摘要
目的探究miRNA-424-5p(miR-424-5p)调控SIRT4表达影响胃癌细胞迁移和侵袭能力的潜在分子机制。方法利用RT-qPCR检测57例胃癌患者肿瘤组织与癌旁组织中miR-424-5p和SIRT4的表达水平。用脂质体法将miR-424-5p inhibitor和miR-424-5p mimic分别瞬时转染入MNK-28和HGC-27胃癌细胞中,RT-qPCR检测细胞中miR-424-5p和SIRT4 mRNA的表达水平,Western Blot检测细胞中SIRT4蛋白的表达量,划痕愈合实验和Transwell侵袭实验检测各组细胞的迁移和侵袭能力。双荧光素酶基因报告实验检测miR-424-5p对SIRT4的靶向调控机制。共转染miR-424-5p和SIRT4进一步验证miR-424-5p和SIRT4对胃癌细胞迁移和侵袭能力的影响。结果胃癌组织中miR-424-5p的表达明显高于癌旁组织(P<0.001),SIRT4的表达水平明显低于癌旁组织(P<0.001),相关性分析表明胃癌组织中miR-424-5p的表达与SIRT4呈负相关(r=-0.382,P=0.034)。此外,miR-424-5p高表达与肿瘤的浸润深度、TNM分期、脉管侵犯和神经侵犯显著相关(均P<0.05)。过表达miR-424-5p能促进胃癌细胞迁移和侵袭能力,而抑制miR-424-5p表达则呈现出相反的效果。过表达miR-424-5p可降低胃癌细胞中SIRT4 m RNA和蛋白的表达水平,相反,抑制miR-424-5p表达则上调胃癌细胞中SIRT4 m RNA和蛋白的表达水平。双荧光素酶报告基因实验显示miR-424-5p能显著影响野生型SIRT4-3’UTR表达载体的荧光素酶活性。回复实验结果显示miR-424-5p和SIRT4能显著影响胃癌细胞的迁移和侵袭能力。结论miR-424-5p在胃癌中高表达,其通过靶向抑制抑癌基因SIRT4的表达促进肿瘤细胞的迁移和侵袭,从而参与胃癌的的发生、发展。
Objective To explore the potential molecular mechanism of miRNA-424-5p(miR-424-5p)in affecting the migration and invasion of gastric cancer(GC)cells by targeting the expression of SIRT4.Methods The expression levels of miR-424-5p and SIRT4 in tumor tissues and adjacent tissues of 57 patients with GC were detected by RT-qPCR.MiR-424-5p inhibitor and miR-424-5p mimic were transiently transfected into MKN-28 and HGC-27 GC cells by liposome method,and then the expression of miR-424-5p and SIRT4 mRNA in cells were detected by RT-qPCR;the expression level of SIRT4 protein in cells was detected by Western Blot,and the migra-tion and invasion ability of each group were detected by Wound-healing assay and Transwell invasion assay.The dual luciferase gene reporter assay was performed to detect the targeted reguLation of miR-424-5p on SIRT4.Co-transfection of miR-424-5p and SIRT4 was performed to further verify the effects of miR-424-5p and SIRT4 on migration and invasion of GC cells.Results The expression of miR-424-5p in GC tissues was significantly higher than that in adjacent tissues(P<0.001),while the expression level of SIRT4 was significantly lower(P<0.001).Further correlation analysis showed the expression of miR-424-5p in GC tissues was negatively correlated with SIRT4(r=-0.382,P=0.034).Furthermore,the high expression of miR-424-5p was significantly associated with the depth of tumor invasion,TNM stage,vascular invasion and nervous invasion(P<0.05,respectively).Overex-pression of miR-424-5p promoted the migration and invasion of GC cells,while inhibition of miR-424-5p expres-sion exhibited the opposite effect.Overexpression of miR-424-5p reduced the expression of SIRT4 mRNA and protein in GC cells.Conversely,inhibition of miR-424-5p expression up-reguLated the expression of SIRT4 mRNA and protein.The dual luciferase reporter gene assay showed that miR-424-5p significantly affected the luciferase activity of the wild-type SIRT4-3'UTR expression vector.Further recovery experiments confirmed that miR-424-5p and SIRT4
作者
陈莉
徐惠丽
王梦漪
张子龙
CHEN Li;XU Huili;WANG Mengyi;ZHANG Zilong(Department of Oncology Central Hospital of Wuhan,Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China;不详)
出处
《实用医学杂志》
CAS
北大核心
2020年第8期1022-1029,共8页
The Journal of Practical Medicine
基金
湖北省自然科学基金面上项目(编号:2019CFB809)。