过表达SNHG5对高糖诱导HK-2细胞凋亡及氧化应激的影响

Effects of Overexpression of SNHG5 on High Glucose-Induced Apoptosis and Oxidative Stress in HK-2 Cells

该文探讨小核仁RNA宿主基因5(small nucleolar RNA host gene 5,SNHG5)对高糖(high glucose,HG)诱导的HK-2凋亡、氧化应激的影响及分子机制。采用30 mmol/L葡萄糖处理HK-2细胞48 h,将pcDNA、pcDNA-SNHG5、anti-miR-阴性对照(negative control,NC)、微小RNA-196a(microRNA-196a,miRNA/miR-196a) inhibitor、pcDNA-SNHG5分别与miR-NC、miR-196a mimic共转染至HK-2细胞中,用30 mmol/L葡萄糖处理48 h。实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)检测SNHG5和miR-196a的表达水平;细胞计数试剂盒8(cell counting kit-8,CCK-8)检测细胞增殖;流式细胞术检测细胞凋亡情况;Western blot法检测蛋白表达水平;丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)试剂盒分别检测MDA含量及SOD、GSH-Px活性;双荧光素酶报告实验检测SNHG5和miR-196a的靶向关系。该研究得出,高糖诱导的HK-2细胞活性以及细胞中的SNHG5表达水平、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)表达水平均降低,miR-196a表达水平、凋亡率、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved cysteinyl aspartate specific proteinase 3,Cleaved-caspase3)表达水平、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)表达水平、MDA含量均升高,SOD、GSH-Px活性降低(P<0.05)。过表达SNHG5或抑制miR-196a表达后,高糖诱导的HK-2的细胞活性显著上升且细胞死亡率降低,Bcl-2表达水平显著上升,Bax、Cleaved-caspase3与MDA含量明显降低,SOD、GSH-Px活性升高(P<0.05)。SNHG5靶向调控miR-196a,miR-196a过表达可逆转SNHG5对高糖诱导的HK-2细胞活性、凋亡和氧化应激的影响。总之,过表达SNHG5可能通过下调miR-196a抑制高糖诱导的HK-2凋亡及氧化应激。

This study investigated the effect and molecular mechanism of SNHG5 (small nucleolar RNA host gene 5) on HG (high glucose)-induced HK-2 apoptosis and oxidative stress.HK-2 cells were treated with 30 mmol/L glucose for 48 h.pcDNA,pcDNA-SNHG5,anti-miR-NC (negative control),microRNA-196a (miRNA/miR-196a) inhibitor,pcDNA-SNHG5 were co-transfected with miR-NC or miR-196a mimic into HK-2 cells,respectively.The expression levels of SNHG5 and miR-196a were detected by RT-qPCR (quantitative real-time PCR);cell proliferation was detected by CCK-8 (cell counting kit-8);flow cytometry was performed to determine cell apoptosis;protein expression detected by Western blot;MDA (malondialdehyde),SOD(superoxide dismutase),glutathione peroxidase GSH-Px (glutathione peroxidase) kits were implemented to monitor the content of MDA and the activities of SOD and GSH-Px;the targeting relationship between SNHG5 and miR-196a was detected by dual-luciferase reporter assay.The study concluded that the expression level of SNHG5,cell viability,and expression level of Bcl-2 (B cell lymphoma/lewkmia-2) in HK-2 cells induced by high glucose decreased,miR-196a expression level,apoptosis rate,expression of activated Cleaved-caspase3 (Cleaved cysteinyl aspartate specific proteinase 3) and Bax (Bcl-2 associated X protein) level and MDA content increased,while SOD and GSH-Px activities decreased (P<0.05).After overexpression of SNHG5 or inhibition of miR-196a expression,high glucose-induced HK-2 cell activity significantly increased and cell death rate decreased,Bcl-2 expression level increased significantly,Bax,Cleaved-caspase3 and MDA contents significantly decreased,SOD,GSH-Px activity increased (P<0.05).SNHG5 can target and regulate miR-196a expression,and miR-196a overexpression can reverse the effects of SNHG5 on high glucose-induced HK-2 cell viability,apoptosis and oxidative stress.In conclusion,overexpression of SNHG5 may inhibit high glucose-induced HK-2 apoptosis and oxidative stress by downregulating miR-196a.