毛壳菌素通过作用于JAK2-STAT3信号通路抑制神经母细胞瘤的增殖及迁移  

Chetomin inhibits the proliferation and migration of neuroblastoma through JAK2-STAT3 signaling pathway

作　　者：唐湘莲[1] 周宇翔[1] 黄召[1] 肖雅玲[1] TANG Xianglian;ZHOU Yuxiang;HUANG Zhao;XIAO Yaling(Department of Pediatric Surgery, Hunan Children′s Hospital, Changsha 410007, China)

机构地区：[1]湖南省儿童医院普外二科,长沙410007

出　　处：《中国小儿血液与肿瘤杂志》2021年第4期198-203,217,共7页Journal of China Pediatric Blood and Cancer

基　　金：湖南省卫生健康委2020年度科研课题(20200320)。

摘　　要：目的探讨毛壳菌素影响神经母细胞瘤SH-SY5Y增殖和迁移的机制。方法应用DMSO作为对照,与毛壳菌素分别与SH-SY5Y细胞进行孵育,应用荧光定量PCR(RT-qPCR)和WB检测毛壳菌素的靶分子HIF-1α的mRNA相对表达水平以及蛋白表达水平。采用流式细胞术和CCK8实验检测细胞的增殖,利用Transwell试验检测细胞的迁移能力。采用流式细胞术和WB检测JAK2-STAT3信号通路的活化情况。结果人神经母细胞瘤SH-SY5Y细胞中HIF-1α的mRNA相对表达量为1.68±0.06,显著高于人正常胶质细胞(0.98±0.05,均P<0.001),SH-SY5Y细胞系HIF-1α的WB检测的蛋白水平相对灰度值为1.81±0.04,显著高于人正常胶质细胞(0.97±0.06,均P<0.001)。毛壳菌素孵育后的SH-SY5Y细胞的Ki-67-high的占比为(35.89±7.23)%,显著低于DMSO对照组[(76.81±5.12)%,P<0.01];Ki-67的平均荧光强度为573.7±80.25,显著低于DMSO对照组(1522±53.41,P<0.001)。CCK8检测细胞的增殖能力,毛壳菌素组在16h,24h和48h的OD450nm值分别为0.25±0.06,0.30±0.02,0.38±0.03,显著低于对照组(0.40±0.05,0.56±0.02,0.77±0.05,P<0.01)。迁移实验结果表明,DMSO对照组的迁移能力显著高于毛壳菌素组,细胞数分别为22816±4267.2和68172±9733.5,P<0.001。WB分析JAK2和STAT3的磷酸化水平,毛壳菌素孵育细胞的p-JAK2和p-STAT3的蛋白相对表达水平显著低于DMSO对照组,差异具有统计学意义(P<0.001)。流式细胞术分析SH-SY5Y细胞的p-JAK2和p-STAT3所占的比例及平均荧光强度,毛壳菌素组显著低于DMSO对照组,差异具有统计学意义(P<0.01)。WB和流式细胞术分别检测IL-6与毛壳菌素共同孵育后的JAK和STAT3的磷酸化水平,IL-6能够逆转毛壳菌素对JAK2和STAT3磷酸化的抑制作用。结论毛壳菌素可能通过抑制HIF-1α,抑制JAK2-STAT3信号通路的活化,从而抑制神经母细胞瘤SH-SY5Y细胞的增殖和迁移。Objective To investigate the mechanism of Chetomin inhabiting the proliferation and migration of neuroblastoma.Methods DMSO was used as control for Chetomin to incubate with SH-SY5Y cells.Real-time quantitative PCR(RT-qPCR)and western blot(WB)were used to detect the mRNA and protein expression levels of HIF-1α,the target molecule of Chetomin.Cell proliferation was detected by flow cytometry and Cell Counting Kit-8(CCK8)assay,and cell migration ability was detected by Transwell assay.The activation of JAK2-STAT3 signaling pathway was detected by flow cytometry and WB.Results The relative mRNA expression of HIF-1αin SH-SY5Y cells was 1.68±0.06,significantly higher than that in normal human glial cells(0.98±0.05,P<0.001),and the relative gray value of HIF-1αprotein levels detected by WB in SH-SY5Y cell line was 1.81±0.04,significantly higher than that in normal human glial cells(0.97±0.06,P<0.001).The Ki-67-high proportion of SH-SY5Y cells incubated with Chetomin was(35.89±7.23)%,which was significantly lower than that of DMSO control group((76.81±5.12)%,P<0.01).The mean fluorescence intensity of Ki-67 was 573.7±80.25,which was significantly lower than that of DMSO control group(1522±53.41,P<0.001).CCK8 assay was used to detect the proliferation ability of SH-SY5Y cells,and the OD450 nm reading values at 16,24 and 48 hours were 0.25±0.06,0.30±0.02,and 0.38±0.03 separately in the Chetomin group,which were significantly lower than those in the control group(0.40±0.05,0.56±0.02,0.77±0.05,P<0.01).The data of migration experiment showed that the migration ability of DMSO control group was significantly higher than that of Chetomin group,with cell numbers of 22816±4267.2 and 68172±9733.5 respectively,P<0.001.The phosphorylation levels of JAK2 and STAT3 were analyzed by WB,and the relative protein expression levels of p-JAK2 and P-STAT3 in the cells incubated with Chetomin were significantly lower than those in the DMSO control group(P<0.001).Flow cytometry analysis of the proportion of p-JAK2 and p-STAT

关 键 词：毛壳菌素 HIF-1Α JAK2-STAT3 神经母细胞瘤 SH-SY5Y 

分 类 号：R73[医药卫生—肿瘤]