PTPRZ1-MET融合基因阳性脑胶质瘤细胞对MET靶向抑制剂耐药机制的初步探究

Preliminary study on the resistance mechanism of PTPRZ1-MET fusion gene-positive glioma cells to MET-targeted inhibitors

目的初步探究PTPRZ1-MET融合基因(ZM)阳性脑胶质瘤细胞(U87-ZM)对MET靶向抑制剂—PLB-1001的耐药机制。方法取U87-ZM细胞,用含有PLB-1001的培养基培养48 h,获得U87-ZM+PLB-1001细胞;培养4个月获得U87-ZM-LT细胞。采用CCK-8法检测U87-ZM和U87-ZM-LT细胞对PLB-1001的敏感性,采用EdU细胞增殖检测法评价U87-ZM+PLB-1001和U87-ZM-LT细胞的增殖活性。采用蛋白质免疫印迹法(WB)评价PLB-1001对U87-ZM和U87-ZM+PLB-1001细胞的抑制效应及各组细胞的细胞周期蛋白D1(Cyclin D1)的表达,采用实时荧光定量PCR(qRT-PCR)法检测各组细胞Cyclin D1 mRNA的表达水平。结果WB结果显示,U87-ZM与U87-ZM+PLB-1001细胞p-MET蛋白的相对表达量分别为1.22±0.06和0(P<0.05),MET总蛋白的相对表达量分别为1.00±0.06和0.98±0.09(P=0.810)。CCK-8检测显示,PLB-1001对U87-ZM细胞和U87-ZM-LT细胞均具有生长抑制作用,且该抑制作用随PLB-1001浓度的增加而增强,其中U87-ZM细胞的半抑制浓度(IC50)为(13.42±0.74)μmol/L,U87-ZM-LT细胞的IC50为(40.76±1.20)μmol/L,差异有统计学意义(P<0.05)。EdU细胞增殖检测显示,U87-ZM、U87-ZM-LT细胞中EdU阳性细胞的百分率分别为(23.10±4.32)%、(21.28±1.22)%,均高于U87-ZM+PLB-1001细胞中的(10.25±3.40)%,差异均有统计学意义(均P<0.05)。WB结果显示,U87-ZM、U87-ZM-LT细胞Cyclin D1蛋白的相对表达量分别为1.08±0.06、0.94±0.15,与U87-ZM+PLB-1001细胞的0.66±0.08比较,差异均具有统计学意义(均P<0.05)。qRT-PCR检测显示,U87-ZM、U87-ZM-LT细胞Cyclin D1 mRNA的相对表达量分别为1.01±0.00、1.14±0.21,与U87-ZM+PLB-100细胞的0.92±0.15比较,差异均无统计学意义(均P>0.05)。结论相较于PLB-1001短期处理的U87-ZM细胞,PLB-1001长期处理的U87-ZM细胞具有更强的DNA合成活性;U87-ZM细胞对MET靶向抑制剂的耐药机制可能与Cyclin D1蛋白增加有关,可针对该通路深入探索逆转肿瘤耐药的可行方案。

Objective To explore the resistance mechanism of PTPRZ1-MET fusion gene(ZM)-positive glioma cells(U87-ZM)to the MET-targeted inhibitor PLB-1001.Methods The cells of U87-ZM were cultured in the medium containing PLB-1001 for 48 hours to obtain U87-ZM+PLB-1001 cells;after 4 months of culture,U87-ZM-LT cells were obtained.The CCK-8 method was used to detect the sensitivity of cells to PLB-1001 in the U87-ZM and U87-ZM-LT groups,and the EdU cell proliferation method was used to evaluate the proliferation activity of the cells in the U87-ZM+PLB-1001 and U87-ZM-LT groups.Western blot(WB)was used to evaluate the inhibitory effect of PLB-1001 on U87-ZM and U87-ZM+PLB-1001 cells and the expression of cyclin D1 protein in each group.The transcriptional levels of Cyclin D1 in each group was detected by real-time fluorescence quantitative PCR(polymerase chain reaction).Results WB results showed that the relative expression of p-MET protein in U87-ZM group and U87-ZM+PLB-1001 group were 1.22±0.06 and 0(P<0.05),and the relative expression of total MET protein was 1.00±0.06 and 0.98±0.09(P=0.810)respectively.The results of CCK-8 showed that PLB-1001 inhibited the growth of U87-ZM cells and U87-ZM-LT cells,and the inhibition increased with the enhancement of PLB-1001 concentration.The half inhibitory concentration(IC50)of U87-ZM cells was 13.42±0.74μmol/L,the IC50 of U87-ZM-LT cells was 40.76±1.20μmol/L,and the difference was statistically significant(P<0.05).The results of EdU showed that the percentages of EdU positive cells in the U87-ZM group and U87-ZM-LT group was(23.10±4.32)%and(21.28±1.22)%respectively,which were higher than in the U87-ZM+PLB-1001 group[(10.25±3.40)%],and the differences were statistically significant(both P<0.05).WB results showed that the relative expression levels of Cyclin D1 protein in U87-ZM and U87-ZM-LT groups were 1.08±0.06 and 0.94±0.15,respectively,which were statistically different compared with 0.66±0.08 in U87-ZM+PLB-1001 group(both P<0.05).PCR detection showed that the relative expression of Cyclin D1 mRNA in the U87-ZM and U87-ZM-LT groups were 1.01±0.00 and 1.14±0.21,respectively,which had no significant difference compared with 0.92±0.15 in the U87-ZM+PLB-100 group(both P>0.05).Conclusions Compared with U87-ZM cells treated with PLB-1001 for a short period of time,U87-ZM cells treated with PLB-1001 for a long period of time have stronger DNA synthesis activity.The resistance mechanism of U87-ZM cells to MET-targeted inhibitors may be related to the increase of Cyclin D1 protein,and a feasible solution for reversing tumor resistance could be explored according to that pathway.