长链非编码RNA膀胱癌相关转录物1对微小RNA-503-5p的靶向关系及对结直肠癌细胞增殖和凋亡的影响

Relationship of long-chain non-coding RNA BLACAT1 to microRNA-503-5p and the effect of colorectal cancer cell proliferation and apoptosis

目的研究长链非编码RNA(lncRNA)膀胱癌相关转录物1(BLACAT1)对微小RNA(miR)-503-5p的靶向关系及结直肠癌细胞增殖和凋亡的影响。方法实时荧光定量PCR(qRT-PCR)检测结肠癌细胞株SW620,LOVO,HT29和人正常结肠黏膜上皮细胞株NCM460中BLACAT1和miR-503-5p的表达。在HT29细胞中转染si-BLACAT1、pcDNA-BLACAT1或miR-503-5p,噻唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞凋亡,蛋白质印迹法(Western blotting)检测细胞核相关抗原Ki-67(Ki-67)、细胞周期蛋白D1(Cyclin D1)、活化的多聚ADP-核糖聚合酶(Cleaved PARP)和活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(Cleaved caspase-3)的表达,starbase预测和双荧光素酶报告分析BLACAT1与miR-503-5p之间的靶向关系。si-BLACAT1和anti-miR-503-5p共转染,观察干扰miR-503-5p对沉默BLACAT1诱导的结直肠癌细胞增殖和凋亡的影响。结果与NCM460细胞比较,SW620、LOVO和HT29中BLACAT1表达量明显增加[(4.93±0.58)、(5.66±0.53)、(6.17±0.66)比(1.03±0.22)],miR-503-5p表达量减少[(0.72±0.11)、(0.67±0.09)、(0.51±0.08)比(1.04±0.14)](P<0.05)。沉默BLACAT1或转染miR-503-5p明显减少HT29细胞的细胞存活率、Ki-67、CyclinD1蛋白表达量,提高细胞凋亡率、Cleaved PARP和Cleaved caspase-3蛋白水平(P<0.05),过表达BLACAT1则反之。BLACAT1靶向miR-503-5p调控其表达。干扰miR-503-5p部分逆转沉默BLACAT1抑制结直肠癌细胞增殖、Ki-67、CyclinD1蛋白表达和诱导结直肠癌细胞凋亡、Cleaved PARP、Cleaved caspase-3蛋白表达的作用。结论lncRNA BLACAT1在表达上调,沉默BLACAT1可通过靶向调控miR-503-5p的表达,来抑制结直肠癌细胞增殖,并诱导细胞凋亡。

Objective To study the targeting relationship of long-chain non-coding RNA(lncRNA)BLACAT1 to microRNA(miR)-503-5p and its effect on colorectal cancer cell proliferation and apoptosis.Methods The expressions of BLACAT1 and miR-503-5p in colon cancer cell lines SW620,LOVO,HT29 and human normal colon mucosal epithelial cell line NCM460 were detected by real-time fluorescent quantitative PCR(qRT-PCR).HT29 cells were transfected with si-BLACAT1,pcDNA-BLACAT1 or miR-503-5p.MTT was used to detect cell proliferation,flow cytometry was used to measure apoptosis,and Western blot was used to determine expression of nuclear-related antigen Ki-67(Ki-67),cyclin D1(Cyclin D1),cleaved Poly ADP-ribose polymerase(Cleaved PARP),and cleaved cysteinyl aspartate specific proteinase-3(Cleaved caspase-3).The starbase Prediction and dual luciferase reports were used to analyze the targeting relationship between BLACAT1 and miR-503-5p.si-BLACAT1 and anti-miR-503-5p were co-transfected to observe the effect of anti-miR-503-5p interfering on the proliferation and apoptosis of colorectal cancer cells induced by silenced BLACAT1.Results Compared with NCM460 cells,the expression of BLACAT1 in SW620,LOVO and HT29 increased[(4.93±0.58),(5.66±0.53),(6.17±0.66)vs.(1.03±0.22)],and the expression of miR-503-5p evidently decreased[(0.72±0.11),(0.67±0.09),(0.51±0.08)vs.(1.04±0.14)](P<0.05).Silencing BLACAT1 or transfecting miR-503-5p greatly reduced the cell survival rate,Ki-67,and CyclinDl protein expression in HT29 cells,and obviously increased the apoptosis rate,Cleaved PARP and Cleaved caspase-3 protein levels(P<0.05),which were the opposite of overexpression of BLACAT1.BLACAT1 targeted miR-503-5p to regulate its expression.Interfering with miR-503-5p partially reversed the effects of silencing BLACAT1 on inhibiting the colorectal cancer cell proliferation,Ki-67,CyclinDl protein expression and inducing colorectal cancer cell apoptosis,Cleaved PARP,and Cleaved caspase-3 protein expression.Conclusion The expression of lncRNA BLACAT1 is up-regulated.Silencing BLACAT1 can inhibit the proliferation of colorectal cancer cells and induce apoptosis by targeting the regulation of miR-503-5p expression.