胰腺癌PANC1细胞microRNA-217靶基因ANLN的鉴定

Identification of microRNA-217 targeted gene ANLN in pancreatic cancer PANC1 cells

目的通过实验验证ANLN为microRNA-217（miR-217）的靶基因。方法使用生物学软件预测miR-217调控的靶基因可能为ANLN。设计并合成mR-217结合的ANLN及突变型ANLN（mutANLN）序列，将其PCR扩增的片段插入表达质粒psiCHECK-2，构建重组质粒psiCHECK-2．ANLN及psiCHECK-2-mutANLN。采用脂质体法将2个重组质粒单独或分别与miR-217、miR．217inhibitor及与人同源性远的miRNA序列（NC）、NCinhibitor共转染胰腺癌PANCl细胞，采用双荧光素酶报告系统检测荧光素酶活性，采用蛋白质印迹法检测ANLN蛋白的表达。结果转染psiCHECK-2-ANLN、psiCHECK-2-ANLN＋miR-217、psiCHECK-2-ANLN＋miR-217inhibitor、psiCHECK-2-ANLN＋NC、psiCHECK-2-ANLN＋NCinhibitor各组问的荧光素酶活性分别为2．221±0．188、0．7694-0．061、3．764±0．371、2．265±0．201、2．242±0．018，差异有统计学意义（F=77．405，P〈0．01），但psiCHECK-2-ANLN组、psiCHECK-2-ANLN＋NC组及psiCHECK-2-ANLN＋NCinhibitor组两两比较差异无统计学意义，而psiCHECK-2-ANLN＋miR-217组的荧光素酶活性较这3组明显下降，psiCHECK-2-ANLN＋miR-217inhibitor组活性较其他4组明显升高（P值均〈0．01）。转染psiCHECK-2-mutANLN各组间荧光素酶活性差异无统计学意义（P=0．053）。psiCHECK-2．ANLN＋miR-217共转染组PANC1细胞的ANLN蛋白表达水平较单纯转染psiCHECK-2-ANLN组细胞的ANLN蛋白表达明显下调。结论在胰腺癌中，ANLN可能是miR-217的直接靶基因。

Objective To identify the miR-217 targeted gene ANLN by experiment. Methods Bioinformatic algorithms were used to predict the potential targets of miR-217. Then, ANLN binding with miR- 217 and mutant ANLN （mutANLN） sequence were designed and synthesized, and their amplified fragments were inserted into plasmid psiCHECK-2, and recombinant plasmid psiCHECK-2-ANLN and psiCHECK-2- mutANLN were reconstructed. The two recombinant plasmids were eo-transfected into pancreatic cancer cell line PANC1 with miR-217, miR-217 inhibitor, NC, NC inhibitor by liposome, respectively. Dual luciferase reporter system was used to determine the luciferase activity, and Western blot was used to measure the expression of ANLN protein. Results The luciferase activities of psiCHECK-2-ANLN, psiCHECK-2-ANLN ＋ miR-217, psiCHECK-2ANLN ＋ miR-217 inhibitor, psiCHECK-2ANLN ＋ NC, psiCHECK-2-ANLN ＋ NC inhibitor were 2. 221±0. 188, 0.769 ± 0. 061, 3. 764± 0. 371, 2. 265±0. 201, 2. 242± 0. 018, and the difference among these groups was statistically significant （F = 77. 405, P 〈 0.001 ） , but the difference amongpsiCHECK-2ANLN group, psiCHECK-2-ANLN ＋ NC group and psiCHECK-2-ANLN ＋ NC inhibitor group was not statistically significant. However, luciferase activities of psiCHECK-2-ANLN ＋ miR-217 group were significantly decreased when compared with other 3 groups, and luciferase activity of psiCHECK-2-ANLN ＋ miR-217 inhibitor group were significantly increased when compared with other 4 groups （all P 〈0. 001 ）. Luciferase activities of groups transfected with psiCHECK-2-mutANLN was not significantly different （P = 0.053）. The expression of ANLN protein in PANC1 with psiCHECK-2-ANLN ＋ miR-217 co-transfection was significantly down-regulated when compared with that with psiCHECK-2-ANLN transfection alone. Conclusions ANLN is one of the direct target genes of miR-217 in PANC1 cells.