长链非编码RNA FBXL19-AS1靶向miR-339-3p对胰腺癌细胞增殖、迁移和侵袭的影响

Effects of long non-coding RNA FBXL19-AS1 targeting miR-339-3p on the proliferation, migration and invasion of pancreatic cancer cells

目的探讨长链非编码RNA(lncRNA)FBXL19-AS1对胰腺癌细胞增殖、迁移和侵袭的影响,明确lncRNA FBXL19-AS1与微小RNA-339-3p(miR-339-3p)的靶向关系。方法收集2017年1月至2019至8月间烟台市烟台山医院73例行手术切除且经病理确诊的胰腺癌患者的癌组织及匹配的癌旁正常胰腺组织,体外培养正常胰腺上皮细胞(hTERT-HPNE)和3株胰腺癌细胞(Capan-1、SW1990、PaTu8988),采用实时荧光定量PCR法检测胰腺癌组织和各株细胞lncRNA FBXL19-AS1和miR-339-3p表达。将胰腺癌Capan-1细胞分为NC组(正常对照组)、si-NC组(转染无意义阴性序列)、si-FBXL19-AS1组(转染FBXL19-AS1小干扰RNA),miR-NC组(转染空质粒对照)、miR-339-3p组(转染miR-339-3p过表达慢病毒载体),si-FBXL19-AS1+anti-miR-339-3p NC组(共转染FBXL19-AS1siRNA和miR-339-3p抑制剂阴性对照序列)、si-FBXL19-AS1+anti-miR-339-3p组(共转染FBXL19-AS1siRNA和miR-339-3p抑制剂)。采用CCK-8法检测各组细胞增殖活性,采用Transwell小室检测细胞迁移和侵袭能力,采用蛋白质免疫印迹法检测细胞cyclinD1、基质金属蛋白酶2(MMP2)、MMP9蛋白表达。采用生物信息学和双荧光素酶报告基因实验分析lncRNA FBXL19-AS1和miR-339-3p的靶向关系。结果胰腺癌组织lncRNA FBXL19-AS1表达量显著高于癌旁正常胰腺组织(2.96±0.21比1.00±0.13,P<0.05),miR-339-3p表达量显著低于癌旁正常胰腺组织(0.37±0.05比1.00±0.11,P<0.05)。Capan-1、SW1990及PaTu8988细胞lncRNA FBXL19-AS1表达量分别为2.43±0.18、1.97±0.13和1.73±0.14,均显著高于hTERT-HPNE细胞的1.00±0.07,miR-339-3p表达量分别为0.42±0.03、0.54±0.03和0.57±0.04,均显著低于hTERT-HPNE细胞的1.00±0.05。其中以Capan-1细胞的lncRNA FBXL 19-AS1表达量最高,miR-339-3p表达量最低。与si-NC组比较,si-FBXL19-AS1组Capan-1细胞450nm处吸光度值(A450值)、迁移细胞数、穿膜细胞数显著下降[0.47±0.03比0.94±0.06、(81.00±7.41)个比(187.00±16.13)个、(67.00±5.41)个比(141.00±9.24)个],cyclinD1、MMP2和MMP9蛋白表达量显著降低(0.44±0.03比0.83±0.05、0.48±0.03比0.92±0.07、0.38±0.02比0.73±0.05)。与miR-NC组比较,miR-339-3p组Capan-1细胞A450值、迁移细胞数、穿膜细胞数显著下降[0.54±0.04比0.94±0.05、(98.00±6.53)个比(193.00±10.02)个、(83.00±6.58)个比(146.00±7.11)个],cyclinD1、MMP2、MMP9蛋白表达量显著降低(0.47±0.03比0.81±0.07、0.43±0.03比0.94±0.06、0.32±0.02比0.71±0.06)。与si-FBXL19-AS1+anti-miR-NC组比较,si-FBXL19-AS1+anti-miR-339-3p组细胞A450值、迁移细胞数、穿膜细胞数显著上升[0.96±0.07比0.48±0.03、(204.00±11.25)个比(83.00±5.11)个、(157.00±8.76)个比(64.00±4.12)个],cyclinD1、MMP2和MMP9蛋白表达量显著升高(0.84±0.06比0.42±0.03、0.96±0.08比0.47±0.08、0.74±0.06比0.37±0.02)。上述差异均有统计学意义(P值均<0.05)。共转染野生型lncRNA FBXL19-AS1和miR-339-3p mimics组Capan-1细胞的荧光素酶活性显著低于共转染野生型lncRNA FBXL19-AS1和miR-NC组(0.47±0.04比1.00±0.10),差异有统计学意义(P<0.05),而共转染突变型lncRNA FBXL19-AS1和miR-339-3p mimics组与共转染突变型lncRNA FBXL19-AS1和miR-NC组Capan-1细胞的荧光素酶活性差异无统计学意义。结论LncRNA FBXL19-AS1在胰腺癌组织及胰腺癌Capan-1细胞株中呈高表达,敲减lncRNA FBXL19-AS1可靶向结合miR-339-3p调节胰腺癌细胞增殖、迁移和侵袭能力,促进胰腺癌的发生和发展。

