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

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 BLA-CAT1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.

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

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 wasused to detect cell proliferation, flow cytometry was used to measure apoptosis, and Western blot was used to determine expression ofnuclear-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 CyclinD1 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, CyclinD1 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.