lncRNA GAS5对胰岛素抵抗模型中卵巢颗粒细胞功能的影响及相关机制研究

Effects and mechanisms of lncRNA GAS5 on ovarian granulosa cells function in insulin resistance model

目的探究lncRNA GAS5在多囊卵巢综合征(PCOS)中的表达及其对胰岛素抵抗(IR)模型中卵巢颗粒细胞功能的影响及其相关机制。方法收集PCOS包含伴IR者(n=20)与不伴IR者(n=20)及非PCOS患者(n=20)的卵巢颗粒细胞。RT-PCR检测患者颗粒细胞中lncRNA GAS5的表达;应用脂质体稳定转染技术将过表达lncRNA GAS5质粒转染入大鼠卵巢颗粒细胞中,采用地塞米松及胰岛素联合诱导方法建立大鼠颗粒细胞的IR模型;己糖激酶及比色法检测模型中葡萄糖及乳酸的消耗量进行鉴定;将大鼠颗粒细胞分为4组,对照组(正常培养无任何特殊处理的颗粒细胞);IR组(胰岛素抵抗条件下的颗粒细胞);IR-pcDNA3.1-NC组(对转染pcDNA3.1-NC后的颗粒细胞进行IR造模处理);IR-pcDNA3.1-GAS5组(对转染pcDNA3.1-GAS5后的颗粒细胞进行IR造模处理)。应用MTT、Annexin V-FITC、流式细胞术及ELISA实验分别检测过表达lncRNA GAS5对IR条件下颗粒细胞的增殖、凋亡、细胞周期和炎症因子TNF-α、IL-6、IL-18表达的影响;Western blot检测过表达lncRNA GAS5对IR条件下颗粒细胞中磷脂酰肌醇-3(PI3K)/蛋白激酶B(AKT)信号通路p-PI3K、p-AKT及细胞周期蛋白Cyclin B1、CDK1和凋亡相关蛋白Bcl-2、cleaved caspase-3表达的影响。采用重复测量方差分析、独立样本t检验和单因素方差分析比较组间的指标水平差异。结果与对照组比较,IR组和IR-pcDNA3.1-NC组颗粒细胞中lncRNA GAS5的表达(1.09±0.17比0.41±0.11、0.38±0.06)降低,而IR-pcDNA3.1-GAS5组lncRNA GAS5表达水平(1.09±0.17比3.10±0.33)升高,差异均有统计学意义(P均<0.05);与IR组比较,IR-pcDNA3.1-GAS5组颗粒细胞增殖能力(52.16±8.83比68.39±11.14)及细胞周期阻滞(G1期:69.05±10.09比43.51±6.09,S期:25.37±5.59比9.44±2.29,G2/M期:7.11±2.81比47.02±6.69)均升高,细胞凋亡率(33.26﹪±3.44﹪比16.01﹪±2.56﹪),促炎因子TNF-α(138.56±27.05比105.30±12.64)、IL-6(123.69±10.08比86.11±8.03)、IL-18的表达水平(41.72±5.61比28.29±8.17)均降低,差异具有统计学意义(P均<0.05)。Western blot检测结果显示,与IR组比较,IR-pcDNA3.1-GAS5组颗粒细胞中p-PI3K/PI3K(0.79±0.06比0.51±0.02)、p-AKT/AKT(0.83±0.03比0.47±0.01)、cleaved caspase-3(0.81±0.03比0.39±0.07)均降低,而Cyclin B1(0.09±0.03比0.55±0.06)、CDK1(0.12±0.05比0.53±0.08)和Bcl-2(0.09±0.02比0.21±0.02)均升高,差异具有统计学意义(P均<0.05)。结论伴有IR的PCOS患者卵巢颗粒细胞中lncRNA GAS5表达显著降低,而过表达颗粒细胞中lncRNA GAS5能够促进IR条件下的细胞增殖和细胞周期进展,并降低其凋亡发生率,抑制颗粒细胞对促炎因子TNF-α、IL-6和IL-18的表达。LncRNA GAS5可能通过PI3K/AKT信号通路发挥上述作用,以保护IR条件下的颗粒细胞功能损伤。

