高剂量甘精胰岛素改变胰腺癌细胞中 microRNAs 的表达谱。
High dose glargine alters the expression profiles of microRNAs in pancreatic cancer cells.
机构信息
Department of Gastroenterology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China.
出版信息
World J Gastroenterol. 2012 Jun 7;18(21):2630-9. doi: 10.3748/wjg.v18.i21.2630.
AIM
To investigate the effect of high dose glargine on the expression profiles of microRNAs in human pancreatic cancer cells.
METHODS
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.
RESULTS
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).
CONCLUSION
These observations suggested that modulation of miRNA expression may be an important mechanism underlying the biological effects of glargine.
目的
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。
方法
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。
结果
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）。
结论
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。
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