miR-494通过激活Wnt/β-catenin信号通路促进胰岛β细胞增殖、抑制其凋亡增加胰岛素分泌

miR-494 promotes islet β cell proliferation, inhibits apoptosis and increases insulin secretion by activating Wnt/β-catenin signaling pathway

目的 探究微小RNA-494 (miR-494)与胰岛β细胞功能和妊娠糖尿病之间的关系。方法 选择20例妊娠糖尿病患者和健康体检者,反转录PCR测定其外周血miR-494的含量;培养INS-1胰岛细胞瘤细胞,分别采用含低糖(3.3 mmol/L)和高糖(16.7 mmol/L)处理后,采用ELISA测定胰岛素含量来评价胰岛细胞基础和高糖刺激的胰岛素分泌能力;采用miR-494模拟物对照、miR-494模拟物、miR-494抑制物对照、miR-494抑制物分别处理INS-1细胞24、48、72 h,收集细胞。采用噻唑蓝(MTT)法检测INS-1细胞的活性,流式细胞术检测细胞凋亡;反转录PCR检测INS-1细胞Wnt3a、β联蛋白(β-catenin)、细胞周期蛋白D1(cyclin D1)和c-Myc的mRNA表达;Western blot法检测细胞Wnt3a、β-catenin、cyclin D1、c-Myc蛋白表达。结果 与正常对照组相比,妊娠糖尿病患者空腹胰岛素、空腹血糖、1 h血糖和2 h血糖显著升高;外周血miR-494含量降低。过表达miR-494的INS-1细胞上清液胰岛素浓度增加,当给予高糖后,过表达miR-494进一步促进胰岛素分泌。过表达miR-494明显促进INS-1细胞活性,抑制INS-1细胞凋亡;miR-494能够显著性促进Wnt3a、β-catenin、cyclin D1、c-Myc的mRNA和蛋白表达,miR-494抑制物处理则结果相反。结论 miR-494通过激活Wnt/β-catenin信号通路促进胰岛β细胞增殖、抑制其凋亡增加胰岛素分泌。

Objective To investigate the relation between the miR-494 expression with pancreatic islets β cell function and gestational diabetes mellitus. Methods Twenty patients with gestational diabetes mellitus and healthy subjects were enrolled. The content of miR-494 in peripheral blood was measured by reverse transcription PCR. INS-1 cells were cultured and treated with low glucose(3.3 mmol/L) and high glucose(16.7 mmol/L), respectively. The insulin concentration was tested by ELISA to evaluate the insulin secretion of islet cells stimulated by high glucose. Cells were collected, after treated with miR-494 mimics control, miR-494 mimics, miR-494 inhibitor control and miR-494 inhibitor for 24 hours, 48 hours and 72 hours, respectively. The activity of INS-1 cells was detected by MTT assay;Apoptosis was detected by flow cytometry. Reverse transcription PCR and Western blot analysis were used to detect the mRNA and protein expression of Wnt3a, β-catenin, cyclin D1 and c-Myc, respectively. Results Compared with the normal control, fasting insulin, fasting blood glucose, 1 hour-blood glucose and 2 hour-blood glucose in patients with gestational diabetes mellitus increased significantly. The content of miR-494 in peripheral blood decreased. The insulin concentration in the supernatant of INS-1 cells overexpressing miR-494 increased. When high glucose was given, the overexpression of miR-494 further promoted insulin secretion. Overexpression of miR-494 significantly promoted INS-1 cell activity and inhibited INS-1 cell apoptosis. miR-494 significantly promoted the protein expressionof Wnt3a, β-catenin, cyclin D1 and c-Myc. miR-494 inhibitor treatment showed the opposite results. ConclusionmiR-494 promotes islet β cell proliferation, inhibits apoptosis and increases insulin secretion by activating Wnt/β-catenin signaling pathway.