ORY-1001靶向赖氨酸特异性去甲基化酶1下调胶质母细胞瘤Notch/HES1通路的表达并发挥抗肿瘤作用

doi:10.12122/j.issn.1673-4254.2024.08.22

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1620-1630.doi: 10.12122/j.issn.1673-4254.2024.08.22

• • 上一篇

ORY-1001靶向赖氨酸特异性去甲基化酶1下调胶质母细胞瘤Notch/HES1通路的表达并发挥抗肿瘤作用

杨红丽¹^,³(), 向亚运², 谭婷婷², 雷阳²()

^1.重庆医科大学附属第一医院神经内科//国家卫健委功能性脑疾病诊治重点实验室，重庆 400000
^2.重庆医科大学附属大学城医院医学科学研究中心//神经内科，重庆 400000
^3.简阳市人民医院急诊科，四川简阳 641400

收稿日期:2024-04-03 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 雷阳 E-mail:2021110163@stu.cqmu.edu.cn;leiyang339@hospital.cqmu.edu.cn
作者简介:杨红丽，主治医师，E-mail: 2021110163@stu.cqmu.edu.cn
基金资助:
重庆市自然科学基金(cstc2020jcyj-msxmX0323)

ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression

Hongli YANG¹^,³(), Yayun XIANG², Tingting TAN², Yang LEI²()

^1.Department of Neurology, First Affiliated Hospital of Chongqing Medical University; National Health Commission Key Laboratory of Diagnosis and Treatment on Brain Functional Diseases, Chongqing 400000, China
^2.Medical Sciences Research Center, Department of Neurology, University-Town Hospital of Chongqing Medical University, Chongqing 400000, China
^3.Department of Emergency Medicine, Jianyang People's Hospital, Jianyang 641400, China

Received:2024-04-03 Online:2024-08-20 Published:2024-09-06
Contact: Yang LEI E-mail:2021110163@stu.cqmu.edu.cn;leiyang339@hospital.cqmu.edu.cn

摘要/Abstract

摘要：

目的探究赖氨酸特异性去甲基化酶1（LSD1）抑制剂ORY-1001对胶质母细胞瘤（GBM）的抑瘤作用和机制。方法从公开数据库（TCGA和HPA）下载相关数据，分析LSD1在GBM和正常脑中的表达情况。将24只雌性BALB/c小鼠，随机分为对照组与ORY-1001组，12只/组。肿瘤细胞种植后，每7 d 用400 µg/kg ORY-1001进行灌胃，检测肿瘤生长和动物生存时间，评估ORY-1001对GBM的抗肿瘤效应。检测A_{490 nm}值测定ORY-1001对GBM细胞活力的影响。Western blotting检测ORY-1001对LSD1表达的影响。利用慢病毒试剂，构建稳定敲降LSD1的GBM细胞（sh-LSD1），并通过动物成瘤实验评估沉默LSD1在活体动物内的作用以检测LSD1药物抑制获得的表型特异性。运用生物信息学方法分析与LSD1相关的基因及通路。Western blotting检测沉默LSD1及不同剂量ORY-1001治疗后Notch/HES1通路的表达。通过慢病毒转染，构建稳定过表达Notch1（oe-Notch1）的U87细胞。测定过表达Notch1后，ORY-1001对GBM动物模型肿瘤生长及生存时间的影响。通过ChIP揭示ORY-1001对Notch/HES1通路的具体调控机制。结果 LSD1在GBM中高表达，并与患者的预后呈负相关（P<0.001）。ORY-1001治疗以及LSD1基因沉默都使荷瘤动物肿瘤负荷减轻（P<0.05）和生存时间延长（P<0.001），对多种GBM细胞的活力均表现出抑制作用（P<0.05）。ORY-1001呈剂量依赖性地抑制LSD1的表达。Notch通路富集了与LSD1抑制相关的差异基因，LSD1沉默及ORY-1001治疗后Notch/HES1通路表达显著下调（P<0.05），逆转了ORY-1001对GBM的抗肿瘤作用（P<0.05）。结论 ORY-1001通过抑制GBM中LSD1的表达而下调Notch/HES1通路，并发挥抗肿瘤效应。

关键词: 赖氨酸特异性去甲基化酶1, 生信分析, 胶质母细胞瘤, Notch/HES1通路, 癌症治疗

Abstract:

