摘要
目的观察miR-185-5p对肺癌细胞迁移、侵袭的影响,分析酮还原酶1家族成员C1(AKR1C1)在miR-185-5p调控肺癌细胞迁移和侵袭中的作用。方法RT-qPCR检测miR-185-5p在组织样本(人肺癌及对应癌旁组织)、细胞系(人肺上皮细胞系BEAS-2B和肺癌细胞系A549、H460)中的表达。按照处理方式不同将A549细胞分为miR-NC组(转染miR-NC)、miR-185-5p组(转染miR-185-5p)、miR-185-5p+pcDNA组(共转染miR-185-5p和pcDNA)、miR-185-5p+pcDNA-AKR1C1组(共转染miR-185-5p和pcDNA-AKR1C1);Transwell和划痕实验检测各组A549细胞迁移和侵袭;在线预测网站Starbase预测miR-185-5p的下游靶基因;双荧光素酶报告基因实验检测A549细胞荧光素酶活性;蛋白免疫印迹实验检测各组A549细胞中AKR1C1、基质金属蛋白酶(MMP)-2、MMP-9的蛋白表达。结果与癌旁组织比较,肺癌组织中miR-185-5p的表达显著降低(P<0.001);与BEAS-2B细胞比较,A549和H460细胞中miR-185-5p的表达亦显著降低(均P<0.001);与miR-NC组比较,miR-185-5p组A549细胞的miR-185-5p表达显著升高,迁移和侵袭细胞数及划痕愈合率均显著降低(均P<0.001),MMP-2、MMP-9蛋白的表达均显著降低(均P<0.001)。Starbase预测显示miR-185-5p与AKR1C1之间存在连续的互补结合序列;与miR-NC组比较,miR-185-5p组AKR1C1-WT细胞的荧光素活性显著降低(P<0.05)。与miR-NC组比较,miR-185-5p组细胞中AKR1C1蛋白表达显著降低(P<0.05);与anti-miR-NC组比较,anti-miR-185-5p组细胞中AKR1C1蛋白表达显著升高(P<0.05)。与癌旁组织比较,肺癌组织中AKR1C1 mRNA和蛋白表达均显著升高(均P<0.001);与BEAS-2B细胞比较,A549细胞中AKR1C1 mRNA和蛋白表达均显著升高(均P<0.001)。与miR-185-5p+pcDNA组比较,miR-185-5p+pcDNA-AKR1C1组miR-185-5p表达显著降低(P<0.001)、迁移和侵袭细胞数及划痕愈合率均显著升高(均P<0.001)、MMP-2和MMP-9的蛋白表达均显著升高(均P<0.001)。结论miR-185-5p可抑制肺癌细胞的迁移、侵袭,该作用与miR-185-5p靶向负性调控AKR1C1有关。
Objective To observe the effect of miR-185-5p on migration and invasion of lung cancer cells,and to analyze the role of aldo-keto reductase 1 family member C1(AKR1C1)in the regulation of miR-185-5p on migration and invasion of lung cancer cells.Methods The expression of miR-185-5p in human lung cancer tissues,paracancerous tissues,lung epithelial BEAS-2B cells and lung cancer A549 and H460 cells was detected by RT-qPCR.Furthermore,the A549 cells were transfected with miR-NC,miR-185-5p,miR-185-5p+pcDNA,or miR-185-5p+pcDNA-AKR1C1.Cell invasion and migration were determined by Transwell and scratch test,respectively.The downstream target genes of miR-185-5p were predicted by Starbase.Double luciferase reporter assay was used to detect the activity of luciferase in A549 cells.The protein expression of AKR1C1,matrix metalloproteinase(MMP)-2 and MMP-9 was detected by Western blotting.Results The expression of miR-185-5p in lung cancer tissues was lower than that in paracancerous tissues,and its expression in A549 and H460 cells was lower than that in BEAS-2B cells(P<0.001).Compared with the miR-NC group,transfection with miR-185-5p up-regulated the expression of miR-185-5p,inhibited the migration,invasion and scratch healing rate of A549 cells,and decreased the expression of MMP-2 and MMP-9(P<0.001).Starbase predicted that there was a continuous complementary binding sequence between miR-185-5p and AKR1C1.The fluorescence activity of wild-type AKR1C1 cells and the expression of AKR1C1 protein in the miR-185-5p group were lower than those in the miR-NC group(P<0.05).The expression of AKR1C1 protein in the anti-miR-185-5p group was higher than that in the anti-miR-NC group(P<0.05).The levels of AKR1C1 mRNA and protein expression in lung cancer tissues were higher than those in paracancerous tissues,and those in A549 cells were higher than those in BEAS-2B cells(P<0.001).Compared with the miR-185-5p+pcDNA group,transfection with miR-185-5p+pcDNA-AKR1C decreased the expression of miR-185-5p,increased the migration,invasion and scratch healing rate,and enhanced the expression of MMP-2 and MMP-9(P<0.001).Conclusion miR-185-5p can inhibit the migration and invasion of lung cancer cells by targeted inhibition of AKR1C1.
作者
贠宇辉
杨三虎
杨锋
YUN Yu-hui;YANG San-hu;YANG Feng(Department of Thoracic Surgery,Tangdu Hospital of Air Force Military Medical University,Xi’an 710000,China)
出处
《南昌大学学报（医学版）》
2022年第6期11-16,共6页
Journal of Nanchang University：Medical Sciences
基金
陕西省医学科学研究重点课题计划(2018JM3272)。
