【摘要】 目的 探讨长链非编码RNA(lncRNA) GAS5通过靶向miR-155调节Th17/Treg失衡在乳糜泻中的作用。方法 收集20例乳糜泻患者(乳糜泻组)及同时期20例无乳糜泻或其他自身免疫性疾病的健康体检者(健康组)的外周血,分选两组的CD4~+T细胞。q RT-PCR法检测乳糜泻患者与对照组CD4~+T细胞中lncRNA GAS5以及miR-155的表达,流式细胞术分析Th17/Treg比例。Pearson统计分析lnc RNA GAS5、miR-155的表达水平与Th17/Treg比例的相关性。体外实验中,将乳糜泻组患者的CD4~+T细胞分为7组:对照组, pcDNA3.1-NC组, pc DNA3.1-GAS5组, miR-NC组, miR-155-mimics组, pc DNA3.1-GAS5+miRNC组, pc DNA3.1-GAS5+miR-155-mimics组。q RT-PCR法检测各组细胞中lnc RNA GAS5和miR-155的表达。Edu染色和Hoest33342染色法分别检测各组CD4~+T细胞的增殖和凋亡。细胞免疫荧光染色检测各组CD4~+T细胞中IL-17和Foxp3的表达。流式细胞术检测CD4~+T细胞中IL-17~+细胞和Foxp3~+细胞的比例。结果 与健康组相比,乳糜泻组lnc RNA GAS5的表达水平降低, miR-155的表达水平增加。lnc RNA GAS5表达与Th17/Treg比例呈负相关性(r=-0.65,P<0.05), miR-155表达与Th17/Treg比例呈正相关性(r=0.70,P<0.05)。乳糜泻组CD4~+T细胞中,与pc DNA3.1-NC组相比, pc DNA3.1-GAS5组中lnc RNA GAS5和Foxp3的表达水平、 CD4~+T细胞的增殖率和Foxp3~+细胞比例均增加(P_均<0.05),而miR-155和IL-17的表达水平、 CD4~+T细胞的凋亡率及IL-17~+细胞比例均降低(P_均<0.05);与miR-NC组相比, miR-155-mimics组中Foxp3的表达水平、 CD4~+T细胞的增殖率和Foxp3~+细胞比例均降低(P_均<0.05),而miR-155和IL-17的表达水平、 CD4~+T细胞的凋亡率及IL-17~+细胞比例均增加(P_均<0.05);与pc DNA3.1-GAS5+miR-NC组比, pcDNA3.1-GAS5+miR-155-mimics组中Foxp3的表达水平、 CD4~+T细胞的增殖率和Foxp3~+细胞比例均降低(P_均<0.05),而miR-155和IL-17的表达水平、 CD4~+T细胞的凋亡率及IL-17~+细胞比例均增加(P_均<0.05)。结论 lncRNA GAS5通过靶向miR-155调节Th17/Treg失衡,影响乳糜泻患者CD4~+T细胞的增殖和凋亡,进而影响IL-17和Foxp3的表达,提示lncRNA GAS5可能是乳糜泻治疗的潜在靶点。更多还原

【Abstract】 Objective To investigate how long non-coding RNA(lncRNA) GAS5 regulates Th17/Treg imbalance through targeting miR-155 in celiac disease(CeD).Methods Peripheral blood was collected from 20 CeD patients(CeD group) and 20 healthy individuals without CeD or other autoimmune diseases(healthy group), CD4~+T cells were isolated from both groups. the expression levels of lncRNA GAS5 and miR-155 in CD4~+T cells from CeD patients and the control group were dected by qRT-PCR. Flow cytometry was used to analyze the Th17/Treg ratio. Pearson statistical analysis was performed to evaluate the correlation between the expression levels of lncRNA GAS5 and miR-155 with the Th17/Treg ratio in CeD patients. In vitro, CD4~+T cells from the CeD patients were divided into 7 groups: control group, pcDNA3. 1-NC group, pcDNA3. 1-GAS5 group, miR-NC group, miR-155-mimics group, pcDNA3. 1-GAS5 + miR-NC group, and pcDNA3. 1-GAS5 + miR-155-mimics group. q RT-PCR was used to detect the expression levels of lncRNA GAS5 and miR-155 in each group. Edu staining and Hoest33342 staining was used to detect the proliferation and apoptosis of CD4~+T cells, respectively. Immunofluorescence staining was used to measure the expression of IL-17 and Foxp3. Flow cytometry was used to detect the proportion of IL-17~+and Foxp3~+cells in each group.Results Compared to those in the healthy group, the expression level of lncRNA GAS5 in the CeD group was decreased, while the expression level of miR-155 was increased. lncRNA GAS5 was negatively correlated with the Th17/Treg ratio(r=-0. 65,P< 0. 05), and miR-155 was positively correlated with the Th17/Treg ratio(r= 0. 70,P<0. 05). In CD4~+T cells from the CeD group, the pcDNA3. 1-GAS5 group showed increased expression levels of lncRNA GAS5 and Foxp3, increased proliferation rate of CD4~+T cells, and increased proportion of Foxp3~+cells(allP< 0. 05), while the expression levels of miR-155 and IL-17, the apoptosis rate of CD4~+T cells, and the proportion of IL-17~+cells were decreased(allP<0. 05) when compared with those in the pcDNA3. 1-NC group. When compared with the miR-NC group, the miR-155-mimics group showed decreased Foxp3 expression level, decreased proliferation rate of CD4~+T cells, and decreased proportion of Foxp3~+cells(allP< 0. 05), while the expression levels of miR-155 and IL-17, the apoptosis rate of CD4~+T cells, and the proportion of IL-17~+cells were increased(allP< 0. 05). Compared with the pcDNA3. 1-GAS5 + miR-NC group, the pcDNA3. 1-GAS5~+miR-155-mimics group showed decreased Foxp3 expression level, decreased proliferation rate of CD4~+T cells, and decreased proportion of Foxp3~+cells(allP< 0. 05), while the expression levels of miR-155 and IL-17, the apoptosis rate of CD4~+T cells, and the proportion of IL-17~+cells were increased(allP< 0. 05).Conclusion lncRNA GAS5 regulates Th17/Treg imbalance by targeting miR-155, affecting the proliferation and apoptosis of CD4~+T cells in CeD patients, thereby influencing IL-17 and Foxp3 expression. This suggests that lncRNA GAS5 may serve as a potential therapeutic target for CeD.更多还原