Objective To explore the inhibitory effect ORY-1001, a lysine-specific histone demethylase 1 (LSD1) inhibitor, on growth of glioblastoma (GBM) and the underlying mechanism. Methods We analyzed LSD1 expressions in GBM and normal brain tissues based on data from TCGA and HPA databases. Female BALB/c mouse models bearing xenografts derived from U87 cells or cells with lentivirus-mediated LSD1 silencing or Notch overexpression were treated with saline or 400 µg/kg ORY-1001 by gavage every 7 days, and GBM formation and survival time of the mice were recorded. The effect of ORY-1001 on GBM cell viability was assessed, and its effect on LSD1 expression was analyzed with Western blotting. The genes and pathways associated with LSD1 were analyzed using bioinformatics methods. Western blotting and qRT-PCR were used to detect Notch/HES1 pathway expression after LSD1 silencing and ORY-1001 treatment. The impact of ORY-1001 on viability of U87 cells with Notch1 silencing or overexpression was assessed, and the regulatory effects of ORY-1001 on Notch/HES1 pathway were analyzed using chromatin immunoprecipitation assay. Results A high expression of LSD1 in GBM was negatively correlated with patient survival (P<0.001). ORY-1001 and LSD1 silencing obviously reduced tumor burden and prolonged the survival time of GBM-bearing mice. ORY-1001 treatment significantly inhibited the viability and dose-dependently decreased LSD1 expression in GBM cells, and such inhibitory effect of ORY-1001 was attenuated by LSD1 silencing. The Notch pathway enriched the differential genes related to LSD1, and Notch/HES1 pathway expression was significantly down-regulated after LSD1 silencing and ORY-1001 treatment. Notch1 overexpression significantly attenuated the anti-tumor effect of ORY-1001 on GBM. Mechanistically, ORY-1001 disrupted the interaction between LSD1 and the Notch pathway target genes including Notch3, HES1 and CR2. Conclusion ORY-1001 down-regulates the Notch/HES1 pathway by inhibiting LSD1 expression to suppress the growth of GBM in mice.

Key words: Lysine-specific histone demethylase 1, bioinformatics analysis, glioblastoma, Notch/HES1 pathway, cancer treatment

杨红丽, 向亚运, 谭婷婷, 雷阳. ORY-1001靶向赖氨酸特异性去甲基化酶1下调胶质母细胞瘤Notch/HES1通路的表达并发挥抗肿瘤作用[J]. 南方医科大学学报, 2024, 44(8): 1620-1630.

Hongli YANG, Yayun XIANG, Tingting TAN, Yang LEI. ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression[J]. Journal of Southern Medical University, 2024, 44(8): 1620-1630.

图/表 5

表1 qRT-PCR引物序列

Tab.1 Sequence of primers used for qRT-PCR

Primer

Sequences 5'-3'

Notch1

Forward

Reverse

CTGAAGAACGGGGCTAACAA

CAGGTTGTACTCGTCCAGCA

HES1

Forward

Reverse

GCAGATGACGGCTGCGCTGA

AAGCGGGTCACCTCGTTCATGC

HES4

Forward

Reverse

GCTCAGCTCAAAACCCTCATC

TTCACCTCCGCCAGACACTCG

HEY1

Forward

Reverse

TGCATACGGC AGGAGGGAAAG

AGTCGAACTCGAAGCGGGTCA

HEY2

Forward

Reverse

AGGCGTCGGGATCGGATAAAT

AAGAGCGTGTGCGTCAAAGTA

GAPDH

Forward

Reverse

GGAGTCCACTGGCGTCTTCACC

GAGGAGTGGGTGTCGCTGTTG

LSD1

Forward

Reverse

AGACGACAGTTCTGGAGGGTA

TCTTGAGAAGTCATCCGGTCA

表2 用于ChIP的启动子引物序列

Tab.2 Primer sequences of Notch target gene promoters used for ChIP

Primer n

Sequences 5'-3'

