天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (5): 358-363.doi: 10.11958/20192806

• 细胞与分子生物学 • 上一篇    下一篇

IRX5促进肝癌细胞的侵袭迁移及其与miR-136-5p靶向关系的验证#br#

代龙光 1，朱丽英 1，2，沈婕 1，钱雯 1，张競之 1，朱金凤 1，许永劼 1，刘歆蕾 1，许雯 1，朱科静 1，张令 1，潘卫 1，3，李兴 1，4△
  

  1. 1贵州医科大学医学检验学院（邮编550004）；2贵州医科大学附属医院临床检验中心血液体液科；3贵州医科大学附属医院贵
    州省产前诊断中心；    4贵州中医药大学

  • 收稿日期:2019-09-11 修回日期:2020-04-07 出版日期:2020-05-15 发布日期:2020-06-24
  • 通讯作者: 李兴 E-mail:542535283@qq.com
  • 作者简介:代龙光（1991），男，硕士在读，主要从事肝癌发生发展的分子机制研究
  • 基金资助:
    贵州省科技厅项目（黔科合基础［2016］1123）；贵州省科技厅学术新苗项目（黔科合平台人才［2017］5718）；贵州省教育厅青年科技人才成长项目（黔教合 KY［2018］176）；贵州省高等学校大学生创新创业训练计划项目（20195200893）；贵州医科大学附属医院博士启动基金（I-2019-04）

Study of IRX5 promotes invasive migration of hepatocellular carcinoma cells and the validation of its targeting relationship with miR-136-5p #br#

DAI Long-guang1, ZHU Li-ying1,2, SHEN Jie1, QIAN Wen1, ZHANG Jing-zhi1, ZHU Jin-feng1, XU Yong-jie1, LIU Xin-lei1, XU Wen1, ZHU Ke-jing1, ZHANG Ling1, PAN Wei1,3, LI Xing1,4△ #br#   

  1. 1 Department of Medical Laboratory, Guizhou Medical University, Guiyang 550004, China; 2 Department of Hematology and Body
    Fluids, Clinical Laboratory Center, the Affiliated Hospital of Guizhou Medical University; 3 The Affiliated Hospital of Guizhou Medical
    University, Guizhou Prenatal Diagnosis Center; 4 Guizhou University of Traditional Chinese Medicine
  • Received:2019-09-11 Revised:2020-04-07 Published:2020-05-15 Online:2020-06-24

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摘要：

摘要：目的 探讨易洛魁族同源盒基因5（IRX5）对肝癌细胞侵袭与迁移的影响，并验证其与miR-136-5p的靶向
关系。方法 取对数生长期肝癌 SMMC7721 细胞，过表达 IRX5 实验设空载质粒（pcDNA3.1）组和过表达 IRX5
（pcDNA3.1-IRX5）组，敲低 IRX5实验设阴性对照（sh-NC）组和敲低 IRX5（sh-IRX5）组。采用 Western blot验证 IRX5
过表达和敲低效率；采用划痕实验和 Transwell 实验检测 IRX5 对肝癌细胞迁移与侵袭的影响；采用 miRanda、
Targetscan生物信息学软件预测 IRX5结合的 miRNA和位点；构建野生型和突变型 IRX5 3'UTR双荧光素酶报告基因
质粒，采用酶切、基因测序方法鉴定重组质粒是否构建成功。取对数生长期 293T 细胞，设野生型质粒+阴性对照
（IRX5-3'UTR-Wt+NC）组和野生型质粒+miR-136-5p（IRX5-3'UTR-Wt+miR-136-5p）组，突变型质粒+阴性对照
（IRX5-3'UTR-Mut+NC）组和突变型质粒+miR-136-5p（IRX5-3'UTR-Mut+miR-136-5p）组，双荧光素酶报告系统检
测各组荧光素酶活性。结果 过表达 IRX5组 IRX5蛋白表达水平、细胞迁移及侵袭数量均高于空载质粒组，敲低
IRX5组IRX5蛋白表达水平、细胞迁移及侵袭数量均低于阴性对照组（均P＜0.05）。成功构建IRX5基因3'UTR野生
型和突变型双荧光素酶报告质粒；双荧光素酶实验结果显示，IRX5-3'UTR-Wt+miR-136-5p组双荧光素酶活性低于
IRX5-3'UTR-Wt+NC 组（P＜0.01），IRX5-3'UTR-Mut+miR-136-5p 组与 IRX5-3'UTR-Mut+NC 组差异无统计学意
义。结论 IRX5可促进肝癌细胞的侵袭与迁移，IRX5是 miR-136-5p的一个靶基因，miR-136-5p可能通过 IRX5的
3'UTR抑制肝癌细胞的侵袭与迁移。

关键词: 肝肿瘤, 细胞运动, 肿瘤侵润, 双荧光素酶, IRX5, miR-136-5p

Abstract:

Abstract: Objective To investigate the effect of IRX5 on the invasion and migration of hepatocellular carcinoma
(HCC) cells and verify the targeted relationship between miR-136-5p and IRX5. Methods The SMMC7721 cells were
divided into empty plasmid (pcDNA3.1) group and overexpress IRX5 (pcDNA3.1-IRX5) group in the overexpression IRX5
experiment. For knockdown IRX5 experiment a negative control (sh-NC) group and knockdown IRX5 (sh-IRX5) group were
set up. The overexpression and knockdown efficiency rates of IRX5 were detected by Western blot assay. The effects of IRX5
on the invasion and migration of HCC cells were detected by wound healing assay and Transwell assay. miRNA and binding
sites of IRX5 were predicted by miRanda and Targetscan. The 3' untranslated regions (UTR) of IRX5 were amplified by PCR
and connected to pGL3-Control vector. To verified recombinant plasmid by enzyme digestion and gene sequencing methods.
The 293T cells were divided into four groups: IRX5-3'UTR-Wt+NC group, IRX5-3'UTR-Wt+miR-136-5p group, IRX5-
3'UTR-Mut+NC group and IRX5-3'UTR-Mut+miR-136-5p group in dual luciferase assay. The luciferase activity was
detected by dual luciferase reporter system. Results The expression level of IRX5 was significantly higher in the
overexpression IRX5 group than that of the empty plasmid group (P＜0.05). Compared with the negative control group, the
expression level of IRX5 was significantly reduced in the knockdown IRX5 group (P＜0.01). IRX5 promoted the invasion
and migration of HCC cells (P＜0.05). The IRX5-3'UTR-Wt and IRX5-3'UTR-Mut were successfully constructed. The
results of dual luciferase assay showed that miR-136-5p can reduce IRX5-3'UTR-Wt luciferase activity (P＜0.01), which
showed no effect on IRX5-3'UTR-Mut luciferase activity (P＞0.05). Conclusion IRX5 promotes the invasion and
migration of HCC cells. IRX5 acts as a target gene of miR-136-5p. miR-136-5p may inhibit the invasion and migration of
HCC cells through the 3'UTR of IRX5.

Key words: liver neoplasms, cell movement, neoplasm invasiveness, dual luciferase, IRX5, miR-136-5p

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