Wnt通路抑制因子1及调控miR-137在无功能性垂体腺瘤中的表达及功能研究

Expression and function of Wnt inhibitor factor 1 and miR-137 in non-functioning adenomas

目的分析Wnt通路抑制因子1(Wif1)及其调控miR-137在无功能性垂体腺瘤中的表达水平及意义,并观察miR-137对GT1-1细胞增殖、凋亡和Wnt信号通路的影响。方法用免疫组织化学法检测72例无功能性垂体腺瘤的侵袭能力,用实时荧光定量聚合酶链反应(RT-PCR)法检测样本中miR-137的表达水平。在GT1-1细胞中瞬时转染miR-137-mimic和miR-137-NC,用MTS检测观察细胞活力变化,用流式细胞术检测细胞凋亡情况,用RT-PCR和蛋白质印迹(Western blot)法检测分泌性卷曲相关蛋白4(sFRP4)和Wif1的表达水平。结果在72例临床样本中,Wif1的平均H-score值为134.50±45.30,根据H-score的中位数分为高、低表达组。临床资料显示,Wif1低表达组的肿瘤具有更强的侵袭行为(21例/36例vs 10例/36例,P=0.01)和更大的肿瘤体积[(15.46±3.54)cm^(3) vs(10.21±2.83)cm^(3),P<0.05]以及更短的肿瘤复发时间[(3.20±1.30)年vs(5.10±1.70)年,P<0.05]。RT-PCR结果显示,miR-17-5p和miR-137在Wif1低表达组分别下调至0.53±0.15(P<0.05)和0.17±0.07(P<0.01),而miR-29、miR-181a-5p和miR-603差异均无统计学意义(均P>0.05)。在GT1-1细胞中瞬时转染miR-137-mimic和miR-137-NC 24,48和72 h后,miR-137-mimic组的细胞活力较miR-137-NC组分别下降至(75.80±4.30)%(P<0.05),(62.50±3.90)%(P<0.01)和(52.40±3.40)%(P<0.01)。转染miR-137-mimic和miR-137-NC 24 h后,miR-137-NC组和miR-137-mimic组的Annexin V阳性细胞率分别为(4.50±1.60)%和(16.40±5.20)%(P<0.01);PI阳性细胞则分别为(2.10±0.80)%和(8.70±2.30)%(P<0.05)。miR-137促进sFRP4和Wif1的表达作用呈时间依赖性增加(P<0.05,P<0.01)。结论Wif1参与调控无功能性垂体腺瘤的生长和侵袭;在GT1-1细胞中,miR-137通过调控sFRP4和Wif1的表达,从而起到抗肿瘤作用。

Objective To analyze the levels of Wnt inhibitor factor 1(Wif1)and miR-137 in non-functioning adenomas and their relationship with clinical features,and measure the cell viability and apoptosis of GT1-1 cell line after miR-137 transfection.Methods Immunochemistry staining measured the H-score of Wif1 and real time PCR(RT-PCR)proved the level of miR-137 in 72 specimens.After transfection of miR-137-mimic and miR-137-NC in GT1-1 cells,the changes of cell viability were observed by MTS assay,apoptosis was detected by flow cytometry,and the expression levels of secreted frizzled related protein 4(sFRP4)and Wif1 were detected by quantitative real-time polymerase chain reaction(RT-PCR)and Western blot.Results The average H-score of Wif1 was 134.50±45.30 in 72 specimens.According to the median of H-score value,72 specimens were divided into high group and low group.There was stronger invasive behavior in low group than that in high group(21 cases/36 cases vs 10 cases/36 cases,P=0.01),lager volume[(15.46±3.54)cm^(3) vs(10.21±2.83)cm^(3),P<0.05]and shorter recurrence time[(3.20±1.30)years vs(5.10±1.70)years,P<0.05].RT-PCR results showed that miR-17-5p and miR-137 were down-regulated to 0.53±0.15(P<0.05)and 0.17±0.07(P<0.01)in the Wif1 low expression group,and there was no significant difference among miR-29,miR-181a-5p and miR-603(all P>0.05).After transient transfection of miR-137-mimic and miR-137-NC in GT1-1 cells for 24,48 and 72 h,the cell viability of miR-137 group decreased to(75.80±4.30)%(P<0.05),(62.50±3.90)%(P<0.01)and(52.40±3.40)%(P<0.01)than those in miR-137-NC group.After transient transfection of miR-137-mimic and miR-137-NC in GT1-1 cells for 24 h,the rates of Annexin V positive cells in the miR-137-NC group and miR-137-mimic group were(4.50±1.60)%and(16.40±5.20)%(P<0.01),and the PI positive cells were(2.10±0.80)%and(8.70±2.30)%(P<0.05).miR-137 upregulated the levels of sFRP4 and Wif1 in timely manner(P<0.05,P<0.01).Conclusion Wif1 is involved in regulating the growth and invasion of non-functioning pituitary adenomas,and miR-137 plays a tumor antagonism role in GT1-1 cells by regulating the expression of sFRP4 and Wif1 in GT1-1 cells.