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WTAP通过调控SIRT1诱导小胶质细胞M1型极化的研究
WTAP promotes microglial M1 polarization by regulating SIRT1
- 陆军军医大学学报 2022年44卷第7期 页码：620-629
- 作者机构：
1.400016 重庆，重庆医科大学附属第一院眼科，眼科学重庆市重点实验室，重庆市眼科研究所
2.402760 重庆，重庆市璧山区人民医院眼科
- 作者简介：
[侯胜平, E-mail: sphou828@163.comHOU Shengping, E-mail: sphou828@163.com
- 基金信息：重庆市教委创新科研团队项目(CXQT19015);重庆市自然科学基金面上项目(cstc2019jcyjmsxmX0120);重庆市留学创新支持计划项目(cx2018010);重庆市教委项目(KJQN202000406)
DOI：10.16016/j.2097-0927.202109187
中图分类号： R322.91;R329.25;R341收稿：2021-09-24，
修回：2021-11-17，
纸质出版：2022-04-15
- 稿件说明：
移动端阅览
周虹秀, 高煦, 侯胜平. WTAP通过调控SIRT1诱导小胶质细胞M1型极化的研究[J]. 陆军军医大学学报, 2022,44(7):620-629.
Hongxiu ZHOU, Xu GAO, Shengping HOU. WTAP promotes microglial M1 polarization by regulating SIRT1[J]. Journal of Army Medical University, 2022, 44(7): 620-629.
周虹秀, 高煦, 侯胜平. WTAP通过调控SIRT1诱导小胶质细胞M1型极化的研究[J]. 陆军军医大学学报, 2022,44(7):620-629. DOI： 10.16016/j.2097-0927.202109187.
Hongxiu ZHOU, Xu GAO, Shengping HOU. WTAP promotes microglial M1 polarization by regulating SIRT1[J]. Journal of Army Medical University, 2022, 44(7): 620-629. DOI： 10.16016/j.2097-0927.202109187.
摘要
目的
2
探讨Wilms肿瘤抑制因子1相关蛋白(Wilms' tumor 1-associating protein，WTAP)在小胶质细胞活化调控中的作用及其可能的分子机制。
方法
2
用100 ng/mL IFN
γ
联合1 μg/mL LPS诱导HMC3小胶质细胞M1极化作为实验组，以未处理的细胞作为空白对照组，采用实时荧光定量PCR和Western blot检测细胞中促炎细胞因子IL-6、IL-1β、TNF-α和N6-甲基腺苷(m6A)相关基因的表达，在HMC3小胶质细胞中转染RNAi慢病毒敲低WTAP，使用实时荧光定量PCR和Western blot检测敲低效率，Western blot检测敲低HMC3小胶质细胞WTAP后促炎细胞因子IL-6、IL-1β和TNF-α蛋白表达，Western blot检测敲低WTAP后HMC3小胶质细胞沉默信息调节因子2相关酶1 (silent mating-type information regulation 2 homolog 1
SIRT1)、COP1、USP18、EP4、促炎转录因子和部分炎症相关通路受体的表达，使用SIRT1激动剂SRT1720和SIRT1抑制剂EX527分别处理空载病毒组和敲低WTAP组HMC3细胞后，Western blot检测c/EBPβ的表达。
结果
2
与空白对照组比较，100 ng/mL IFN
γ
联合1 μg/mL LPS诱导的HMC3细胞IL-6、IL-1β、TNF-α表达上调(
P
<
0.05)。M1极化的HMC3小胶质细胞中WTAP高表达(
P
<
0.05)。敲低WTAP后，HMC3细胞促炎细胞因子IL-6、IL-1β和TNF-α表达降低(
P
<
0.05)，SIRT1表达升高(
P
<
0.05)，且促炎转录因子c/EBPβ表达降低(
P
<
0.05)。SIRT1激动剂SRT1720使空载病毒组相对高表达的c/EBPβ降低(
P
<
0.01)。SIRT1抑制剂EX527使敲低WTAP的HMC3细胞相对低表达的c/EBPβ升高(
P
<
0.05)。
结论
2
WTAP促进HMC3小胶质细胞M1极化，其作用机制可能与通过调控SIRT1表达进而影响c/EBPβ信号通路有关。
Abstract
Objective
2
To investigate the role and the possible molecular mechanism of RNA methylase
Wilms' tumor 1-associating protein (WTAP)
in microglial activation.
Methods
2
HMC3 cells was induced to M1 polarization by treatment of 100 ng/mL
IFN
γ
combined with 1 μg/mL LPS and then served as experimental group
and the untreated cells served as control group. The expression of pro-inflammatory cytokines IL-6
IL-1β
TNF-α and N6-methyladenosine (m6A) at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting
respectively. After HMC3 cells were transfected with RNAi lentivirus to knock down WTAP
the knockdown efficiency was detected by real-time quantitative PCR and Western blotting. Western blot assay was employed to measure the expression levels of pro-inflammatory cytokines IL-6 IL-1β and TNF-α in WTAP knockdown HMC3 cells. Western blotting was also adopted to detect the protein levels of silent mating-type information regulation 2 homolog 1 (SIRT1)
COP1
USP18
EP4
pro-inflammatory transcription factors and some inflammatory pathway receptors in the knockdown cells. SIRT1 agonist SRT1720 and SIRT1 inhibitor EX527 were employed to treat HMC3 cells in the blank-virus group and the knockdown WTAP group
respectively
and the expression level of C/EBPβ was detected by Western blotting.
Results
2
Treatment of 100 ng/mL IFN
γ
combined with 1 μg/mL LPS induced significant up-regulation of IL-6
IL-1β and TNF-α expression in HMC3 cells as compared with the untreated cells (
P
<
0.05). WTAP was highly expressed in M1 polarized HMC3 microglia (
P
<
0.05). WTAP knockdown resulted in obviously decreased expression levels of pro-inflammatory cytokines IL-6
IL-1β and TNF-α (
P
<
0.05)
increased level of SIRT1 (
P
<
0.05)
and reduced pro-inflammatory transcription factor C/EBPβ (
P
<
0.05). SIRT1 agonist SRT1720 reduced the relatively high expression of C/EBPβ in the blank-virus group (
P
<
0.01). While
the inhibitor EX527 increased the relative low expression of C/EBPβ in HMC3 cells with low WTAP knockdown (
P
<
0.05).
Conclusion
2
WTAP promotes M1 polarization in HMC3 cells
possibly through regulating the expression of SIRT1 and thus affecting the C/EBPβ signaling pathway.
关键词
Keywords
references
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