Tianjin Medical Journal

Tianjin Medical Journal ›› 2020, Vol. 48 ›› Issue (5): 358-363.doi: 10.11958/20192806

• Cell and Molecular Biology • Previous Articles     Next Articles

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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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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