目的探究脂肪含量和肥胖相关蛋白(fat mass and obesity-associated protein,FTO)和丝氨酸-苏氨酸激酶蛋白激酶D2(serine-threonine kinase protein kinase D2,PRKD2)在糖尿病肾病(diabetic kidney disease,DKD)进展中的调控作用和调节...目的探究脂肪含量和肥胖相关蛋白(fat mass and obesity-associated protein,FTO)和丝氨酸-苏氨酸激酶蛋白激酶D2(serine-threonine kinase protein kinase D2,PRKD2)在糖尿病肾病(diabetic kidney disease,DKD)进展中的调控作用和调节机制。方法采用35 mmol/L葡萄糖对足细胞(MPC5细胞)进行高糖刺激24h构建DKD体外模型。采用FTO过表达载体(pcDNA-FTO)和PRKD2过表达载体(pcDNA-PRKD2),或空载体(vector)转染高糖诱导的MPC5细胞。通过RT-qPCR检测FTO和PRKD2过表达效率;MeRIP检测PRKD2 mRNA的N6-甲基腺苷(N6-methyladenosine,m6A)修饰水平;ELISA检测Caspase-3活性、IL-6,TNF-α和单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)分泌量;流式细胞术分析细胞凋亡率;Western blot评估FTO和PRKD2蛋白水平,以及SIRT1/HIF-1α通路关键蛋白表达水平;Pearson分析FTO和PRKD2水平的相关性。结果与无高糖诱导对照组比较,高糖诱导的足细胞中FTO蛋白(0.51±0.04 vs 1.00±0.03)和PRKD2蛋白(0.45±0.03 vs 1.01±0.04)水平显著下调,差异具有统计学意义(t=13.17,16.76,均P<0.001)。高糖诱导的足细胞中FTO蛋白水平和PRKD2蛋白水平呈正相关(r2=0.7051,P<0.001)。与vector组相比,pcDNA-FTO组PRKD2 mRNA的m6A水平(0.56±0.09 vs1.01±0.13)降低,PRKD2 mRNA水平(3.16±0.14 vs 1.03±0.02)显著升高,差异具有统计学意义(t=51.37,11.82,均P<0.001)。与control组(IL-6:512.76±61.85 pg/ml,TNF-α:28.17±2.83 pg/ml,MCP-1:157.31±17.69 pg/ml)和vector组(IL-6:498.41±87.51 pg/ml,TNF-α:26.35±5.47 pg/ml,MCP-1:165.52±16.87 pg/ml)比较,pcDNA-PRKD2组IL-6(301.86±21.85 pg/ml),TNF-α(11.06±4.12 pg/ml),MCP-1分泌量(81.45±9.03pg/ml)显著减少,差异具有统计学意义(F=7.51,10.47,61.97,均P<0.01)。与control组(Caspase-3:689.65±79.5U/L,细胞凋亡率:22.31%±2.69%)和vector组(Caspase-3:715.91±113.58 U/L,细胞凋亡率:21.07%±3.28%)比较,pcDNA-PRKD2组Caspase-3活性(437.64±104.76 U/L)和细胞凋亡率(8.41%±3.15%)下降,差异具有统计学意义(F=2.35,79展开更多
In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscop...In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from GI phase to S phase and inhibit glioma cell growth.展开更多
基金supported by the Science and Technology Foundation Program of Jiangsu Province(Tumorigenic nucleostemin genes and adenovirus-based RNA interference targeting to brain tumor stem cell the rapy),No.BK2007072
文摘In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from GI phase to S phase and inhibit glioma cell growth.
