Hu Zhaoyang, Wu Hongjin, Li Ying, Hou Qiang, Wang Yan, Li Shuang, Xia Bing, Wu Shixiu
Hangzhou Cancer Institute, Hangzhou Cancer Hospital, Hangzhou City, China.
Anticancer Drugs. 2015 Jun;26(5):531-9. doi: 10.1097/CAD.0000000000000216.
The study aimed to clarify the relationship between β-elemene, a long noncoding RNA (lncRNA), and human telomerase reverse transcriptase (hTERT) in esophageal carcinoma ECA-109 cells. The proliferation of ECA-109 cells was measured using a CCK-8 kit and flow cytometry. PCR microarray and real-time RT-PCR were designed to determine lncRNA expression in ECA-109 cells before and after treatment with β-elemene. Western blot was used to detect the hTERT level after the differentially expressed lncRNAs in ECA-109 cells were interfered with small interfering RNA (siRNA). On treatment with β-elemene, the proliferation of ECA-109 cells was notably inhibited, and about 85% of the lncRNAs showed higher expression levels in ECA-109 cells than in those untreated cells, from which, CDKN2B-AS1 was screened out. A specific siRNA (si-CDKN2B-AS1) that targets the β-elemene-mediated lncRNA CDKN2B-AS1 was designed, synthesized, and applied to treat ECA-109 cells. Its interference efficiency reached as high as 89.6%. When ECA-109 cells were transfected with the siRNA, the hTERT level was increased by 84.7%. The CCK-8 assay showed that the proliferation of ECA-109 cells treated with β-elemene was significantly promoted after siRNA transfection (P<0.01). It was also shown by flow cytometry that, compared with the scramble-treated group (negative control), the proliferation index value of ECA-109 cells in the si-CDKN2B-AS1 treatment group was notably increased (25.7 vs. 51.7%) and the TERT protein level was increased by 67.25% after the cells were treated with si-CDKN2B-AS1. The chemotherapeutic drug β-elemene suppressed the proliferation of esophageal carcinoma ECA-109 cells by regulating the inhibition of hTERT expression by lncRNA CDKN2B-AS1.
该研究旨在阐明长链非编码RNA(lncRNA)β-榄香烯与食管癌ECA-109细胞中人端粒酶逆转录酶(hTERT)之间的关系。使用CCK-8试剂盒和流式细胞术检测ECA-109细胞的增殖情况。设计PCR微阵列和实时逆转录PCR以确定β-榄香烯处理前后ECA-109细胞中lncRNA的表达。在ECA-109细胞中差异表达的lncRNAs被小干扰RNA(siRNA)干扰后,使用蛋白质免疫印迹法检测hTERT水平。用β-榄香烯处理后,ECA-109细胞的增殖受到显著抑制,约85%的lncRNAs在ECA-109细胞中的表达水平高于未处理细胞,从中筛选出CDKN2B-AS1。设计、合成了一种靶向β-榄香烯介导的lncRNA CDKN2B-AS1的特异性siRNA(si-CDKN2B-AS1),并将其应用于处理ECA-109细胞。其干扰效率高达89.6%。当用siRNA转染ECA-109细胞时,hTERT水平提高了84.7%。CCK-8检测表明,siRNA转染后,用β-榄香烯处理的ECA-109细胞的增殖得到显著促进(P<0.01)。流式细胞术也显示,与乱序处理组(阴性对照)相比,si-CDKN2B-AS1处理组中ECA-109细胞的增殖指数值显著增加(25.7%对51.7%),并且在用si-CDKN2B-AS1处理细胞后,TERT蛋白水平提高了67.25%。化疗药物β-榄香烯通过调节lncRNA CDKN2B-AS1对hTERT表达的抑制作用来抑制食管癌ECA-109细胞的增殖。