摘要
目的 观察黄芪、党参、龟板对人红系K562细胞γ珠蛋白mRNA表达及m6A甲基化的影响。方法 采用体外培养K562细胞株,分为黄芪党参龟板高(a)/低(b)剂量组(10、2.5 mg·mL^(-1))、黄芪党参高(c)/低(d)剂量组(10、2.5 mg·mL^(-1))、龟板高(e)/低(f)剂量组(10、2.5 mg·mL^(-1))、丁酸钠组(500μmol·L^(-1))及空白对照组,药物干预96 h。采用实时荧光定量PCR法检测K562细胞γ珠蛋白、METTL3、ALKBH5、FTO mRNA表达水平;比色法检测细胞m6A甲基化总体水平;Western Blot法检测细胞METTL3、FTO、ALKBH5蛋白表达水平。结果 与空白对照组比较,中药组(a、b、c、d、f组)K562细胞γ珠蛋白mRNA表达明显上调(P<0.05,P<0.01);中药组(a、b、c、f组)K562细胞m6A甲基化总体水平明显下降(P<0.05,P<0.01),丁酸钠组K562细胞m6A甲基化水平显著升高(P<0.01);各中药组K562细胞ALKBH5、FTO mRNA表达均明显上调(P<0.05,P<0.01),中药组(a、c、d、e组)K562细胞METTL3 mRNA表达明显上调(P<0.05);中药组(a、c组)K562细胞FTO蛋白表达显著上调(P<0.01)。结论 中药黄芪、党参、龟板可能通过上调m6A去甲基化酶FTO的表达,进而下调红系K562细胞内的m6A甲基化水平;其诱导γ珠蛋白基因表达治疗儿童β地中海贫血的分子机制可能与调节m6A甲基化有关。
Objective To observe the effects of Astragali Radix,Codonopsis Radix and Testudinis Carapax et Plastrum on the mRNA expression and m~6A methylation of γ-globin in human erythroid k562 cells.Methods The K562 cells line was cultured in vitro and divided into Astragali Radix+Codonopsis Radix+Testudinis Carapax et Plastruma high-(a)and low-(b)dose groups(10,2.5 mg·mL^(-1)),Astragali Radix+Codonopsis Radix high-(c)and low-(d)dose groups(10,2.5 mg·mL^(-1)),Testudinis Carapax et Plastruma high-(e)and low-(f)dose groups(10,2.5 mg·mL^(-1)),sodium butyrate group(500μmol·L^(-1))and blank control group.All the groups were given drug intervention for 96 hours.The mRNA expression levels of γ-globin,METTL3,ALKBH5 and FTO in K562 cells were detected by real-time fluorescence quantitative PCR;the overall level of m~6A methylation in cells was detected by colorimetric method;the protein expression levels of METTL3,FTO and ALKBH5 in cells were detected by Western Blot.Results Compared with the blank control group,the mRNA expression of γ-globin in K562 cells in the Chinese medicine groups(a,b,c,d,f)was significantly up-regulated(P<0.05,P<0.01);the overall m~6A methylation level in K562 cells in the Chinese medicine groups(a,b,c,f)was significantly decreased(P<0.05,P<0.01),and the m~6A methylation level in K562 cells in the sodium butyrate group was significantly increased(P<0.01);the mRNA expressions of ALKBH5 and FTO in K562 cells were significantly up-regulated in all Chinese medicine group(P<0.05,P<0.01),and the mRNA expression of METTL3 in K562 cell was significantly upregulated in Chinese medicine groups(a,c,d,e)(P<0.05),the protein expression of FTO in K562 cells was significantly up-regulated in Chinese medicine group(a,c)(P<0.01).Conclusion The Chinese medicines Astragali Radix,Codonopsis Radix and Testudinis Carapax et Plastrum may up-regulate the expression of m~6A demethylase FTO and thereby down-regulate m~6A methylation level in human erythroid k562 cells;the molecular mechanism of their induction of γ-globin
作者
卢焯明
徐方蔚
陈珏璇
LU Zhuoming;XU Fangwei;CHEN Juexuan(Guangzhou Women and Children's Medical Center,Guangzhou 510623 Guangdong,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2022年第11期1472-1477,共6页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
广东省中医药局科研项目(20211301)
广州市妇女儿童医疗中心内科部基金项目(NKE-PRE-2019-007)。
关键词
β地中海贫血
黄芪
党参
龟板
Γ珠蛋白
m^(6)A甲基化
K562细胞
β-thalassemia
Astragali Radix
Codonopsis Radix
Testudinis Carapax et Plastrum
γ-globin
m^(6)A methylation
K562 cells
