摘要
目的探讨lncRNA PVT1调控巨噬细胞向M1型极化对胰腺癌细胞侵袭的影响及机制。方法培养单核细胞株THP-1,通过感染NC shRNA慢病毒、PVT1 shRNA慢病毒、miR-NC慢病毒、miR-409-5p慢病毒、miR-409-5p抑制物慢病毒及嘌呤霉素筛选的方法得到稳定转染的THP-1细胞,检测lncRNA PVT1、miR-409-5p、音猬因子(SHH)的表达水平;进行M1型极化诱导后检测TNF-α、IL-1β、IL-6、诱导型一氧化氮合酶(iNOS)的表达水平。稳定转染的THP-1细胞进行M1型极化诱导后与胰腺癌细胞株PANC-1细胞共培养,检测PANC-1细胞的侵袭数目。结果稳定转染PVT1 shRNA后,THP-1细胞中lncRNA PVT1、SHH表达明显降低(NC shRNA组、PVT1 shRNA组lncRNA PVT1分别为1.00±0.12、0.43±0.05;SHH分别为1.00±0.13、0.47±0.05)(均P<0.05),miR-409-5p、TNF-α、IL-1β、IL-6和iNOS表达明显增加(NC shRNA组、PVT1shRNA组的miR-409-5p分别为1.00±0.08、1.83±0.19;TNF-α分别为1.00±0.11、1.93±0.25,IL-1β分别为1.00±0.08、1.77±0.21,IL-6分别为1.00±0.09、2.31±0.20;iNOS分别为1.00±0.13、2.09±0.17)(均P<0.05),共培养体系中PANC-1细胞的侵袭数目明显减少(P<0.05);稳定转染miR-409-5p后,THP-1细胞中SHH表达明显降低(P<0.05),miR-409-5p、TNF-α、IL-1β、IL-6、i NOS表达明显增加(P<0.05),共培养体系中PANC-1细胞的侵袭数目明显减少(P<0.05);稳定转染PVT1 shRNA与miR-409-5p抑制物后,THP-1细胞中SHH表达明显增加,miR-409-5p、TNF-α、IL-1β、IL-6、iNOS表达明显降低(均P<0.05),共培养体系中PANC-1细胞的侵袭数目明显增加(P<0.05)。结论敲低lncRNA PVT1能够促进巨噬细胞M1型极化并抑制胰腺癌细胞侵袭,这一调控作用可能与靶向miR-409-5p/SHH轴有关。
Objective To study the effect and mechanism of knocking down lncRNA PVT1 regulating M1polarization of macrophage on invasion of pancreatic cancer cells.Methods THP-1 monocyte cell line was cultured,the stably transfected THP-1 cells were obtained by infecting NC shRNA lentivirus,PVT1 shRNA lentivirus,miR-NC lentivirus,miR-409-5p lentivirus,miR-409-5p inhibitor lentivirus and puromycin screening.The expression of lncRNA PVT1,miR-409-5p and SHH were detected.After M1 polarization induction,the expression of tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β),interleukin 6(IL-6),inducible nitric oxide synthase(iNOS)were detected.After M1 polarization induction,the stable transfected THP-1 cells and pancreatic cancer cell line PANC-1 were co-cultured in transwell and the number of invaded PANC-1 cells was detected.Results After stable transfection of PVT1 shRNA,the expression of lncRNA PVT1(1.00±0.12 vs.0.43±0.05)and SHH(1.00±0.13 vs.0.47±0.05)in THP-1 cells significantly decreased(all P<0.05),and the expression of miR-409-5p(1.00±0.08 vs.1.83±0.19),TNF-α(1.00±0.11 vs.1.93±0.25),IL-1β(1.00±0.08 vs.1.77±0.21),IL-6(1.00±0.09 vs.2.31±0.20),iNOS(1.00±0.13 vs.2.09±0.17)significantly increased,and the number of invaded PANC-1 cells number significantly decreased in the co-culture system(all P<0.05).After stable transfection of miR-409-5p,the expression level of SHHin THP-1 cells significantly decreased(P<0.05),and the expression of miR-409-5p,TNF-α,IL-1β,IL-6 and iNOS significantly increased(all P<0.05),and the number of invaded PANC-1 cells significantly decreased in the co-culture system(P<0.05).After stable transfection of PVT1 shRNA and miR-409-5p inhibitor,the expression level of SHH in THP-1 cells significantly increased(P<0.05),and the expression of miR-409-5p,TNF-α,IL-1β,IL-6 and iNOS significantly increased(all P<0.05),and the number of invaded PANC-1 cells significantly decreased in the co-culture system(P<0.05).Conclusion Knocking down lncRNA PVT1 promotes macrophage M1polarization and inhibits invasion of pancreatic cancer cells,which may be related with targeting miR-409-5p/SHH axis.
作者
石宇
严晓娣
沈超艳
姚宁华
钱静
SHI Yu;YAN Xiao-di;SHEN Chao-yan;YAO Ning-hua;QIAN Jing(Department of Tumor Radiotherapy,Afiliated Hospital of Nantong University,Nantong,Jiangsu Province 226001,China)
出处
《解剖学研究》
CAS
2023年第5期417-424,共8页
Anatomy Research
基金
南通市科技项目(JC2021148)。
