长链非编码RNANEAT1对HaCaT细胞中miR-485-5p和STAT3表达的影响

EffectofIncRNAA NEAT1 on the expression of miR-485-5p/STAT3 in HaCaT cells

目的:探讨LncRNA NEAT1对HaCaT细胞中miR-485-5p和STAT3表达的调控及对HaCaT细胞增殖、凋亡的影响。方法:采用PCR、Western印迹法检测HaCat细胞和正常人表皮角质形成细胞(NHEK细胞)中LncRNANEAT1、miR-485-5p及STAT3 mRNA和蛋白表达情况。荧光原位杂交技术(FISH)和双荧光素酶报告基因检测LncRNA NEAT1、miR-485-5p和STAT3之间的靶向调控关系。再通过RNA干扰技术分别沉默HaCat细胞中LncRNANEAT1、STAT3表达,以及转染miR-485-5p模拟剂(mimic)和抑制剂(inhibitor)分别上、下调miR-485-5p,然后应用PCR、Western Blot、流式细胞术、CCK8等实验技术分别检测LncRNANEAT1、miR-485-5p、STAT3表达及细胞增殖、凋亡情况。结果:与NHEK细胞组相比,LncRNA NEAT1和STAT3在HaCaT细胞组中高表达,而miR-485-5p在HaCaT细胞中低表达;miR-485-5p与LncRNA NEAT1共定位于HaCaT细胞质,且miR-485-5p与LncRNA NEAT1 3′-UTR、STAT3 3′-UTR之间存在互补结合序列;共转染转野生型psiCHECK-LncRNA NEAT1-3′UTR-WT、psiCHECK-STAT3-3′UTR-WT重组质粒时,miR-485-5pmimic组相对萤光素酶活性明显低于miR-NC组。而共转染突变型psiCHECK-LncRNANEAT1-3′UTR-MUT、psiCHECK-STAT3-3′UTR-MUT重组载体质粒时,miR-485-5p mimic组和miR-NC组萤光素酶活性无明显差异;沉默LncRNA NEAT1或STAT3均可抑制HaCaT细胞增殖及促进其凋亡;下调miR-485-5p可促进HaCaT细胞增殖及抑制其凋亡,而上调miR-485-5p则与之相反;下调miR-485-5p可减弱沉默LncRNANEAT1对HaCaT细胞功能的影响。结论:LncRNANEAT1可通过充当竞争性内源RNA(competing endogenous RNA,ceRNA)吸附miR-485-5p增强STAT3表达来促进HaCaT细胞增殖并抑制其凋亡。

Objective:To investigate the effect of lncRNA NEAT1 on the expression of miR-485-5p and STAT3,as well as the proliferation and apoptosis of HaCaT cells.Methods:First,the expression of NEAT1,miR-485-5p and STAT3 in HaCat cells and NHEK cells was detected by qPCR and Western Blotting Afterward,FISH and double luciferase assays were used to detect the regulatory relationships between IncRNA NEATI,miR-485-5p and STAT3.The expression of IncRNA NEAT1 and STAT3 in Ha-Cat cells was silenced by RNA interference technology,and miR-485-5p mimic and inhibitor were transfected to up-regulate and down-regulate miR-485-5p respectively.Then,the expression of IncRNA NEAT1,miR-485-5p and STAT3 was detected by qPCR and Western Blotting,and the cell proliferation and apoptosis were detected by flow cytometry and CCK8.Results:Compared with the NHEK cell group,lncRNA NEAT1 and STAT3 were highly expressed in HaCaT cells,while miR-485-5p was lowly expressed.miR-485-5p and IncRNA NEAT1 were co-located in the cytoplasm of HaCaT cells,and there were complementary binding sequences between miR-485-5p and IncRNA NEATI 3'-UTR and STAT33'-UTR.Afer co-transfection with wild-type psiCHECKIncRNA NEAT1-3'UTR-WT and psiCHECK-STAT3-3'UTR-WT recombinant plasmids,the relative luciferase activity of the miR-485-5p mimic group was significantly lower than that of the miR-NC group.After co-transfection with the mutant psiCHECK-LncRNA NEATI-3'UTR-MUT and psiCHECK-STAT3-3'UTR-MUT recombinant vector plasmids,there was no significant difference in luciferase activity between the miR-485-5p mimic group and the miR-NC group.Silencing lncRNA NEAT1 or STAT3 inhibited the proliferation of HaCaT cells and promoted their apoptosis.Down-regulation of miR-485-5p promoted the proliferation of HaCaT cells and inhibited their apoptosis,while up-regulation of miR-485-5p achieved opposite results.Down-regulation of miR-485-5p attenuated the effect of silencing LncRNA NEATI on the function of HaCaT cells.Conclusion:LncRNA NEAT1 can promote the proliferation of HaCaT cells and inhibit their apoptosis by acting as ceRNA sponge miR-485-5p to enhance the expression of STAT3.