摘要
目的探讨胃癌细胞中微RNA(miR)-103a-3p与Kazal基序富含半胱氨酸的逆转诱导蛋白(RECK)表达的关系及其对胃癌细胞迁移、侵袭能力的影响。方法将对数生长期人胃癌细胞系MKN-45细胞随机分为阴性对照(NC)组、miR-103a-3p组、RECK组、miR-103a-3p+RECK组。NC组细胞共转染miR-103a-3p-NC(miR-NC)和pCDH空表达载体,miR-103a-3p组细胞共转染miR-103a-3p模拟物(miR-103a-3p mimics)和pCDH空表达载体,RECK组细胞共转染miR-NC和含RECK的pCDH表达载体(pCDH-RECK),miR-103a-3p+RECK组细胞共转染miR-103a-3p mimic和pCDH-RECK。采用划痕试验检测各组细胞的迁移能力,采用Transwell实验检测各组细胞的侵袭能力,采用Western blot法检测各组细胞中RECK蛋白相对表达量。应用Starbase数据库通过生物信息分析预测miR-103a-3p与RECK的3′非翻译区(3′-UTR)是否存在结合位点,构建含有预测结合位点的野生型(Wt)、突变型(Mt)RECK 3′-UTR片段的荧光素酶报告基因载体Wt-RECK和Mt-RECK,使用脂质体2000将Wt-RECK或Mt-RECK载体与miR-NC或miR-103a-3p mimics共转染入MKN-45细胞,将转染后的细胞设为miR-NC+Wt-RECK组、miR-103a-3p+Wt-RECK组、miR-NC+Mt-RECK组、miR-103a-3p+Mt-RECK组,使用双荧光素酶检测试剂盒检测4组细胞的相对荧光素酶活性。结果4组细胞的划痕愈合率、侵袭细胞数比较差异有统计学意义(F=16.044、60.975,P<0.05);RECK组细胞划痕愈合率显著低于NC组,miR-103a-3p组细胞划痕愈合率显著高于RECK组,miR-103a-3p+RECK组细胞划痕愈合率显著低于miR-103a-3p组(P<0.05);miR-103a-3p组、miR-103a-3p+RECK组与NC组细胞划痕愈合率比较差异无统计学意义(P>0.05);RECK组与miR-103a-3p+RECK组细胞划痕愈合率比较差异无统计学意义(P>0.05)。RECK组侵袭细胞数显著少于NC组,miR-103a-3p组侵袭细胞数显著多于NC组(P<0.05);miR-103a-3p组、miR-103a-3p+RECK组侵袭细胞数显著多于RECK组(P<0.05);miR-103a-3p+RECK组侵袭细胞数显著少于miR-103a-3p组(P<0.05);NC组与miR-103a-3p+RECK组侵袭细胞数比较差异无统计学意义(P>0.05)。4组细胞中RECK蛋白相对表达量比较差异有统计学意义(F=33.473,P<0.05);RECK组细胞中RECK蛋白相对表达量显著高于NC组,miR-103a-3p组、miR-103a-3p+RECK组细胞中RECK蛋白相对表达量显著低于RECK组(P<0.05);NC组与miR-103a-3p组、miR-103a-3p+RECK组细胞中RECK蛋白相对表达量比较差异无统计学意义(P>0.05);miR-103a-3p组与miR-103a-3p+RECK组细胞中RECK蛋白相对表达量比较差异无统计学意义(P>0.05)。Starbase数据库预测显示,miR-103a-3p与RECK的3′UTR存在结合位点。miR-NC+Wt-RECK组、miR-NC+Mt-RECK组、miR-103a-3p+Mt-RECK组细胞的相对荧光素酶活性显著高于miR-103a-3p+Wt-RECK组(P<0.05);miR-NC+Wt-RECK组、miR-NC+Mt-RECK组、miR-103a-3p+Mt-RECK组细胞的相对荧光素酶活性比较差异无统计学意义(P>0.05)。结论miR-103a-3p可能通过靶向抑制RECK的表达而促进胃癌细胞的侵袭和迁移。
Objective To investigate the relationship between the expression of microRNA(miR)-103a-3p and the reversion inducing cysteine rich protein with Kazal motifs(RECK)in gastric cancer cells and their effects on the migration and invasion of gastric cancer cells.Methods MKN-45 cells of human gastric cancer cell line at logarithmic growth stage were randomly divided into negative control(NC)group,miR-103a-3p group,RECK group,miR-103a-3p+RECK group.The cells in the NC group were co-transfected with miR-103a-3p negative control(miR-NC)and pCDH empty vector;the cells in the miR-103a-3p group were co-transfected with miR-103a-3p mimics and pCDH empty vector;the cells in the RECK group were co-transfected with miR-NC and pCDH vector containing RECK(pCDH-RECK);the cells in the miR-103a-3p+RECK group were co-transfected with miR-103a-3p mimic and pCDH-RECK.The migration ability of cells in each group was detected by scratch test,the invasion ability of cells in each group was detected by Transwell test,and the relative expression of RECK protein of cells in each group was detected by Western blot.The Starbase database was used to predict whether there was a binding site between miR-103a-3p and the 3′untranslated region(3′-UTR)of RECK through bioinformatics analysis,and the luciferase reporter gene vectors Wt-RECK and Mt-RECK containing wild-type(Wt)and mutant-type(Mt)RECK 3′-UTR fragments with predicted binding sites were constructed.Wt-RECK or Mt-RECK vectors and miR-NC or miR-103a-3p mimics were co-transfected into MKN-45 cells by using liposome 