沉默Krüppel样因子5对电离辐射后大鼠小肠上皮IEC-6细胞生物学功能的影响

Translated title of the contribution: Effects of Krüppel-like factor 5 gene silencing on biological functions of rat intestinal epithelial cells IEC-6 after radiation

Wenyu Zhao, Siming Yang, Zhimin Yang, Xiaotang Xiao, Zengfu Shang, Ming Li

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: To investigate the effects of down-regulation of Krüppel-like factor 5 (KLF5) on biological functions of rat intestinal epithelial cells IEC-6 in response to ionizing radiation. Methods: Rat intestinal epithelial IEC-6 cells were irradiated with 0, 2, 4, 8, 12, 16 Gy of X-rays and 3 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. IEC-6 cells were irradiated to 12 Gy and 0, 0.5, 1, 2, 3, 5, 7 and 24 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. shRNA sequences targeting rat KLF5 gene were designed, synthesized and inserted into the lentiviral vector. The recombinant lentiviral vectors were packaged in human embryonic kidney 293T cells, and the lentivirus titers were determined. IEC-6 cells were infected with the recombinant lentivirus, and the transfection efficiency was observed under fluorescence microscope. Real-time PCR and Western blot were used to detect the expressions of KLF5 mRNA and protein in the transfected cells 72 h post transfection. The consequent experiments included four groups: negative control group, shKLF5 group, radiation group and radiation + shKLF5 group. The cell viability was observed in KLF5 silencing cells irradiated with 8 Gy by using CCK-8 assay. The cell cycle distribution and apoptosis were detected in KLF5 silencing cells irradiated with 8 Gy by flow cytometry. Immunofluorescence staining was applied to visualize the γ-H2AX foci in nucleus of KLF5 silencing cells irradiated with 2 Gy. Results: The expression of KLF5 increased with the different doses. The expression of KLF5 increased first, then decreased and peaked at 5 h post-irradiation with 12 Gy. KLF5 shRNA lentiviral vectors were successfully constructed. The mRNA and protein level of KLF5 were down-regulated in recombinant lentivirus transfected IEC-6 cells 72 h after transfection. Knockdown of KLF5 markedly induced G2/M phase arrest (t=11.56, P<0.05), proliferation inhibition, more apoptosis rate [radiation group: (7.42±0.49)%, radiation + shKLF5 group: (12.49±0.63)%, t=10.98, P<0.05], and more γ-H2AX foci in nucleus post-irradiation than negative control (t=22.07, 23.89, 11.24, 59.97, 20.85, P<0.05). Conclusions: The KLF5 knockdown intestinal epithelial cell line was successfully established. The down-regulation of KLF5 expression could induce cell arrest at G2/M, suppress the proliferation of irradiated cells and improve the cell apoptosis, enhance DNA double strand breaks and prolong DNA damage repair.

Translated title of the contributionEffects of Krüppel-like factor 5 gene silencing on biological functions of rat intestinal epithelial cells IEC-6 after radiation
Original languageChinese (Traditional)
Pages (from-to)500-506
Number of pages7
JournalChinese Journal of Radiological Medicine and Protection
Volume40
Issue number7
DOIs
StatePublished - Jul 25 2020
Externally publishedYes

Keywords

  • Apoptosis
  • Ionizing radiation
  • Krüppel-like factor 5
  • Radiation induced intestinal injury

ASJC Scopus subject areas

  • Radiology Nuclear Medicine and imaging
  • Radiological and Ultrasound Technology

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