卡格列净通过抑制长链非编码RNA CO9缓解高糖环境下肾小管上皮细胞凋亡

Canagliflozin improved renal tubular epithelial cell apoptosis by inhibiting lncRNA CO9 under high glucose culture in vitro

目的探讨高糖环境下卡格列净通过抑制lncRNA CO9缓解肾小管上皮细胞凋亡的作用及其机制。方法体外培养人近端肾小管上皮细胞系(HK-2),将其分为正常葡萄糖(NG)组、高渗(HO)组、高糖(HG)组、高糖+二甲基亚砜组(DMSO组)、高糖+卡格列净组(CANA组)、高糖+阴性对照小干扰RNA组(si-NC组)、高糖+lncRNA CO9小干扰RNA组(si-CO9组)、高糖+空载质粒组(PEX-NC组)、高糖+lncRNA CO9过表达质粒组(PEX-CO9组)、高糖+卡格列净+lncRNA CO9过表达质粒组(CANA+PEX-CO9组)。采用细胞计数试剂-8(CCK-8)法与四唑盐(MTT)比色法检测细胞增殖率;Western blotting检测凋亡相关蛋白[B细胞淋巴瘤2相关X蛋白(Bax)、B细胞淋巴瘤2(Bcl-2)、半胱氨酸蛋白酶-3(Caspase-3)、活化型Caspase-3]与核因子(NF)-κB p50亚基及其前体p105蛋白的相对表达量,计算Bax/Bcl2蛋白相对表达量比值;流式细胞术检测细胞凋亡率;实时荧光定量PCR(qRT-PCR)检测lncRNA CO9与NF-κB1 mRNA的相对表达量。采用荧光原位杂交检测lncRNA CO9亚细胞定位,基因本体论(GO)、京都基因与基因组百科全书(KEGG)分析预测其功能。收集临床正常对照组(NC组,5例)与糖尿病肾脏病组患者(DKD组,5例)肾脏样本,检测其近端肾小管上皮细胞lncRNA CO9的表达。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果与NG组相比,HG组细胞增殖率降低,Bax/Bcl2蛋白相对表达量比值、Caspase-3与活化型Caspase-3蛋白相对表达量、细胞凋亡率、lncRNA CO9均显著上调;而与HG组相比,CANA组细胞凋亡率下降,lncRNA CO9表达下调(P<0.05)。lncRNA CO9定位于细胞质。GO与KEGG分析显示,lncRNA CO9与细胞凋亡通路相关。在DKD组肾脏组织近端肾小管上皮细胞lncRNA CO9表达较NC组上调(P<0.05)。相较于HG组,si-CO9组凋亡蛋白相对表达量、凋亡率及NF-κB1 mRNA、NF-κB p50亚基及其前体p105蛋白相对表达量下降,PEX-CO9组则增高(P<0.05)。与CANA组相比,CANA+PEX-CO9组的凋亡蛋白相对表达量、凋亡率与NF-κB1 mRNA、NF-κB p50亚基及其前体p105蛋白相对表达量上升(P<0.05)。结论卡格列净可通过抑制lncRNA CO9的表达缓解高糖诱导的肾小管上皮细胞凋亡。

Objective To investigate the protective effect and potential mechanism of canagliflozin on apoptosis of renal tubular epithelial cell cultured with high glucose through long non-coding RNA(lncRNA)CO9.Methods Human kidney cells line-2(HK-2)were cultured and divided into normal glucose(NG)group,high osmolar(HO)group,high glucose(HG)group,high glucose+dimethyl sulfoxide(DMSO)group(DMSO group),high glucose+canagliflozin(CANA)group(CANA group),HG+negative control small interfering RNA(si-NC)group(si-NC group),HG+lncRNA CO9 small interfering RNA(si-CO9)group(si-CO9 group),HG+negative control plasmid(PEX-NC)group(PEX-NC group),HG+lncRNA CO9 overexpression plasmid(PEX-CO9)group(PEX-CO9 group),HG+canagliflozin+PEX-CO9 group(CANA+PEX-CO9 group).Cell proliferation rate was detected by cell counting kit-8(CCK-8)assay and methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay,relative protein expression of apoptotic protein[B cell lymphoma-2-associated X(Bax),B cell lymphoma-2(Bcl-2),Caspase-3,and Cleaved-caspase-3]and nuclear factor(NF)-κB p50 subunit and precursor p105 protein was detected by Western blotting,Bax/Bcl2 relative protein expression value was calculated,and cell apoptosis rate was detected by flow cytometry.Expression of lncRNA CO9 and NF-κB1 mRNA was verified by fluorescence quantitative real-time PCR(qRT-PCR).The subcellular localization of lncRNA CO9 was detected by fluorescence in situ hybridization,and its function was predicted by gene ontology(GO)and Kyoto encyclopedia of genes and genome(KEGG)analysis.Kidney samples from clinical normal controls(NC group,n=5)and patients with diabetic kidney disease(DKD group,n=5)were collected to detect lncRNA CO9 expression in proximal renal tubular epithelial cells.The t-test was used for comparison between the two groups,and the one-way analysis of variance was used for the comparison among multiple groups.Results Compared with NG group,the cell viability in HG group was decreased,the protein expression of Bax/Bcl2,Caspase-3,Cleaved-caspase-3 and the cell apoptosis rate were significantly higher,and the expression level of lncRNA CO9 was significantly increased;while compared with HG group,the cell apoptosis rate and the expression level of lncRNA CO9 in CANA group were both decreased(P<0.05).LncRNA CO9 was located in the cytoplasm.GO and KEGG analysis showed that the main biological processes of lncRNA CO9 were associated with apoptosis pathway.LncRNA CO9 expression in proximal renal tubular epithelial cells was up-regulated in DKD group compared with NC group(P<0.05).Compared with HG group,the expression level of apoptotic protein,apoptosis rate and the expression of NF-κB1 mRNA and NF-κB p50 subunit and precursor p105 protein decreased in si-CO9 group,while increased in PEX-CO9 group(P<0.05).Compared with CANA group,the level of apoptotic protein,apoptosis rate and the expression of NF-κB1 mRNA and NF-κB p50 subunit and precursor p105 protein were significantly increased in CANA+PEX-CO9 group(P<0.05).Conclusion Canagliflozin alleviates renal tubular epithelial cell apoptosis induced by high glucose by inhibiting the expression of lncRNA CO9.