lncRNA BLACAT1在下咽鳞状细胞癌中介导自噬调控肿瘤细胞的化疗敏感性

lncRNA BLACAT1 mediating autophagy in hypopharyngeal squamous cell carcinoma to modulate the chemosensitivity of tumor cells

目的探究长链非编码RNA(lncRNA)膀胱癌相关转录因子1(BLACAT1)对下咽鳞状细胞癌(HSCC)细胞株顺铂(DDP)敏感性的影响及其作用机制。方法采用qRT-PCR检测40例HSCC患者的癌组织、癌旁组织、HSCC细胞株FaDu及DDP耐药细胞株FaDu/DDP中lncRNA BLACAT1的表达水平,CCK-8检测经不同浓度DDP处理后FaDu细胞和FaDu/DDP细胞的存活率。分别转染siRNA-BLACAT1序列和siRNA阴性对照至FaDu/DDP细胞中,记为si-BLACAT1组和si-NC组,以未转染的FaDu/DDP细胞作为对照组,qRT-PCR验证转染效果,CCK-8检测转染后经不同浓度DDP处理的FaDu/DDP细胞存活率,流式细胞术检测细胞凋亡率,透射电子显微镜观察细胞自噬情况,免疫荧光染色测定LC3B荧光斑点的形成情况,Western blot检测细胞自噬相关蛋白的表达。结果HSCC组织中lncRNA BLACAT1 mRNA相对表达量显著高于癌旁组织(P<0.01);FaDu/DDP细胞中lncRNA BLACAT1 mRNA相对表达量高于FaDu细胞(P<0.05)。si-BLACAT1组细胞lncRNA BLACAT1 mRNA相对表达量低于对照组和si-NC组(P<0.05);当DDP作用浓度在6.25μmol/L及以上时,FaDu/DDP细胞存活率高于FaDu细胞(P<0.05),si-BLACAT1+DDP组细胞存活率低于DDP组和si-NC+DDP组(P<0.05)。与对照组比较,DDP组和si-NC+DDP组FaDu/DDP细胞凋亡率升高(P<0.05);与DDP组和si-NC+DDP组比较,si-BLACAT1+DDP组细胞凋亡率也升高(P<0.05)。对照组有一些自噬体和自噬溶酶体形成,DDP组和si-NC+DDP组自噬溶酶体形成较多,而si-BLACAT1+DDP组细胞中未观察到自噬体及自噬溶酶体。DDP组和si-NC+DDP组细胞内LC3B荧光斑点数较对照组增加(P<0.05);si-BLACAT1+DDP组细胞内LC3B荧光斑点数较DDP组和si-NC+DDP组均减少(P<0.05)。与对照组比较,DDP组和si-NC+DDP组FaDu/DDP细胞中LC3-Ⅱ/LC3-Ⅰ水平上调,Beclin-1蛋白表达量上升,p62蛋白表达量下降(P<0.05);与DDP组和si-NC+DDP组比较,si-BLACAT1+DDP组细胞中LC3-Ⅱ/LC3-Ⅰ水平下调,Beclin-1蛋白表达量下降,p62蛋白表达量上升(P<0.05)。结论lncRNA BLACAT1在HSCC组织及DDP耐药细胞株FaDu/DDP中高表达,下调lncRNA BLACAT1能够提高HSCC细胞对DDP的敏感性,其作用机制可能通过抑制自噬水平实现。

Objective To explore the effect of long non-coding RNA(lncRNA)bladder cancer associated transcript 1(BLACAT1)on the sensitivity of hypopharyngeal squamous cell carcinoma(HSCC)cell lines to cisplatin(DDP)and its mechanism.Methods qRT-PCR was used to detect the expression levels of lncRNA BLACAT1 in the cancer tissues and adjacent tissues of 40 HSCC patients,HSCC cell line FaDu and DDP-resistant cell line FaDu/DDP,CCK-8 was used to detect the survival rate of FaDu cells and FaDu/DDP cells after treated by different concentrations of DDP.The siRNA-BLACAT1 sequence and the siRNA negative control were transfected into the FaDu/DDP cells respectively,which were designated as the si-BLACAT1 group and the si-NC group,and the untransfected FaDu/DDP cells were designated as the control group;qRT-PCR was used to verify the transfection effect,CCK-8 was used to detect the survival rate of FaDu/DDP cells treated by different concentrations of DDP after transfection,flow cytometry was used to detect the apoptosis rate,transmission electron microscopy was used to observe the autophagy,immunofluorescence staining was used to determine the formation of LC3B fluorescent spots,Western blot was used to detect the expression of autophagy-related proteins.Results The relative expression of lncRNA BLACAT1 mRNA in the HSCC tissues was significantly higher than that in the adjacent tissues(P<0.01);the relative expression of lncRNA BLACAT1 mRNA in the FaDu/DDP cells was higher than that in the FaDu cells(P<0.05).The relative expression of lncRNA BLACAT1 mRNA in the si-BLACAT1 group was lower than that in the control group and the si-NC group(P<0.05);when the concentrations of DDP were 6.25μmol/L and above,the survival rate of FaDu/DDP cells was higher than that of the FaDu cells(P<0.05),the survival rate in the si-BLACAT1+DDP group was lower than that in the DDP group and the si-NC+DDP group(P<0.05).Compared with the control group,the apoptosis rate of FaDu/DDP cells in the DDP group and the si-NC+DDP group increased(P<0.05);compared with the DDP group and the si-NC+DDP group,the apoptosis rate of the si-BLACAT1+DDP group also increased(P<0.05).A number of autophagosomes and autophagolysosomes were observed in the control group,more autophagolysosomes were observed in the DDP group and the si-NC+DDP group,while autophagosomes and autophagolysosomes were not observed in the si-BLACAT1+DDP group.Compared with the control group,the number of intracellular LC3B fluorescent spots in the DDP group and the si-NC+DDP group increased(P<0.05);compared with the DDP group and the si-NC+DDP group,the number of intracellular LC3B fluorescent spots in the si-BLACAT1+DDP group decreased(P<0.05).Compared with the control group,the levels of LC3-Ⅱ/LC3-Ⅰin FaDu/DDP cells were up-regulated,Beclin-1 protein expression increased,and p62 protein expression decreased in the DDP group and the si-NC+DDP group(P<0.05);compared with the DDP group and the si-NC+DDP group,the level of LC3-Ⅱ/LC3-Ⅰwas down-regulated,Beclin-1 protein expression decreased,and p62 protein expression increased in the si-BLACAT1+DDP group(P<0.05).Conclusion The lncRNA BLACAT1 is highly expressed in the HSCC tissues and the DDP-resistant cell line FaDu/DDP.The down-regulation of lncRNA BLACAT1 can increase the sensitivity of HSCC cells to DDP,and its mechanism may be achieved via inhibiting the level of autophagy.