miR-23a/PTEN轴在结直肠癌发生发展中的作用机制

Role of miR-23a/PTEN axis in the occurrence and development of colorectal cancer

目的分析微小RNA-23a(miR-23a)与磷酸酶及张力蛋白同源物基因(PTEN)调控轴在结直肠癌(colorectal cancer,CRC)LS513细胞中的表达情况,并探讨二者对CRC发生发展的影响。方法将LS513细胞分为空白对照组(NG组,细胞不做特殊处理)、阴性对照组(NC组,加入5μl Lipofectamine3000和50 pmol/μl NC)、抑制miR-23a表达组(miR-23a-inhibitor组,转染miR-23a-inhibitor序列)。采用实时荧光定量PCR法检测LS513细胞miR-23a、PTEN mRNA水平;MTT法检测LS513细胞增殖能力;Transwell实验检测细胞迁移、侵袭能力;蛋白质印迹法检测增殖、迁移侵袭及磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)通路相关蛋白水平变化。结果与NG组、NC组比较,miR-23a-inhibitor组LS513细胞中miR-23a水平显著降低(P<0.05),PTEN mRNA及蛋白表达水平均显著升高(P<0.05)。MTT法检测结果显示与NG组、NC组比较,miR-23a-inhibitor组增殖抑制率显著升高[(16.62±3.21)%vs(16.65±3.26)%vs(47.55±13.61)%](P<0.05),PCNA蛋白表达显著降低(P<0.05)。与NG组、NC组比较,miR-23a-inhibitor组LS513细胞迁移数[(287.25±58.32)vs(285.37±58.43)vs(136.87±36.53)]、侵袭数[(242.53±50.95)vs(239.87±50.62)vs(103.77±30.06)]均显著减少(P<0.05),且MMP-9、E-cadlerin蛋白表达显著降低(P<0.05)。与NG组、NC组比较,miR-23a-inhibitor组LS513细胞中p-PI3K/PI3K、p-ATK/ATK蛋白表达量显著降低(P<0.05)。结论抑制miR-23a表达可能上调PTEN基因表达抑制PI3K/Akt通路活化,进而抑制LS513细胞增殖、迁移及侵袭。

Objective To analyze the expression of microRNA-23a(miR-23a)and phosphatase and tensin homolog detected on chromosome ten(PTEN)regulatory axis in LS513 cells,and to explore the effects of miR-23a and PTEN on the occurrence and development of CRC.Methods LS513 cells were divided into blank control group(NG group):cells were not specially treated,negative control group(NC group):5μl Lipofectamine3000 and 50 pmol/μl NC were added,Inhibition of miR-23a expression group(miR-23a-inhibitor group):miR-23a-inhibitor sequence was transfected.The mRNA levels of miR-23a and PTEN in LS513 cells were detected by real-time fluorescence quantitative PCR(qRT-PCR);the proliferation of LS513 cells was detected by MTT method;Transwell assay was used to detect cell migration and invasion;Western blotting was used to detect proliferation,migration,invasion and phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT)signaling pathway related proteins.Results Compared with those in NG group and NC group,the level of miR-23a in miR-23a inhibitor group was significantly lower(P<0.05),and the expression levels of PTEN mRNA and protein were significantly higher(P<0.05).The results of MTT showed that,compared with those in NG group and NC group,the proliferation inhibition rate in miR-23a-inhibitor groupwas significantly higher(P<0.05),and the expression of PCNA protein was significantly lower(P<0.05).Compared with those in NG group and NC group,the migration number and invasion number of LS513 cells in miR-23a-inhibitor group were significantly lower(P<0.05),and the expression of MMP-9 and E-cadherin protein was significantly lower(P<0.05).Compared with those in NG group and NC group,the expression of p-pi3k/PI3K and p-atk/ATK protein in miR-23a-inhibitor group was significantly lower(P<0.05).Conclusion Inhibition of miR-23a expression may up-regulate PTEN gene expression,inhibit PI3K/Akt pathway activation,and then inhibit the proliferation,migration and invasion of LS513 cells.