HES1

-2200

Forward

Reverse

AGG TCA CCC AGA GTC AGG AA

CCA GCG TCT TGT TTG ATG TG

HES1

TSS

Forward

Reverse

CGT GTC TCC TCC TCC CAT T

GAG AGG TAG ACG GGG GAT TC

HES1

+500

Forward

Reverse

TCA ACA CGA CAC CGG ATA AA

TCA GCT GGC TCA GAC TTT CA

HES1

+2600

Forward

Reverse

GGC TTT TGG TGG AAT TTG AA

TCA TGG AGG ATT GGT GAA AAG

Notch3

-5000

Forward

Reverse

TAG CCC CTG GTC AGT CAT TC

GGT GCA TCG TAT CAG GAG GT

Notch3

TSS

Forward

Reverse

TGG CCT CAG TTT CCA GAG TT

CAC ACC CAA CCT CGT GAA C

Notch3

+3000

Forward

Reverse

GTC TCA GCA CAC CCC ATT CT

AAC CAC AAA GCA GGG GAA G

Notch3

+28600

Forward

Reverse

GGG GGC TAA AGA CAC AAA CA

GTT CCT TCT CTC CCC ACT CC

DTX1

-1700

Forward

Reverse

TGT GAA TGA CAT GGC AGA GG

TGA ATC TCC TGC CAG TAC CC

DTX1

+30000

Forward

Reverse

ACA TGC CAG ACA GCA GAA CA

AAC CTT CCA GAC CCT GTG TG

CR2

+5000

Forward

Reverse

GCC GGA AGG ATG TTC TTG TA

CAG GGA AGG CCA TGA AAA TA

CR2

+21000

Forward

Reverse

CCC CAC AGT GCT TAC GAT CT

AAG CCA GGA TTG CAG TCA AC

图1 药物抑制LSD1延长GBM动物模型的存活时间

Fig.1 LSD1 inhibition prolongs the survival of GBM-bearing mice. A: LSD1 mRNA expression in glioblastoma and normal brain tissues. B: Expression of LSD1 in glioblastoma and normal brain tissues (Scale bar=200 μm). C: Kaplan-Meier survival analysis showing negative correlation between LSD1 expression and patient survival. D: ROC analysis of specificity and sensitivity of LSD1 expression for predicting GBM. E: General comparison of subcutaneous tumors in treated group and control group on day 21 after tumor cell engraftment. F: Tumor growth in the treated and control group (n=12). G: Survival curves of treated group and control group. H: Body weight curves of ORY-1001-treated group and control group. I: Cell viability of U87 cells treated with ORY-1001. J: Cell viability of U251 treated with ORY-1001. K: Cell viability of A172 treated with ORY-1001. L: Changes of LSD1 enzyme activity. *P<0.05, **P<0.01, ***P<0.001 vs the control group, vehicle.

图2 LSD1基因靶向反映LSD1对GBM的药理抑制作用

Fig.2 Genetic targeting of LSD1 mirrors LSD1 pharmacological inhibition in GBM. A, B: Western blotting of the expression of LSD1 in ORY-1001-treated U87 cells. C, D: Western blotting of H3K4me2 expression in in ORY-1001-treated U87 cells with H3 expression as the internal reference. E: qPCR for detecting relative expression of LSD1 mRNA in sh-LSD1 and sh-NC cells. F: LSD1 expression of sh-LSD1 and sh-NC cells verified by Western blotting. G: Quantification of LSD1 expression. H: Cell viability of sh-LSD1-U87 cells compared with sh-NC-U87 cells. I, J: Cleaved caspase-3 expression upon LSD1 silencing. K: Effect of different ORY-1001 concentrations on sh-LSD1 and sh-NC cells. L: The subcutaneous tumor growth curves of LSD1-silenced group and control group. M: Survival curves of mice injected with LSD1-silenced and control GBM cells. N: Relative expression of LSD1 mRNA in LSD1-silenced group and control group at the time of death. *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol, sh-NC.

图5 ORY-1001影响LSD1与GBM中Notch靶基因启动子区域的结合

Fig.5 ORY-1001 affects the binding of LSD1 to the Notch target gene promoter regions in GBM. ChIP analysis was performed on the chromatin from GBM U87 cells treated with ORY-1001 (16 nmol/L) for 24 h. A: LSD1 binds to the promoter sequence of the Notch target gene (red). B: Changes of LSD1 occupancy in Notch target gene promoter region after ORY-1001treatment. *P<0.05, **P<0.01 vs Vehicle group.

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ORY-1001靶向赖氨酸特异性去甲基化酶1下调胶质母细胞瘤Notch/HES1通路的表达并发挥抗肿瘤作用

ORY-1001 inhibits glioblastoma cell growth by downregulating the Notch/HES1 pathway via suppressing lysine-specific demethylase 1 expression

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