miR-629-5p对胃癌细胞生物学行为的影响及其靶基因MTRF1L的鉴定

Effects of miR-629-5p on biological behavior of gastric cancer cells and identification of its target gene MTRF1L

摘要:

目的：研究miR-629-5p对胃癌细胞生物学行为的影响并鉴定其靶基因。方法：通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测miR-629-5p在16例胃癌组织和3种胃癌细胞中的表达水平；将细胞实验分为2个组：实验组（IN）和对照组（NC）；转染miR-629-5p抑制剂下调胃癌细胞MKN-45中miR-629-5p的表达；CCK-8实验和平板克隆实验检测MKN-45增殖；Transwell小室实验和划痕实验检测MKN-45迁移和侵袭；细胞流式技术检测MKN-45凋亡；通过miRWalk2.0工具预测miR-629-5p的靶基因，Western blot检测MKN-45中线粒体样转录释放因子1（mitochondrial translational release factor 1 like，MTRF1L）蛋白表达。结果：与癌旁组织[1.574（0.197，4.503）]相比，胃癌组织中miR-629-5p相对表达量[2.081（0.231，7.216）]明显增加（P=0.003）；与正常胃黏膜细胞（1.017±0.226）相比，胃癌细胞中miR-629-5p相对表达量（MKN-28：3.040±0.506；MKN-45：5.924±0.403；SCG-7901：3.377±0.372）均明显增加（P=0.000）。下调miR-629-5p表达后，胃癌细胞MKN-45的增殖减慢（IN：64±12；NC：102±12；P=0.018），迁移（IN：106±9；NC：194±29；P=0.008）和侵袭（IN：25±7；NC：60±9；P=0.007）能力减弱，凋亡细胞数增加[IN：（19.257±2.440）%；NC：11.687±2.237；P=0.017]。miR-629-5p预测的靶基因共有46个，下调miR-629-5p的表达后，MTRF1L表达量明显增加（IN：0.923±0.101；NC：0.556±0.026；P=0.004）。结论：miR-629-5p在胃癌中表达上调，可促进MKN-45的增殖，增强MKN-45迁移和侵袭能力，抑制MKN-45凋亡；MTRF1L可能是miR-629-5p的靶基因之一。

Abstract:

Objective：To study the effects of miR-629-5p on the biological behavior of gastric cancer cells and to identify its target gene. Methods：Quantitative real-time PCR（qRT-PCR） was used to detect the expression of miR-629-5p in 16 gastric cancer tis－sues and 3 gastric cancer cell lines. The cell experiment was divided into two groups：the experimental group（IN） and control group （NC）. Transfecting miR-629-5p inhibitor was used to down-regulate the miR-629-5p expression of MKN-45.CCK-8 assay and plate cloning assay was used to detect cell proliferation. Transwell chamber assay and wound healing assay was used to detect invasion and migration of MKN-45，and flow cytometry assay was used to detect apoptosis of MKN-45. MiRWalk2.0 was used to predict target genes of miR-629-5p，and Western blot was used to detect the expression of mitochondrial translational release factor 1 like（MTRF1L） in MKN-45. Results：The expression of miR-629-5p was significantly increased in gastric cancer tissues[2.081（0.231，7.216）] compared with that of paired normal gastric tissue[1.574（0.197，4.503）]（P=0.003）. The expression of miR-629-5p was sig－nificantly increased in gastric cancer cell lines（MKN-28：3.040±0.506；MKN-45：5.924±0.403；SCG-7901：3.377±0.372） compared with that of GES-1（1.017±0.226）（P=0.003）. After down-regulation of miR-629-5p expression，the proliferation of MKN-45 slowed down（IN：64±12；NC：102±12；P=0.018），the abilities of mi－gration（IN：106±9；NC：194±29；P=0.008） and invasion（IN：25±7；NC：60±9；P=0.007） decreased，and the number of apoptotic cells increased[IN：（19.257±2.440）%；NC：11.687±2.237；P=0.017]. There were 46 target genes predicted by miR-629-5p，one of which is MTRF1L. The down-regulation of miR-629-5p increased the expression of MTRF1L（IN：0.923±0.101；NC：0.556±0.026；P=0.004）. Conclusion：The expression of miR-629-5p is up-regulated in gastric cancer tissues and cell lines. miR-629-5p can promote the proliferation，enhance the migration and invasion abilities，and inhibit the apoptosis of MKN-45. MTRF1L may be one of miR-629-5 target genes.