过表达circRNA_LARP4对鼻咽癌细胞的肿瘤干细胞样特性和线粒体膜电位的影响

Effect of Overexpression of circRNALARP4 on Tumor Stem-like Properties and Mitochondrial Membrane Potential of Nasopharyngeal Carcinoma Cells

目的探讨过表达circRNA_LARP4(circ_LARP4)对鼻咽癌CNE2和5-8F细胞肿瘤干细胞样特性及线粒体膜电位的影响。方法鼻咽癌CNE2和5-8F细胞转染circ_LARP4过表达载体,qRT-PCR检测circ_LARP4过表达效率。克隆形成实验检测克隆形成率,肿瘤细胞成球实验检测成球数和成球直径,流式细胞术检测线粒体膜电位变化,试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量,Western blot检测Ki67、P21、八聚体结合转录因子4(octamer-binding transcription factor 4,OCT4)、Nanog、ABCG2、Bcl-2、Bcl-2相关X蛋白(Bcl-2-associated X,Bax)的蛋白表达水平。结果与Control组比较,circ_LARP4组CNE2和5-8F细胞中circ_LARP4表达水平显著上调(均P<0.05),克隆形成率显著降低(均P<0.05),成球率和成球直径显著减少(均P<0.05),线粒体JC-1红/绿荧光比显著降低(均P<0.05),Ki67蛋白水平显著下调,p21蛋白水平显著上调(均P<0.05),OCT4、Nanog和ABCG2蛋白水平显著降低(均P<0.05),Bax/Bcl-2显著增高(均P<0.05),SOD含量显著降低,MDA含量显著升高(均P<0.05)。通过Starbase 2.0和dbDEMC2初步筛选出circ_LARP4在鼻咽癌中的可能靶分子miR-135b-5p和miR-195-5p,并初步发现二者可以逆转circ_LARP4对细胞增殖干性和凋亡的影响(P<0.05)。结论过表达circRNA_LARP4能够降低鼻咽癌CNE2和5-8F细胞的肿瘤干细胞样特性及线粒体膜电位。

Objective To investigate the effect of overexpression of circRNA_LARP4 on the tumor stem cell-like characteristics and mitochondrial membrane potential of nasopharyngeal carcinoma CNE2 and 5-8F cells.Methods Nasopharyngeal carcinoma CNE2 and 5-8F cells were transfected with circ_LARP4 overexpression vector,and qRT-PCR was used to detect the overexpression efficiency of circ_LARP4.Colony formation assay was used to detect the rate of colony formation.Tumor cell spheroidization test was adapted to detect the number and diameter of spheres.Flow cytometry was used to detect the change of mitochondrial membrane potential.The kits were used to detect superoxide dismutase(SOD)and malondialdehyde(malondialdehyde,MDA)content.The protein levels of Ki67,P21,octamer-binding transcription factor 4(octamer-binding transcription factor 4,OCT4),Nanog,ABCG2 Bcl-2 and Bcl-2 related X protein(Bcl-2-associated X,Bax)were tested by Western blotting.Results Compared with the control group,the expression levels of circ_LARP4 in CNE2 and 5-8F cells in the circ_LARP4 group were significantly upregulated(P<0.05).The colony formation rate was significantly reduced(P<0.05),and the number and diameter of spheres were significantly reduced(P<0.05).Mitochondrial JC-1 red/green fluorescence ratio was significantly reduced(P<0.05),Ki67 protein level was significantly downregulated,p21 protein level was significantly upregulated(P<0.05).OCT4,Nanog and ABCG2 protein levels were significantly reduced(P<0.05),Bax/Bcl-2 protein level was significantly increased(P<0.05).SOD content was significantly reduced,and MDA content was significantly increased(P<0.05).Starbase 2.0 and dbDEMC2 were investigated,miR-135b-5p and miR-195-5p were preliminarily characterized as the possible targets of circ_LARP4 in nasopharyngeal carcinoma,and it was preliminarily found that miR-135b-5p and miR-195-5p could reverse the effects of circ_LARP4 on cell proliferation stemness and apoptosis(P<0.05).Conclusion Overexpression of circRNA_LARP4 can reduce the tumor stem cell-like characteristics and mitochondrial membrane potential of nasopharyngeal carcinoma CNE2 and 5-8F cells.