2000;the transfected cells were set as miR-NC+Wt-RECK group,miR-103a-3p+Wt-RECK group,miR-NC+Mt-RECK group and miR-103a-3p+Mt-RECK group;the relative luciferase activity of cells in the four groups was detected by double luciferase detection kit.Results There were significant differences in the scratch healing rate and the number of invasive cells among the four groups(F=16.044,60.975;P<0.05);the scratch healing rate of cells in the RECK group was significantly lower than that in the NC group,the scratch healing rate of cells in the miR-103a-3p group was significantly higher than that in the RECK group,and the scratch healing rate of cells in the miR-103a-3p+RECK group was significantly lower than that in the miR-103a-3p group(P<0.05);there was no significant difference in the scratch healing rate of cells between the miR-103a-3p group,miR-103a-3p+RECK group and NC group(P>0.05);there was no significant difference in the scratch healing rate of cells between the RECK group and the miR-103a-3p+RECK group(P>0.05).The number of invasive cells in the RECK group was significantly less than that in the NC group,and the number of invasive cells in the miR-103a-3p group was significantly more than that in the NC group(P<0.05);the number of invasive cells in the miR-103a-3p group and miR-103a-3p+RECK group was significantly more than that in the RECK group(P<0.05);the number of invasive cells in the miR-103a-3p+RECK group was significantly less than that in the miR-103a-3p group(P<0.05);there was no significant differences in the number of invasive cells between the NC group and the miR-103a-3p+RECK group(P>0.05).There were significant difference in the relative expression levels in RECK protein in cells among the four groups(F=33.473,P<0.05);the relative expression level of RECK protein of cells in the RECK group was significantly higher than that in the NC group,and the relative expression level of RECK protein of cells in the miR-103a-3p group and miR-103a-3p+RECK group was significantly lower than that in the RECK group(P<0.05);there was no significant difference in the relative expression level of RECK protein in cells between the NC group and the miR-103a-3p group,miR-103a-3p+RECK group(P>0.05);there was no significant difference in the relative expression level of RECK protein in cells between the miR-103a-3p group and the miR-103a-3p+RECK group(P>0.05).The prediction of the Starbase database showed that there was a binding site between miR-103a-3p and 3′-UTR of RECK.The relative luciferase activity of cells in the miR-NC+Wt-RECK group,miR-NC+Mt-RECK group and miR-103a-3p+Mt-RECK group was significantly higher than that in the miR-103a-3p+Wt-RECK group(P<0.05);there was no significant difference in the relative luciferase activity of cells among the miR-NC+Wt-RECK group,miR-NC+Mt-RECK group and miR-103a-3p+Mt-RECK group(P>0.05).Conclusion miR-103a-3p may promote the invasion and migration of gastric cancer cells by inhibiting the expression of RECK.
作者
杨静
陈雨娜
吕长坤
万雪莲
YANG Jing;CHEN Yuna;LYU Changkun;WAN Xuelian(College of Medical Technology,Shangqiu Medical College,Shangqiu 476005,Henan Province,China)
出处
《新乡医学院学报》
CAS
2023年第1期18-23,共6页
Journal of Xinxiang Medical University
基金
2019年河南省高等职业学校青年骨干教师培养计划项目(编号:2019GZGG093)。
