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

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,q RT-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 factor1 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。

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-Sp 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 （MTRFIL） 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 thnt of GES-1 （1.017±0.226） （P=-0.003）. After dnwn-regulatinn of miR-629-5n exnression. the nrolifernfion nf MKN--4.5 slonwed 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.