基于RNA-seq测序技术研究新藤黄酸抑制黑色素瘤增殖的作用机制  

Mechanism of gambogenic acid inhibiting the proliferation of melanoma based on RNA-seq sequencing technology

作　　者：刘春 王萌[2] 程卉 李庆林[2] LIU Chun;WANG Meng;CHENG Hui;LI Qinglin(College of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,Anhui,China;Key Laboratory of Xin,an Medical Education Ministry,Anhui University of Traditional Chinese Medicine,Hefei 230038,Anhui,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽中医药大学新安医学教育部重点实验室,安徽合肥230038

出　　处：《中国临床药理学与治疗学》2023年第2期155-163,共9页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：安徽高校自然科学研究重点项目(2022AH050529);安徽中医药大学自然科学研究重点项目(2021zrzd08)。

摘　　要：目的:通过分析新藤黄酸(gambogenic acid,GNA)对黑色素瘤移植瘤模型小鼠mRNA表达谱的影响,探讨GNA治疗黑色素瘤可能的作用机制。方法:体外通过MTT法测细胞存活率和倒置显微镜下观察细胞形态来研究GNA对于黑色素瘤细胞的抑制作用。体内实验中通过对比HE(hematoxylin-eosin)染色和免疫组化(Ki-67)的结果观察GNA对黑色素瘤小鼠移植瘤生长的影响,并记录瘤重和瘤重比。RNA-seq测序技术对GNA中剂量组与模型组进行测序,并对筛选出的mRNA进行GO和KEGG分析,最后利用实时定量荧光PCR验证差异表达基因的筛选结果。结果:不同剂量的GNA作用于黑色素瘤小鼠模型后,模型小鼠的肿瘤组织内出现大面积坏死,肿瘤生长受到明显地抑制。mRNA测序共鉴定出36个差异表达的mRNA,其中30个上调,6个下调,根据GO和KEGG分析的基因组邻接预测mRNA的可能功能。采用实时荧光定量PCR技术进一步验证所选差异mRNA的表达,结果显示Cidec、Mylk4、Ces1d和Igkv9-123的mRNA表达上调,Ryr3和Hapln1的mRNA表达下调。结论:GNA可以体内外抑制黑色素瘤细胞的增殖,其机制与调控细胞因子-细胞因子受体相互作用、NF-κB、MAPK等通路上的mRNA表达有关。AIM:By analyzing the effect of gambogenic acid(GNA)on the mRNA expression profile of melanoma xenograft model mice,the possible mechanism of GNA in the treatment of melanoma was explored.METHODS:The inhibitory effect of GNA on melanoma cells was studied by measuring the cell survival rate by MTT method in vitro and observing the cell morphology under an inverted microscope.In the in vivo experiment,the effect of GNA on the growth of xenografted tumors in melanoma mice was observed by comparing the results of HE(hematoxylin-eosin)staining and immunohistochemistry(Ki-67),and the tumor weight and tumor weight ratio were recorded.RNA-seq sequencing technology was used to sequence the GNA medium-dose group and the model group,and the screened mRNAs were analyzed by GO and KEGG,and finally the screening results of differentially expressed genes were verified by real-time quantitative fluorescent PCR.RESULTS:After different doses of GNA acted on the melanoma mouse model,a large area of necrosis occurred in the tumor tissue of the model mouse,and the tumor growth was significantly inhibited.A total of 36 differentially expressed mRNAs were identified by mRNA sequencing,of which 30 were up-regulated and 6 were down-regulated.The possible functions of the mRNAs were predicted according to the genomic adjacency analyzed by GO and KEGG.The expression of the selected differential mRNAs was further verified by real-time quantitative PCR technology.The results showed that the mRNA expressions of Cidec,Ces1d,Mylk4,and Igkv9-123 were up-regulated,and the mRNA expressions of Ryr3 and Hapln1 were down-regulated.CONCLUSION:GNA can inhibit the proliferation of melanoma cells in vitro and in vivo,and its mechanism is related to the regulation of cytokine-cytokine receptor interaction,NF-κB,MAPK,and other pathways of mRNA expression.

关 键 词：黑色素瘤 新藤黄酸 RNA测序 MRNA 

分 类 号：R965.2[医药卫生—药理学]