Objective To investigate the expression of lncRNA GAS5 in patients with polycystic ovary syndrome(PCOS)as well as the effects and mechanisms of lncRNA GAS5 on the function of ovarian granulosa cells in insulin resistance(IR)model.Methods A total of 60 women were enrolled,and the ovarian granulosa cells were collected,20 of whom with PCOS and IR,20 with PCOS without IR and 20 without PCOS or IR.The expression levels of lncRNA GAS5 in ovarian granulosa cells were detected by RT-PCR.The overexpression of lncRNA GAS5 plasmid was transfected into rat ovarian granulosa cells by liposome stable transfection technology.The IR model of granulosa cells was established by dexamethasone and insulin induction;The consumption of glucose and lactic acid in the model was detected by hexokinase and colorimetry.Rats'granulosa cells were treated with or without insulin,pcDNA3.1-NC and pcDNA3.1-GAS5,and named Control group,IR group,IR-pcDAN3.1-NC group,and IR-pcDAN3.1-GAS5 group,respectively.MTT,Annexin V-FITC,flow cytometry and ELISA were used to detect the effects of lncRNA GAS5 on cell proliferation,apoptosis,cell cycle and the expression of inflammatory factors TNF-α,IL-6 and IL-18 in granulosa cells under IR.Western blot was used to detect the effects of overexpression of lncRNA GAS5 on p-PI3K,p-AKT and cyclin B1 CDK1,Bcl-2 and cleaved caspase-3 in granulosa cells under IR.Two-way repeated-measures ANOVA,t-test and one-way ANOVA were used to compare the differences between the two groups.Results compared with the control group,the expression of lncRNA GAS5 in ovarian granulosa cells of PCOS patients was significantly decreased(1.09±0.17 vs 0.41±0.11,P<0.05),while the expression level of lncRNA GAS5 in granulosa cells of IR model was significantly increased in IR-pcDNA3.1-GAS5 group(1.09±0.17 vs 0.38±0.06,P<0.05).Compared with IR group,granulocytic cell proliferation ability and cell cycle arrest in IR-PCDNA3.1-GAS5 group were(52.16±8.83 vs 68.39±11.14)(G1 phase:69.05±10.09 vs 43.51±6.09),(S phase:25.37±5.59 vs 9.44±2.29,G2/M phase:7.11±2.81 vs 47.02±6.69)increased,apoptosis rate(33.26﹪±3.44﹪vs 16.01﹪±2.56﹪),pro-inflammatory cytokine TNF-α(138.56±27.05 vs 105.30±12.64),IL-6(123.69±10.08 vs 86.11±8.03),IL-18 expression level(41.72±5.61 vs 28.29±8.17)were significantly decreased,(P<0.05);Western blot showed that compared with the IR group,overexpression of lncRNA GAS5 could significantly reduce the ratio of p-PI3K/PI3K(0.79±0.06 vs 0.51±0.02),p-AKT/AKT(0.83±0.03 vs 0.47±0.01),and the protein level of cleaved caspase-3(0.81±0.03 vs 0.39±0.07),while the protein level of Cyclin B1(0.09±0.03 vs 0.55±0.06),CDK1(0.12±0.05 vs 0.53±0.08)and Bcl-2(0.09±0.02 vs 0.21±0.02)were increased(P<0.05).Conclusion In current study,we found that the expression of lncRNA GAS5 in ovarian granulosa cells of PCOS patients with insulin resistance was significantly decreased.In rats'model,overexpression of lncRNA GAS5 in granulosa cells could promote cell proliferation and cell cycle progression,and reduce the incidence of apoptosis,and inhibit the expression of pro-inflammatory cytokines TNF-α,IL-6,and IL-18 in granulosa cells under insulin resistance,which maybe related with PI3K/AKT signaling pathway.