摘要
目的探讨长链非编码RNA钾电压门控通道亚家族Q成员1重叠转录物1(Kcnq1ot1)通过调节miR-92a-1-5p影响胰岛β细胞胰岛素分泌的作用机制。方法自2020年6至12月在中山大学附属第三医院招募初诊2型糖尿病(T2DM)患者25例,同时招募年龄匹配且无糖尿病的受试者21例作为对照组。收集受试者的年龄、体重指数(BMI),检测空腹血糖(FPG)和糖化血红蛋白(HbA1c),采用实时荧光定量PCR(qRT-PCR)检测血清中Kcnq1ot1表达量,并分析其与FPG和HbA1c的相关性。12只5周龄雄性C57BL/6J小鼠采用随机数字表法分为高脂饮食(HFD)组(n=6)和正常饮食(CD)组(n=6),喂养20周后,提取原代胰岛细胞RNA。将体外培养的Min6细胞分为牛血清白蛋白(BSA)组和棕榈酸(PA,400μmol/L)组;按照是否转染Kcnq1ot1 siRNA分为si-NC组和si-Kcnq1ot1组;按照是否转染miR-92a-1-5p的模拟物或抑制剂分为:模拟物对照组、模拟物组、抑制剂对照组和抑制剂组;按照转染Kcnq1ot1/NC siRNA后是否加入抑制剂分为si-NC+抑制剂对照组、si-Kcnq1ot1+抑制剂对照组、si-NC+抑制剂组和si-Kcnq1ot1+抑制剂组。采用qRT-PCR检测Kcnq1ot1、miR-92a-1-5p和胰岛素转录本1(Ins1)、胰岛素转录本2(Ins2)、胰岛素受体(Insr)、NK6同源盒蛋白1(Nkx6.1)、胰岛素受体底物1(Irs1)和神经生成素3(Ngn3)mRNA水平;Western blotting检测胰岛素和Insr蛋白表达水平;葡萄糖刺激胰岛素释放实验检测胰岛素分泌水平;双荧光素酶报告基因实验验证Kcnq1ot1与miR-92a-1-5p之间的直接作用关系。各组间指标的差异比较采用独立样本t检验、方差分析,采用Pearson相关分析法分析受试者Kcnq1ot1表达量与FPG和HbA1c的相关性。结果T2DM患者血清中Kcnq1ot1的表达量较对照组显著下调(P<0.01),且Kcnq1ot1的表达量与FPG和HbA1c呈负相关关系(r=-0.52、-0.49,均P<0.05)。与CD组相比,HFD组小鼠胰岛中Kcnq1ot1表达量下调(P<0.001)。在Min6细胞中,与BSA组相比,PA组细胞中Kcnq1ot1�
Objective To investigate the mechanism of long noncoding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1(Kcnq1ot1)affecting insulin secretion of pancreatic β cells by regulating miR-92a-1-5p.Methods From June to December 2020,25 patients with newly diagnosed type 2 diabetes mellitus(T2DM)were recruited in the Third Affiliated Hospital of Sun Yat-sen University,and 21 age-matched subjects without diabetes were recruited as a control group.Age,body mass index(BMI),fasting plasma glucose(FPG)and glycated hemoglobin A1c(HbA1c)were collected,and the expression of Kcnq1ot1 in serum was detected by quantitative real-time PCR(qRT-PCR),and its correlation with FPG and HbA1c was analyzed.5-week-old male C57BL/6J mice were randomly divided into high-fat diet(HFD)group(n=6)and chow diet(CD)group(n=6).After 20 weeks of feeding,the RNA of primary pancreatic islet cells was extracted.MIN6 cells cultured in vitro were divided into bovine serum albumin(BSA)group and palmitic acid(PA,400μmol/L)group.According to whether Kcnq1ot1 siRNA was transfected or not,the cells were divided into si-NC group and si-Kcnq1ot1 group.According to whether miR-92a-1-5p mimics or inhibitors were transfected or not,the cells were divided into mimic control group,mimics group,inhibitor control group and inhibitors group.According to whether the inhibitors was added after transfection of Kcnq1ot1/NC siRNA,the cells were divided into si-NC+inhibitor control group,si-Kcnq1ot1+inhibitor control group,si-NC+inhibitors group,si-Kcnq1ot1+inhibitors group.qRT-PCR was used to detect the expression level of Kcnq1ot1,miR-92a-1-5p,insulin transcript 1(Ins1),insulin transcript 2(Ins2),insulin receptor(Insr),NK6 homeobox protein 1(Nkx6.1),insulin receptor substrate 1(Irs1)and neurogenin 3(Ngn3).Western blotting was used to detect the expression level of insulin and Insr.Glucose stimulated insulin secretion assay was used to detect the level of insulin.Double luciferase reporting gene assay was used to verify the direct relationsh
作者
陈亚兰
李晏丽
林倍思
麦晓东
彭粤跃
陈丹蕊
苏燕娜
徐芬
李万根
许雯
Chen Yalan;Li Yanli;Lin Beisi;Mai Xiaodong;Peng Yueyue;Chen Danrui;Su Yanna;Xu Fen;Li Wangen;Xu Wen(Department of Endocrinology and Metabolism,Third Affiliated Hospital of Sun Yat-sen University,Guangdong Provincial Key Laboratory of Diabetology,Guangzhou 510630,China;Department of Endocrinology,Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China)
出处
《中华糖尿病杂志》
CAS
CSCD
北大核心
2022年第5期473-481,共9页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金(81800682)
广州市科技计划项目(202102010175)
广州市卫生健康科技项目(20201A011079)。
