Department of Gastroenterology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China.
World J Gastroenterol. 2012 Jun 7;18(21):2630-9. doi: 10.3748/wjg.v18.i21.2630.
To investigate the effect of high dose glargine on the expression profiles of microRNAs in human pancreatic cancer cells.
Real-time polymerase chain reaction array (RT-PCR) was applied to investigate miRNAs differentially expressed in Sw1990 cells treated with or without 100 IU/L glargine. Stem-loop RT-PCR was used to confirm the results of the array assay in Sw1990 and Panc-1 cells. The effects of miR-95 on cell growth, apoptosis, invasion and migration abilities were respectively examined by CCK8 assay, apoptosis assay, Matrigel invasion and migration assay in Sw1990 and Panc-1 cells. Nude mice xenograft models with Sw1990 cells were built to investigate pancreatic cancer growth in vivo after transfection by the lentivirus pGLV3-GFP- miR-95.
Ten miRNAs were significantly up-regulated and 2 miRNAs down-regulated in glargine treated Sw1990 cells when compared with non-treated cells (2.48-fold changes on average, P < 0.01). miR-95, miR-134 and miR-34c-3p are the top three miRNAs regulated by glargine (3.65-fold, 2.67-fold and 2.60-fold changes respectively, P < 0.01) in Sw1990 cells. Stem-loop RT-PCR confirmed that high dose glargine up-regulated the expression of miR-95 and miR-134 in both Sw1990 and Panc-1 cells. The most obvious change is the apparent increase of miR-95. Forced expression of miR-95 significantly increased cell proliferation (Sw1990: 2.510 ± 0.129 vs 2.305 ± 0.187, P < 0.05; Panc-1: 2.439 ± 0.211 vs 2.264 ± 0.117, P < 0.05), invasion (Sw1990: 67.90 ± 12.33 vs 47.30 ± 5.89, P < 0.01; Panc-1: 37.80 ± 8.93 vs 30.20 ± 5.14, P < 0.01), migration (Sw1990: 101 ± 6.00 vs 51.20 ± 8.34, P < 0.01; Panc-1: 91.80 ± 9.22 vs 81.50 ± 7.47, P < 0.01) and inhibited cell apoptosis (Sw1990: 22.05% ± 1.92% vs 40.32% ± 1.93%, P < 0.05; Panc-1: 20.17% ± 0.85% vs 45.60% ± 1.43%, P < 0.05) when compared with paired negative controls, whereas knockdown of miR-95 obtained the opposite effect. Nude mice xenograft models confirmed that miR-95 promoted the growth of pancreatic cancer in vivo when compared with negative control (tumor volume: 373.82 ± 23.67 mL vs 219.69 ± 17.82 mL, P < 0.05).
These observations suggested that modulation of miRNA expression may be an important mechanism underlying the biological effects of glargine.
研究高剂量甘精胰岛素对人胰腺癌细胞中 microRNA 表达谱的影响。
应用实时聚合酶链反应（PCR）阵列（RT-PCR）技术研究 Sw1990 细胞经或未经 100 IU/L 甘精胰岛素处理后差异表达的 miRNAs。采用茎环 RT-PCR 法在 Sw1990 和 Panc-1 细胞中验证阵列检测结果。通过 CCK8 法、凋亡实验、Matrigel 侵袭和迁移实验分别检测 miR-95 对 Sw1990 和 Panc-1 细胞生长、凋亡、侵袭和迁移能力的影响。构建 Sw1990 细胞裸鼠异种移植模型，研究转染慢病毒 pGLV3-GFP-miR-95 后体内胰腺癌细胞的生长情况。
与未处理细胞相比，甘精胰岛素处理的 Sw1990 细胞中有 10 个 miRNAs 显著上调，2 个 miRNAs 下调（平均 2.48 倍变化，P < 0.01）。miR-95、miR-134 和 miR-34c-3p 是甘精胰岛素（3.65 倍、2.67 倍和 2.60 倍变化，P < 0.01）在 Sw1990 细胞中调节作用最强的前 3 个 miRNAs。茎环 RT-PCR 证实高剂量甘精胰岛素上调了 Sw1990 和 Panc-1 细胞中 miR-95 和 miR-134 的表达。最明显的变化是 miR-95 的明显增加。强制表达 miR-95 显著增加细胞增殖（Sw1990：2.510 ± 0.129 比 2.305 ± 0.187，P < 0.05；Panc-1：2.439 ± 0.211 比 2.264 ± 0.117，P < 0.05）、侵袭（Sw1990：67.90 ± 12.33 比 47.30 ± 5.89，P < 0.01；Panc-1：37.80 ± 8.93 比 30.20 ± 5.14，P < 0.01）、迁移（Sw1990：101 ± 6.00 比 51.20 ± 8.34，P < 0.01；Panc-1：91.80 ± 9.22 比 81.50 ± 7.47，P < 0.01），并抑制细胞凋亡（Sw1990：22.05% ± 1.92% 比 40.32% ± 1.93%，P < 0.05；Panc-1：20.17% ± 0.85% 比 45.60% ± 1.43%，P < 0.05），与配对阴性对照相比，而 miR-95 的敲低则获得了相反的效果。裸鼠异种移植模型证实 miR-95 促进了体内胰腺癌的生长（肿瘤体积：373.82 ± 23.67 mL 比 219.69 ± 17.82 mL，P < 0.05）。
这些观察结果表明，miRNA 表达的调节可能是甘精胰岛素生物学效应的重要机制。
