activity was decreased,the apoptosis rate was increased,the intracellular autophagy lysosomes were increased,the LC3-Ⅱ/LC3-Ⅰratio was increased,the relative expression of Beclin-1 p
作者
古丽娜尔·吐尔地
朱成斌
赵辉
迪丽努尔·尼加提
Tuerdi Gulinaer;Zhu Chengbin;Zhao Hui;Nijiati Dilinuer(Radiotherapy Center,People's Hospital of Xinjiang Uygur Autonomous Region,Wulumuqi 830001,China)
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2023年第4期353-359,共7页
Chinese Journal of Radiation Oncology
基金
新疆维吾尔自治区自然科学基金(2020D01C239)。
关键词
癌
鳞状细胞
双链RNA结合蛋白
NF45
辐射敏感性
自噬
Carcinoma,squamous cell
Double-stranded RNA-binding protein
Nuclear factor 45
Radiosensitivity
Autophagy
