尿路上皮癌相关基因1对磷脂酰肌醇3激酶-丝氨酸/苏氨酸激酶信号通路和葡萄糖摄取的影响

Effect of urothelial carcinoma associated 1 on the phosphatidylinositol 3-kinase-serine/threonine kinase signal pathway and glucose uptake

目的：观察长链非编码RNA（lncRNA）尿路上皮癌相关基因1（UCA-1）对人肾上皮293T细胞的磷脂酰肌醇3激酶-丝氨酸/苏氨酸蛋白激酶（PI3K-Akt）信号通路及糖代谢的影响，并探索其中可能的分子机制。方法将293T细胞分为5组，即空白对照组、空质粒的阴性对照组、无义乱序RNA的阴性对照组（si-scr组）、过表达UCA-1组（UCA-1组）及沉默UCA-1组（si-UCA-1组）。通过转染UCA-1过表达质粒或UCA-1特异性小干扰RNA（siRNA）来干预293T细胞中的UCA-1水平。Western blotting法检测PI3K-Akt信号通路关键蛋白的表达变化，实时荧光定量聚合酶链反应（PCR）和荧光素酶报告系统检测UCA-1对第10号染色体缺失的磷酸酶及张力蛋白同源基因（PTEN）的RNA水平和转录活性的影响，并通过2-脱氧-3H-D-葡萄糖掺入法检测细胞的葡萄糖摄取能力。两组之间比较用t检验，多组之间比较采用单因素方差分析。结果（1）与对照组比较，UCA-1组PTEN的mRNA水平明显下降（1.07±0.05比0.24±0.05，t=14.257，P〈0.01），而转染UCA-1特异性siRNA细胞中PTEN的mRNA水平明显上升（si-scr组比si-UCA-1组,1.02±0.03比12.47±0.17, t=15.816, P〈0.01）。（2）阴性对照组与UCA-1组比较，转染UCA-1过表达质粒PTEN的启动子转录活性降低：1.07±0.05比0.23±0.03（t=15.192, P〈0.01），而转染UCA-1特异性siRNA后PTEN的启动子转录活性升高[si-scr组与si-UCA-1比较,1.02±0.03比12.53±0.43（t=14.527, P〈0.01）]。（3）过表达UCA-1组细胞的葡萄糖摄取能力升高至对照组的（3.78±0.39）倍（t=6.711，P〈0.05），同时转染PTEN过表达质粒可逆转这种作用（P〉0.05）。结论 UCA-1可通过抑制PTEN激活293T细胞PI3K-Akt信号通路，并促进其葡萄糖摄取能力。

Objective To observe the effect of long noncoding RNA（lncRNA）-urothelial carcinoma associated 1 （UCA-1） on the phosphatidylinositol 3-kinase-serine/threonine kinase （PI3K-Akt） signal pathway of 293T adipocyte and glucose uptake activity, and to explore the possible molecular mechanism. Methods Renal 293T epithelial cells were divided 5 groups： blank control group, empty plasmid as negative control group, nonsense disorderly sequence RNA negative control group （si-scramble, si-scr）, over expression UCA-1 group （group UCA-1）, and the silent UCA-1 group （si-UCA-1）. UCA-1 was overexpressed or silenced by plasmids or specific siRNAs transfection in 293T. Then the effect of UCA-1 on the PI3K-Akt signal pathway of 293T cells was measured by Western blotting, and the RNA level and transcription activity of PTEN （phosphatase and tensin homolog deleted on chromosome ten） were detected by real-time fluorescence quantitative PCR and luciferase reporter assay, respectively. 2-deoxy-^3H-D-glucose incorporation assay was performed to measure the glucose uptake activity of 293T cells. The t test was performed in comparison between the two groups, while one way variance analysis for multiple groups comparison. Results Compared with the control group, the mRNA of PTEN and transcription activity in group UCA-1 was decreased（1.07 ± 0.05 vs 0.24 ± 0.05, 1.07 ± 0.05 vs 0.23 ± 0.03, t=14.257, 15.192, respectively, both P〈0.01）; Compared with the si-UCA-1group, the level of mRNA of PTEN and transcription activity in si-scr group dramatically increased（1.02±0.03 vs 12.47±0.17, 1.02±0.03 vs 12.53± 0.43, t=15.816, 14.527, both P〈0.01）. The glucose uptake activity was also increased in UCA-1 group （3.78 ± 0.39）times than the control group（ t=6.711,P〈0.05）, and the restore expression of PTEN could reverse the regulation of UCA-1 on the glucose uptake activity（P〉0.05）. Conclusion UCA-1 can activate the PI3K-Akt signal pathway of 293T cells and enhance the glucose uptake activity by inhibiting PTEN expression.