LncRNA LUCAT1激活自噬对肺腺癌A549干细胞影响的研究
Effect of LncRNA LUCAT1 activating autophagy on lung adenocarcinoma A549 stem cells
王鑫 1姜梅 1李乐瑶 1李雷 1王海伦 2李亚军2
作者信息
- 1. 遵义医科大学第三附属医院肿瘤科,遵义市第一人民医院,贵州遵义 563000;遵义医科大学第三附属医院中心实验室,遵义市第一人民医院,贵州遵义 563000
- 2. 遵义医科大学第三附属医院肿瘤科,遵义市第一人民医院,贵州遵义 563000
- 折叠
摘要
目的 探讨LncRNA LUCAT1在肺腺癌A549干细胞(A549/SC)中与细胞自噬调控的关系,及其对A549/SC增殖和迁移的影响.方法 通过qRT-PCR检测A549/SC和肺腺癌A549亲本细胞(A549/MN)中LncRNA LUCAT1的表达情况.慢病毒转染构建稳定敲低LncRNA LUCAT1的A549/SC细胞株(sh-LUCAT1)及对照株(NC-sh LUCAT1);CCK8实验、克隆形成实验、Transwell迁移实验检测抑制LUCAT1表达后细胞增殖和迁移的变化.通过蛋白质印迹法检测各转染组细胞中干性和自噬相关蛋白的表达变化.2组样本比较采用独立样本t检验,采用单因素方差分析对组间差异比较结果进行检验.结果 无血清悬浮培养法可富集得到A549/SC.与A549/MN相比,A549/SC中干性相关蛋白CD44、Nanog和Bmi-1表达水平均升高,均P＜0.05.同时,A549/SC中自噬小体数量明显高于A549/MN(P＜0.01),并且A549/SC中自噬相关蛋白Beclin 1和LC3 Ⅱ/LC3 Ⅰ较A549/MN表达增高,而p62蛋白表达较其亲本细胞偏低,均P＜0.05.A549/SC中LUCAT1的mRNA相对表达量明显高于A549/MN(56.04±6.52 vs 1.04± 0.38),t=14.600,P＜0.001.抑制LUCAT1表达后,A549/SC的增殖、克隆形成及迁移能力均较对照组细胞明显减弱(P＜0.01),同时干性相关蛋白 CD44(t=3.006,P=0.040)、Nanog(t=6.666,P=0.003)、Bmi-1(t=7.294,P=0.002)和自噬相关蛋白 Beclin 1(t=5.460,P=0.005)、LC3Ⅱ/LC3 Ⅰ(t=4.748,P=0.009)表达下降,p62 蛋白(t=2.929,P=0.043)表达升高,差异均有统计学意义.结论 在A549/SC中存在高表达的LncRNA LUCAT1,LUCAT1可激活自噬从而促进A549/SC的增殖和迁移.
Abstract
Objective To investigate the regulatory relationship between LncRNA LUCAT1 and autophagy in lung adeno-carcinoma A549 stem cells(A549/SC),and its effect on proliferation and migration of A549/SC.Methods The expres-sion of LncRNA LUCAT1 in A549/SC and A549 monolayer(A549/MN)was detected by qRT-PCR.A549/SC was ob-tained by infected with LV-LUCAT1 lentivirus to construct A549/SC cell lines(sh-LUCAT1)and control lines(NC-sh LUCAT1)with stable knockdown of LUCAT1,and verify their transfection efficiency.CCK8 assay,clone formation as-say and transwell migration assay were used to detect the changes of cell proliferation and migration after inhibiting LUC-AT1 expression.The expression of stemness-related protein(CD44,Nanog,Bmi-1)and autophagy-related protein(Beclin 1,LC3 Ⅱ/LC3 Ⅰ,p62)were detected by Western blot.Two sets of samples were compared by using independent sam-ple t-tests,and one way ANOVA was used to test the results of inter group differences.Results Serum free suspension culture method can enrich and obtain high expression stemness-related protein of A549/SC(P＜0.05).Meanwhile,the number of autophagosomes in A549/SC was significantly higher than that in A549/MN(P＜0.01),and the expression of autophagy-related proteins Beclin 1 and LC3Ⅱ/LC3Ⅰ in A549/SC was higher than that in A549/MN,while the expression of p62 protein was lower than that of A549/MN.The relative expression of LncRNA LUCAT1 in A549/SC was signifi-cantly higher than that in A549/MN(56.04±6.52 vs 1.04±0.38,t=14.600,P＜0.001).After inhibiting the expres-sion of LUCAT1,the proliferation,clone formation,and migration abilities of A549/SC were significantly reduced com-pared to the control group cells(P＜0.01);while the expression of stemness related proteins:CD44(t=3.006,P=0.040),Nanog(t=6.666,P=0.003),Bmi-1(t=7.294,P=0.002),and the autophagy-related proteins:Beclin1(t=5.460,P=0.005),LC3 Ⅱ/LC3 Ⅰ(t=4.748,P=0.009),was lower than that of the control group,while the protein expression of p62 increased compared to the control group(t=2.929,P=0.043),and the results indicated that the differences were significant.Conclusion The lung adenocarcinoma A549 stem cells exhibite high expression of LncRNA LUC AT1,which can activate autophagy and promote the proliferation and migration ability of A549/SC.
关键词
肺癌/肺腺癌A549干细胞/肺癌相关转录本1/自噬Key words
lung cancer/lung adenocarcinoma A549 stem cells/LncRNA LUCAT1/autophagy引用本文复制引用
基金项目
国家自然科学基金(82060544)
贵州省自然科学基金(黔科合基础-ZK2023一般486)
遵义市第一人民医院研究与试验发展(R&D)计划(院科字202010号)
出版年
2024
NETL
NSTL
万方数据