论文部分内容阅读
目的:探讨长链非编码RNA(lncRNA)肿瘤易感性候选基因9(CASC9)对胰腺癌BxPC-3细胞增殖和凋亡的影响,明确微小RNA-195-5p(miR-195-5p)与lncRNA CASC9的靶向关系。方法:收集2017年4月至2018年5月间湖北省襄阳市中西医结合医院40例行手术切除并经组织病理学确诊的胰腺癌组织及相应癌旁正常胰腺组织,选取4株胰腺癌细胞(AsPC-1、HPAC、BxPC-3、PANC1)和正常胰腺导管上皮细胞HPDE6-C7,采用实时定量PCR法检测胰腺癌组织和各株细胞lncRNA CASC9表达。将BxPC-3细胞分为si-CASC9组(转染靶向lncRNA CASC9的siRNA)、si-control组(转染与lncRNA CASC9不匹配的siRNA)和CASC9组(转染lncRNA CASC9过表达质粒)、CASC9/miR-195-5p组(共转染CASC9过表达质粒和miR-195-5p mimics)。采用MTT法检测各组细胞增殖活性,采用蛋白质免疫印迹法检测凋亡相关蛋白Bax、Bcl-2表达,采用生物信息学和荧光素酶实验分析CASC9和miR-195-5p的靶向关系。结果:胰腺癌组织lncRNA CASC9表达量显著高于癌旁正常胰腺组织(4.70±1.25比2.15±0.82,n P=0.04),胰腺癌细胞株AsPC-1、HPAC、BxPC-3、PANC1细胞lncRNA CASC9表达量分别为1.43±0.12、1.86±0.13、2.03±0.14、1.73±0.15,均显著高于HPDE6-C7细胞的1.00±0.10(n P值均<0.001),其中以BxPC-3细胞的表达量最高。si-CASC9组细胞较si-control组细胞的增殖活性显著下降(细胞培养第3天0.57±0.05比0.72±0.04,n P=0.01;第4天0.75±0.07比0.95±0.07,n P=0.02);Bax表达上调(1.39±0.13比1.07±0.11,n P=0.03),Bcl-2表达显著下调(1.44±0.11比1.71±0.12、n P=0.04)。CASC9/miR-195-5p组细胞增殖活性较CASC9组显著下降(n P值<0.005);Bax表达水平显著高于CASC9组(0.68±0.04比0.56±0.03,n P=0.01),Bcl-2表达显著低于CASC9组(1.05±0.03比1.47±0.04,n P<0.001)。n 结论:lncRNA CASC9靶向结合miR-195-5p可逆转CASC9促进胰腺癌细胞增殖和抑制胰腺癌细胞凋亡的作用。“,”Objective:To investigate the effect of long non-coding RNA (lncRNA) tumor susceptibility candidate gene 9 (CASC9) on the proliferation and apoptosis of pancreatic cancer cell BxPC-3, and to identify the targeting relationship between miR-195-5p and CASC9.Methods:40 pairs of pancreatic cancer tissues and adjacent normal pancreas tissues resected by surgery and diagnosed by histopathology in Xiangyang Hospital of Integrated Traditional and Western Medicine from April 2017 to May 2018 were collected. Four pancreatic cancer cells (AsPC-1, HPAC, BxPC-3, PANC1) and normal pancreatic ductal epithelial cells HPDE6-C7 were used in experiments. The expression level of CASC9 in pancreatic cancer tissues and cell lines were detected by real-time quantitative PCR. The BxPC-3 cells were divided into si-CASC9 group (transfected with siRNA against CASC9), si-control group (transfected with siRNA that did not match CASC9), CACS9 group (transfected with CASC9 overexpressed plasmid), and CASC9/miR-195-5p group (co-transfected with CASC9 overexpressed plasmid and miR-195-5p mimics). Cell proliferation activity was detected by MTT assay. Western blot was used to detect the protein expression of Bax and Bcl-2. The targeting relationship between CASC9 and miR-195-5p was identified by bioinformatics analysis and luciferase assay.Results:The expression level of CASC9 in pancreatic cancer tissues was significantly higher than that in adjacent normal tissues (4.7±1.25 n vs 2.15±0.82, n P=0.04), and the expression levels of CASC9 in pancreatic cancer cell lines AsPC-1, HPAC, BxPC-3, and PANC1 cells were 1.43±0.12, 1.86±0.13, 2.03±0.14, and 1.73±0.15, respectively, which were significantly higher than that in HPDE6-C7 cells (1.00±0.10, n P<0.001). The expression in BxPC-3 cells was the highest. The proliferation activity of cells in si-CASC9 group decreased significantly compared with that in si-control group (on day 3 0.57±0.05n vs 0.72±0.04, n P=0.01; and on day 4 0.75±0.07 n vs 0.95±0.07, n P=0.02). Bax expression was up-regulated (1.39±0.13 n vs 1.07±0.11, n P=0.03), while Bcl-2 expression was significantly down-regulated (1.44±0.11 n vs 1.71±0.12, n P=0.04). The cell proliferation activity of CASC9/miR-195-5p group was significantly decreased compared with that of CASC9 group (n P<0.005). The expression level of Bax was significantly higher than that of CASC9 group (0.68±0.04n vs 0.56±0.03, n P=0.01), and the expression level of Bcl-2 was significantly lower than that of CASC9 group (1.05±0.03 n vs 1.47±0.04, n P<0.001).n Conclusions:miR-195-5p can reverse the effect of CASC9 on promoting proliferation and inhibiting apoptosis of pancreatic cancer cells by targeting lncRNA CASC9.