靶向下调PKM2表达对骨肉瘤细胞增殖与侵袭影响

Effect of reducing PKM2 expression on proliferation and invasion of osteosarcoma cells

目的骨肉瘤是常见的原发性骨恶性肿瘤,M型丙酮酸激酶(pyruvate kinase M2,PKM2)被证实与多种肿瘤进展具有相关性。本实验前期已从组织水平证明了PKM2与骨肉瘤的相关性,本研究从细胞水平探讨PKM2表达对骨肉瘤增殖与侵袭的影响,挖掘PKM2在骨肉瘤早期诊断、治疗靶点和预后评估的价值。方法将携带靶向下调序列PKM-homo-609的siRNA转染骨肉瘤MG63细胞设为实验组(si609组),转染无关序列的MG63细胞为阴性对照组(NC组),正常培养的MG63细胞为空白对照组(KB组),通过蛋白质印迹法检测PKM2在蛋白水平的表达变化。CCK8实验、平板克隆实验检测下调PKM2表达后对骨肉瘤细胞MG63增殖能力的影响。Transwell实验检测下调PKM2后对骨肉瘤细胞MG63侵袭能力的影响。结果 si609组(S-W=0.997,P=0.962)、NC组(S-W=0.750,P=0.342)和KB组(S-W=0.998,P=0.794)的PKM2相对表达量均符合正态分布,si609组(0.48±0.15)分别与NC组(1.40±0.06)、KB组(1.42±0.16)比较,差异均有统计学意义,均P<0.05。CCK8增殖试验中,48hsi609组(S-W=0.783,P=0.324)、NC组(S-W=0.916,P=0.439)和KB组(S-W=1,P=1)吸光度(A)值均符合正态分布,si609组分别与NC组、KB组比较,差异均有统计学意义,均P<0.05。72hsi609组(S-W=1.000,P=1.000)、NC组(S-W=0.855,P=0.253)和KB组(S-W=0.812,P=0.144)A值均符合正态分布。3组组间比较,48h(F=12.250,P=0.008)和72h(F=114.590,P<0.001)差异有统计学意义。平板克隆实验中,si609组(S-W=0.997,P=0.900)、NC组(S-W=0.871,P=0.298)和KB组(S-W=0.991,P=0.817)的细胞克隆计数均符合正态分布;3组细胞克隆计数差异有统计学意义,F=65.73,P<0.001;进一步两两比较,si609组分别与KB组、NC组比较,差异均有统计学意义,均P<0.001。Transwell实验中,si609组(S-W=0.948,P=0.650)、NC组(S-W=0.813,P=0.212)和KB组(S-W=0.91,P=0.30)的穿膜细胞数均符合正态分布;3组穿膜细胞数差异有统计学意义(F=45.056,P<0.001),且si609组分别与KB组和NC组比较,差异均有统计学意义,均P<0.001。结论下调PKM2表达,骨肉瘤细胞增殖与侵袭能力均下降,说明PKM2能促进骨肉瘤细胞的增殖和侵袭。

OBJECTIVE Osteosarcoma is the most common primary bone malignant tumor.The correlation between PKM2 and osteosarcoma has been proved by previous studies of this project from the tissue level.This study explored the effect of PKM2 expression on proliferation and invasion of osteosarcoma at the cellular level,providing a theoretical basis for PKM2 to become a potential target for early diagnosis,treatment and prognosis of osteosarcoma.METHODS Osteosarcoma MG63 cells transfected with siRNA carrying a targeted down-regulation sequence(PMKM-HOMo-609)were used as the experimental group(Si609 group),transfected with unrelated sequence was used as the negative control group(NC group),and normally cultured MG63 cells were used as the blank control group(KB group).Western blot was used to detect the expression changes of PKM2.CCK8 assay and plate cloning assay were used to detect the effect of reducing PKM2 on the proliferation capacity of osteosarcoma cell MG63.Transwell assay was performed to detect the effect of reducing PKM2 on the invasion ability of osteosarcoma cell line MG63.RESULTS The relative PKM2 expression in si609 group(S-W =0.997,P=0.962),NC group(S-W =0.750,P=0.342)and KB group(S-W =0.998,P=0.794)all conform to normal distribution.Expression of PKM2 were significantly decreased in si609 group(0.48±0.15),and the difference was statistically significant(P<0.05)compared with KB group(1.42±0.16)and NC group(1.40±0.06).In the CCK8 proliferation test,the absorbance(A)values of si609 group(S-W =0.783,P=0.324),NC group(S-W=0.916,P=0.439)and KB group(S-W=1.000,P=1.000)all conform to the normal distribution at 48 h.At 72 h,The A values of si609 group(S-W=1.000,P=1.000),NC group(S-W=0.855,P=0.253)and KB group(S-W=0.812,P=0.144)all conform to the normal distribution.Three groups were compared for 48 h(F=12.250,P=0.008)and 72 h(F=114.590,P<0.001),with statistically significant differences.In the plate cloning experiment,the cell clone counts of si609 group(S-W=0.997,P=0.900),NC group(S-W =0.871,P=0.298)and KB group(S-W =0.991,P=0.817)all conform to normal distribution.There were significant differences in cell clone count among the 3 groups(F=65.73,P<0.001).Further pairwise comparison showed that si609 group was statistically significant compared with KB group and NC group respectively(P<0.001).In Transwell experiment,the number of transmembrane cells in si609 group(S-W =0.948,P=0.650),NC group(S-W =0.813,P=0.212)and KB group(S-W=0.914,P=0.309)were all in line with normal distribution.The number of transmambrane cells in the 3 groups was significantly different(F=45.056,P<0.001),and the si609 group was statistically different from the KB group and the NC group(P<0.001).CONCLUSION Reducing of PKM2 expression significantly can decrease the proliferation and invasion ability of osteosarcoma cells,indicating that PKM2 can promote the proliferation and invasion of osteosarcoma cells.