Objective To investigate the effects of long non-coding RNA(lncRNA)FBXL19-AS1 on the proliferation,migration and invasion of pancreatic cancer cells,and to determine the targeting relationship of lncRNA FBXL19-AS1 and microRNA-339-3p(miR-339-3p).Methods From January 2017 to August 2019,73 cancer tissues and matched normal pancreatic tissues adjacent to cancer from patients pathologically diagnosed as pancreatic cancer who underwent surgical resection in Yantai Hospital of Yantai were collected.Normal pancreatic epithelial cells(hTERT-HPNE)and 3 pancreatic cancer cell lines(Capan-1,SW1990,PaTu8988)were cultured in vitro.The real-time fluorescent quantitative PCR was used to detect the expression of lncRNA FBXL19-AS1 and miR-339-3p in pancreatic cancer tissues and cell lines.The Capan-1 cells were divided into the NC group(normal control group),si-NC group(transfected with meaningless negative sequence),si-FBXL19-AS1 group(transfected with FBXL19-AS1 small interfering RNA),miR-NC group(transfected with empty plasmid control),miR-339-3p group(transfected with miR-339-3p overexpression lentiviral vector),si-FBXL19-AS1+anti-miR-339-3p NC group(cotransfected with FBXL19-AS1siRNA and miR-339-3p inhibitor negative control sequence)and si-FBXL19-AS1+anti-miR-339-3p group(cotransfected with FBXL19-AS1siRNA and miR-339-3p inhibitor).CCK-8 method was used to detect cell proliferation activity.Transwell chamber was used to detect cell migration and invasion ability,and western blotting method was used to detect cell cyclinD1,matrix metalloproteinase 2(MMP2)and MMP9 protein expression.Bioinformatics and dual luciferase reporter gene experiments were used to analyze the targeting relationship between lncRNA FBXL19-AS1 and miR-339-3p.Results The expression of lncRNA FBXL19-AS1 in pancreatic cancer tissue was significantly higher than that in normal pancreatic tissue adjacent to cancer(2.96±0.21 vs 1.00±0.13,P<0.05),and the expression of miR-339-3p was significantly lower than that in normal pancreatic tissue adjacent to cancer(0.37±0.05 vs 1.00±0.11,P<0.05).The expression of lncRNA FBXL19-AS1 in Capan-1,SW1990 and PaTu8988 cells were 2.43±0.18,1.97±0.13 and 1.73±0.14,respectively,which were significantly higher than that of hTERT-HPNE cells 1.00±0.07.The expression of miR-339-3p were 0.42±0.03,0.54±0.03 and 0.57±0.04,respectively,which were all significantly lower than 1.00±0.05 of hTERT-HPNE cells.Among them,the expression of lncRNA FBXL 19-AS1 in Capan-1 cells was the highest,and the miR-339-3p was the lowest.Compared with the si-NC group,the absorbance value(A450)of Capan-1 cells in the si-FBXL19-AS1 group,the number of migrating cells,and the number of transmembrane cells were significantly decreased(0.47±0.03 vs 0.94±0.06,81.00±7.41 vs 187.00±16.13,67.00±5.41 vs 141.00±9.24),the protein expression of cyclinD1,MMP2 and MMP9 was significantly reduced(0.44±0.03 vs 0.83±0.05,0.48±0.03 vs 0.92±0.07,0.38±0.02 vs 0.73±0.05).Compared with the miR-NC group,the A450,the number of migrating cells,and the number of transmembrane cells of Capan-1 cells in the miR-339-3p group were significantly decreased(0.54±0.04 vs 0.94±0.05,98.00±6.53 vs 193.00±10.02,83.00±6.58 vs 146.00±7.11,the protein expression of cyclinD1,MMP2 and MMP9 was significantly reduced(0.47±0.03 vs 0.81±0.07,0.43±0.03 vs 0.94±0.06,0.32±0.02 vs 0.71±0.06).Compared with the si-FBXL19-AS1+anti-miR-NC group,the A450,the number of migrating cells and the number of transmembrane cells in the si-FBXL19-AS1+anti-miR-339-3p group increased significantly(0.96±0.07 vs 0.48±0.03,204.00±11.25 vs 83.00±5.11,157.00±8.76 vs 64.00±4.12,P all<0.05),the protein expression of cyclinD1,MMP2 and MMP9 increased significantly(0.84±0.06 vs 0.42±0.03,0.96±0.08 vs 0.47±0.08,0.74±0.06 vs 0.37±0.02,P all<0.05).The luciferase activity of Capan-1 cells cotransfected with WT-FBXL19-AS1 and miR-339-3p mimics was significantly lower than that of the cotransfected with WT-FBXL19-AS1 and miR-NC(0.47±0.04 vs 1.00±0.10,P all<0.05).The difference of the luciferase of Capan-1 cells in the cotransfected MUT-FBXL19-AS1 and miR-339-3p mimics group and the cotransfected MUT-FBXL19-AS1 and miR-NC group was not statistically significant.Conclusions LncRNA FBXL19-AS1 was highly expressed in pancreatic cancer tissues and Capan-1 pancreatic cancer cell lines.Knockdown of lncRNA FBXL19-AS1 can target miR-339-3p to regulate the proliferation,migration and invasion of pancreatic cancer cells,and promote the occurrence and development of pancreatic cancer.