长链非编码RNA TapSAKI对缺氧损伤肾小管上皮细胞的影响

Effect of long non - coding RNA TapSAKI on the renal tubular epithelial cells induced by hypoxia

目的研究长链非编码RNA（LncRNA） TapSAKI对缺氧损伤肾小管上皮细胞HK-2细胞的影响。方法将体外培养的HK-2细胞分为常氧组（A组）、缺氧损伤组（B组）、缺氧损伤＋control siRNA组（C组）、缺氧损伤＋TapSAKI siRNA组（D组）。采用实时荧光定量PCR（qPCR）检测TapSAKI表达变化；CCK-8检测各组细胞的增殖情况；流式细胞仪检测细胞周期和细胞凋亡；Western blot检测周期相关蛋白CyclinD1、CDK2和凋亡相关蛋白Bcl-2、Bax水平。结果qPCR结果显示，与A组比较，B组TapSAKI基因表达增加（48.92±0.31比1.00±0.01，P〈0.05）；与C组比较，D组TapSAKI基因表达降低（4.70±0.60比48.31±0.29，P〈0.05）。CCK-8结果显示，与A组比较，B组的增殖降低（P〈0.05）；与C组比较，D组TapSAKI siRNA干扰后增殖升高（P〈0.05）。流式细胞仪检测结果表明，与A组比较，B组凋亡率升高[（26.38±1.21）%比（6.45±0.46）%，P〈0.05]；与C组比较，D组凋亡率显著降低[（10.98±0.88）%比（21.59±1.30）%，P〈0.05]。Western blot检测Bax、Bcl-2的蛋白表达，与A组比较，B组的Bax表达升高，Bcl-2表达减低（1.304±0.082比0.411±0.002、0.390±0.007比1.027±0.022，均P〈0.05）；与C组比较，D组Bax表达减低，Bcl-2表达升高，差异均有统计学意义（0.655±0.819比1.419±0.087，0.819±0.034比0.437±0.014，均P〈0.05）。与A组比较，B组G0/G1期细胞所占比例显著增加（69.82±1.14比34.46±0.82，P〈0.05），S期细胞占的比例显著减少（11.6±0.60比42.23±1.46，P〈0.05）。与A组比较，B组CyclinD1和CDK2蛋白表达减低（0.659±0.062比1.723±0.084，0.414±0.015比0.87±0.031，均P〈0.05）；与C组比较，D组G0/G1期细胞所占比例明显减少（30.77±0.33比61.81±1.50，P〈0.05），S期细胞占的比例明显增加（40.32±0.72比17.92±0.71，P〈0.05），CyclinD1和CDK2蛋白表达增加（2.049±0.027比0.626±0.024，0.89±0.104比0.424±0.012，均P〈0.05）。结论在缺氧条件下，LncRNA TapSAKI在肾小管上皮细胞表达丰富，引起肾小管上皮细胞增殖缓慢，凋亡增加，提示LncRNA TapSAKI可通过影响细胞的增殖和凋亡参与疾病的恶化。

Objective To study the effect of long non - coding（LncRNA） TapSAKI on HK - 2 cells of renal tubular epithelial ceils induced by hypoxia. Methods The cultured HK -2 cells were divided into the normal oxygen group （group A） ,hypoxia injury group （group B） ,hypoxia injury ± control siRNA group （group C） ,and hypoxia injury ＋ TapSAKI siRNA group （ group D）. The real - time quantitative polymerase chain reaction （qPCR） was used to detect the change in TapSAKI expression; proliferation was measured by CCK- 8 assay; cell cycle and cell apoptosis were measured by flow cytometry;Western blot was used to detect cycle related CyclinD1 protein, CDK2 and apoptosis related protein Bcl - 2 and Bax. Results From the result of qPCR, compared with group A, the expression of TapSAKI gene in group B increased significantly （48.92 ±0.31 vs. 1.00 ±0.01 ,P 〈0.05）. Compared with group C,the expression of TapSAKI gene in group D decreased significantly（4.70 ±0. 60 vs. 48.31 ±0.29,P 〈0.05）. The result of CCK - 8 showed that the proliferation in group B significantly decreased compared with group A （ P 〈 0.05 ）. Compared with group C ,the proliferation in group D significantly increased （ P 〈 0.05 ）. The flow cytometer test results showed that the apoptosis rate in group B was higher than that in group A [ （26.38 ± 1.21 ）% vs. （6.45 ± 0.46）% ,P 〈 0.05 ]. Comparing with group C, the apoptosis rate in group D decreased significantly [ （ 10.98 ± 0.88 ） % vs. （21.59 ± 1.30） %, P 〈 0. 05 ]. Compared with group A, Bax expression in group B increased, and Bcl - 2 expression decreased （ 1. 304 ± 0. 082 vs. 0. 411 ± 0. 002,0. 390 ± 0. 007 vs. 1. 027 ± 0. 022, all P 〈 0.05 ）. In group D, the expression of Bax was lower than that in group C, and the expression of Bcl -2 increased, both of which were statistically significant （0.655 ± 0.819 vs. 1. 419 ± 0.087,0. 819 ± 0. 034 vs. 0. 437 ± 0. 014, all P 〈 0.05 ）. Compared with group A, the proportion of G0/G1 phase cells in group B significantly increased （69.82 ± 1. 14 vs. 34.46 ± 0.82, P 〈 0.05 ）, and the proportion of S phase cells decreased significantly （ 11.6 ± 0.60 vs. 42.23 ± 1.46, P 〈 0.05）. Compared with group A, CyclinD1 and CDK2 protein expressions in group B decreased （0. 659 ± 0.062 vs. 1. 723 ± 0. 084,0.414 ± 0. 015 vs. 0. 87 ± 0.031, all P 〈 0.05 ）. Compared with group C ,group D G0/G1 phase cells significantly decreased （30.77 ± 0.33 vs. 61.81 ±1.50, P 〈 0. 05 ）, the proportion of S phase cells significantly increased （ 40.32 ± 0.72 vs. 17.92 ± 0.71, P 〈 0.05 ）, CyclinD1 and CDK2 protein expressions increased （2. 049 ± 0. 027 vs, 0. 626 ± 0. 024,0.89 ± 0. 104 vs. 0. 424 ± 0. 012, all P 〈 0.05 ）. Conclusions Under hypoxic conditions, LncRNA TapSAKI in renal tubular epithelial cells was abundant, which may inhibit renal tubular epithelial cell proliferation and accelerate apoptosis. It is suggested that LncRNA TapSAKI may play a role in the deterioration of ceil proliferation and apoptosis.