沉默 PKM2对人肺腺癌细胞系及移植瘤的放射增敏研究

A study of radiosensitizing effect of PKM2 silencing in lung adenocarcinoma cells and xenografts

目的：研究沉默丙酮酸激酶 M2型（PKM2）对人肺腺癌细胞系（A549细胞）的放射敏感性及移植瘤的放射协同作用，并探索相关机制。方法将 PKM2基因干扰质粒 pshRNA.PKM2稳定转染至 A549细胞，同时设立空载质粒转染组和未转染组。采用蛋白印迹法检测 A549细胞 pshR.NA.PKM2的沉默效率及微管相关蛋白1轻链3（LC3）表达水平，克隆形成实验、移植瘤生长延迟法检测沉默 PKM2后对 A549细胞、移植瘤放射增敏效应，透射电镜观察 A549细胞及移植瘤自噬形成，免疫组化法检测移植瘤中 PKM2的表达水平。行 Student′s t 检验组间差异，裸鼠体重及移植瘤体积采用连续变量的方差分析。结果成功获得转染 pshRNA.PKM2的 A549稳定细胞株。 pshRNA.PKM2组可显著下调细胞和移植瘤 PKM2的蛋白表达水平（P＝0.001、0.000），对 A549细胞的 SER 为1.47，移植瘤的 SER 为2.00。干扰 PKM2可增加放射所诱导的自噬形成并增加 LC3.Ⅱ／Ⅰ的比值（ P＝0.0001）。结论沉默 PKM2调节自噬可能提高 A549细胞及移植瘤的放射敏感性，其有望成为NSCLC 有效放射增敏靶点，但有待进一步研究确认。

Objective To investigate the impacts of pyruvate kinase M2 isoform （PKM2） silencing on the radiosensitivity of lung adenocarcinoma cell line （A549 cells） and the radiation synergy of xenografts, and to explore their mechanisms. Methods Plasmid pshRNA.PKM2 for interference with PKM2 expression was transfected into A549 cells, and empty vector.transfected cells and untransfected cells were set as con.trols. The silencing efficiency of pshRNA.PKM2 and the expression level of microtubule.associated protein 1 light chain 3（LC3） were measured by Western blot assay. The radiosensitizing effects in A549 cells and xen.ografts after PKM2 silencing were determined by colony.forming assay and xenografts growth curves. Autoph.agy formation in A549 cells and xenografts was analyzed by transmission electron microscopy, and the ex.pression level of PKM2 in xenografts was measured by immunohistochemistry. Comparison between groups was made by Student′s t.test, and the body weights of nude mice and xenograft volumes were subjected to a.nalysis of variance for continuous variables. Results Stable A549 cell lines transfected with pshRNA.PKM2 were successfully produced. Transfection with pshRNA.PKM2 significantly down.regulated PKM2 expression in A549 cells and xenografts （P= 0. 001;P= 0. 000）. The sensitizer enhancement ratios for A549 cells and xenografts were 1. 47 and 2. 00, respectively. Interference with PKM2 expression enhanced radiation.in duced autophagy formation and significantly increased the ratio of LC 3 . II / I （ P= 0.000 1 ） . Conclusions Silencing of PKM2 expression may enhance the radiosensitivity of A549 cells and xenografts by regulation of autophagy, which holds promise for becoming an effective radiosensitizing target for non.small cell lung canc.er, but still needs to be confirmed by further studies.