Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2.
Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2.
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KLF9的干预通过上调DDAH 2缓解妊娠期糖尿病的发展。
DOI：
10.1080/21655979.2021.2005929
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发表时间：
2022-01
期刊：
影响因子：
4.9
通讯作者：
Li K
中科院分区：
文献类型：
--
作者：
Chen W;Wang H;Liu J;Li K
妊娠期糖尿病（GDM）是指在没有糖尿病诊断的孕妇中发现葡萄糖耐受不良的情况。Kruppel-like factor 9 （KLF9）在GDM中的作用尚未被研究，这是我们研究的目的。用高糖（HG）诱导HTR8/SVneo细胞，用链脲佐菌素（STZ）处理妊娠小鼠，分别建立体外和体内GDM模型。采用实时荧光定量PCR、免疫组化染色、Western blot检测KLF9的表达水平。分别采用细胞计数试剂盒-8 （CCK-8）、TUNEL、酶联免疫吸附试验（ELISA）和氧化应激检测试剂盒检测细胞活力、凋亡、炎症和氧化应激。利用生物信息学工具预测KLF9与二甲基精氨酸二甲氨基水解酶2 （DDAH2）的相互作用，并通过荧光素酶报告酶测定和染色质免疫沉淀（ChIP）证实了KLF9与DDAH2的相互作用。GDM患者胎盘组织及hg诱导的HTR8/SVneo细胞中KLF9表达升高。在hg诱导的HTR8/SVneo细胞中，沉默KLF9可提高细胞活力，减少细胞凋亡，抑制炎症和氧化应激。KLF9可以结合DDAH2启动子，负向调控DDAH2的表达。抑制DDAH2部分减弱了KLF9沉默对细胞凋亡、炎症和氧化应激的作用。KLF9沉默对体内血糖和胰岛素浓度的抑制作用也被DDAH2敲低所消除。综上所述，我们提供的证据表明，KLF9的干扰可能通过上调DDAH2来减轻细胞凋亡、炎症和氧化应激，从而阻碍GDM的发展，这可能为GDM的靶向治疗提供指导。缩写：KLF9：类克虏伯因子9；DDAH2：二甲基精氨酸二甲基氨基水解酶2；GDM：妊娠期糖尿病；ELISA：酶联免疫吸附法；CCK-8：细胞计数试剂盒-8；ChIP：染色质免疫沉淀；Sh：短发夹；HG：高葡萄糖；PBS：磷酸盐缓冲盐水；DAPI: 4,6 -二氨基-2-苯基吲哚；il - 6:白细胞介素- 6;TNF-α：肿瘤坏死因子-α；ROS：活性氧；MDA:丙二醛;SOD：超氧化物歧化酶；wt:野生型;傻瓜:突变体
Gestational diabetes mellitus (GDM) is a situation where glucose intolerance is found in pregnant women without a previous diagnosis of diabetes. The role of Kruppel-like factor 9 (KLF9) has not been investigated in GDM, which constituted the aim of our study. HTR8/SVneo cells were induced by high glucose (HG) and pregnant mice were treated with streptozocin (STZ) to establish GDM model in vitro and in vivo, respectively. The expression level of KLF9 was detected by real-time PCR, immunohistochemical staining, and Western blot. Cell viability, apoptosis, inflammation, and oxidative stress were investigated by cell counting kit-8 (CCK-8), TUNEL, enzyme-linked immunosorbent assay (ELISA) and oxidative stress detection kits, respectively. The interaction of KLF9 with dimethylarginine dimethylaminohydrolase 2 (DDAH2) was predicted by bioinformatic tools and confirmed by luciferase reporter assay and chromatin immunoprecipitation (ChIP). The expression of KLF9 was increased in the placental tissues of GDM patients and HG-induced HTR8/SVneo cells. Silencing of KLF9 increased cell viability, reduced cell apoptosis, and suppressed inflammation and oxidative stress in HG-induced HTR8/SVneo cells. KLF9 could bind to DDAH2 promoter and negatively regulate DDAH2 expression. Inhibition of DDAH2 partly weakened the effects of KLF9 silencing on cell apoptosis, inflammation, and oxidative stress. The suppressive effects of KLF9 silencing on blood glucose and insulin concentration in vivo were also abolished by DDAH2 knockdown. In conclusion, we provided evidence that interference of KLF9 could hinder the development of GDM by alleviating cell apoptosis, inflammation, and oxidative stress through upregulating DDAH2, which might instruct the targeting therapies against GDM. Abbreviations: KLF9: Kruppel-like factor 9; DDAH2: dimethylarginine dimethylaminohydrolase 2 ; GDM: gestational diabetes mellitus; ELISA: enzyme-linked immunosorbent assay; CCK-8: cell counting kit-8; ChIP: chromatin immunoprecipitation; sh: short hairpin; HG: high glucose; PBS: phosphate-buffered saline; DAPI: 4, 6-diamidino-2-phenylindole; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; wt: wild-type; mut: mutant
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