长非编码RNA-p53诱导转录本通过靶向miR-1297调控高糖处理HK-2细胞的迁移和上皮间质转化

LINC-PINT regulated the migration and epithelial-mesenchymal transition of HK-2 cells treated with high glucose by targeting miR-1297

目的探讨长非编码RNA-p53诱导转录本(long intergenic non-protein coding RNAp53 induced transcript,LINC-PINT)对高糖(high glucose,HG)处理人肾小管上皮细胞HK-2迁移和上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及其分子机制。方法分别用5.5 mmol/L、30 mmol/L葡萄糖处理HK-2细胞,记为正常糖(normal glucose,NG)组、高糖(high glucose,HG)组。将HG组HK-2细胞分为HG+si-NC组、HG+si-LINC-PINT组、HG+miR-NC组、HG+miR-1297组、HG+si-LINC-PINT+anti-miR-NC组、HG+si-LINC-PINT+anti-miR-1297组。RT-qPCR检测LINC-PINT和miR-1297的表达水平,采用Transwell和Western blot检测HK-2细胞迁移和相关蛋白的表达,双荧光素酶报告实验鉴定LINC-PINT和miR-1297的靶向关系。结果与NG组比较,HG组HK-2细胞中,LINC-PINT[(1.00±0.05)比(2.87±0.19)]和miR-1297[(1.02±0.07)比(0.43±0.04)]的表达水平分别显著升高和降低,迁移细胞数[(89.37±4.56)比(212.35±13.28)]以及基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)[(0.31±0.02)比(0.79±0.07)]、Vimentin[(0.22±0.01)比(0.58±0.03)]、α-平滑肌肌动蛋白(α-smooth actin,α-SMA)[(0.19±0.02)比(0.53±0.05)]和纤维粘连蛋白(fibrimine,FN)[(0.12±0.01)比(0.34±0.04)]的表达水平显著升高,E-钙黏蛋白(E-cadherin)[(0.69±0.07)比(0.27±0.02)]的表达水平显著降低(P<0.05)。抑制LINC-PINT表达或过表达miR-1297显著降低迁移细胞数[(197.28±10.25)比(137.53±7.59),(216.58±13.87)比(145.29±6.82)]、MMP-2[(0.83±0.05)比(0.47±0.04),(0.78±0.05)比(0.42±0.04)]、波形蛋白[(0.62±0.05)比(0.36±0.02),(0.56±0.04)比(0.32±0.03)]、α-SMA[(0.51±0.03)比(0.28±0.02),(0.56±0.03)比(0.30±0.02)]和FN[(0.37±0.03)比(0.23±0.03),(0.36±0.03)比(0.25±0.02)]的表达水平,显著升高E-cadherin[(0.24±0.02)比(0.58±0.03),(0.23±0.02)比(0.55±0.04)]的表达水平(P<0.05)。LINC-PINT靶向调控miR-1297的表达(P<0.05)。下调miR-1297逆转了抑制LINC-PINT表达对HG处理HK-2细胞迁移和EMT的影响(P<0.05)。结论LINC-PINT通过靶向miR-1297调控HG处理HK-2细胞的迁移和EMT。

Objective To explore the effect of long intergenic non-protein coding RNA-p53 induced transcript(LINC-PINT)on the migration and epithelial-mesenchymal transition(EMT)of human renal tubular epithelial HK-2 cells treated with high glucose(HG)and elucidate its molecular mechanism.Methods HK-2 cells were treated with 5.5 and 30 mmol/L glucose and recorded as NG and HG groups.HK-2 cells in HG group were divided into the groups of HG+si-NC,HG+si-LINC-PINT,HG+miR-NC,HG+miR-1297,HG+si-LINC-PINT+anti-miR-NC and HG+si-LINC-PINT+antimiR-1297.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was used for detecting the expression levels of LINC-PINT and miR-1297.Transwell and Western blot for detecting the migration of HK-2 cells and the expression of related proteins.Dual luciferase reporter assay for examining the targeting relationship between LINC-PINT and miR-1297.Results Compared with NG group,the expression levels of LINC-PINT[(1.00±0.05)vs(2.87±0.19)]and miR-1297[(1.02±0.07)vs(0.43±0.04)]significantly spiked and dropped obviously in HG group;number of migrated cells[(89.37±4.56)vs(212.35±13.28)]and expression levels of MMP-2[(0.31±0.02)vs(0.79±0.07)],vimentin[(0.22±0.01)vs(0.58±0.03)],α-SMA[(0.19±0.02)vs(0.53±0.05)]and FN[(0.12±0.01)vs(0.34±0.04)]rose obviously while expression level of E-cadherin declined markedly[(0.69±0.07)vs(0.27±0.02),P<0.05].Suppression of LINC-PINT expression or overexpression of miR-1297 significantly lowered the number of migrated cells[(197.28±10.25)vs(137.53±7.59),(216.58±13.87)vs(145.29±6.82)]and the expression levels of MMP-2[(0.83±0.05)vs(0.47±0.04),(0.78±0.05)vs(0.42±0.04)],vimentin[(0.62±0.05)vs(0.36±0.02),(0.56±0.04)vs(0.32±0.03)],α-SMA[(0.51±0.03)vs(0.28±0.02),(0.56±0.03)vs(0.30±0.02)]and Fn[(0.37±0.03)vs(0.23±0.03),(0.36±0.03)vs(0.25±0.02)]and significantly boosted the expression level of E-cadherin[(0.24±0.02)vs(0.58±0.03),(0.23±0.02)vs(0.55±0.04)](P<0.05).LINC-PINT targeted the expression of miR-1297(P<0.05).A down-regulation of miR-1297 reversed the effect of suppressing the expression of LINC-PINT on the migration and EMT of HK-2 cells treated with HG(P<0.05).Conclusion LINC-PINT regulates the migration and EMT of HK-2 cells treated with HG through targeting miR-1297.