科研之友
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摘要
Objective: To investigate the synergistic regulation of hypoxia-inducible factor (HIF) 1α and 2α on proliferation and chemotherapy resistance of human glioma cells in vitro. Methods: The primary tissues were collected from the tumor specimens obtained during operations at Department of Neurosurgery, the First Affiliated Hospital of Army Military Medical University. The expression of HIF1α and HIF2α in tumor tissues was detected by immunohistochemistry. The expression of HIF1α and HIF2α was detected by immunofluorescence and Western blotting after 72 h of hypoxia culture. CRISPR /Cas9 was used to knock out the expression of HIF1α and HIF2α in the cells, and the cells were divided into HIF1α and HIF2α knockout alone groups, HIF1α+ HIF2α knockout group and vector control group. After 72 h of hypoxic culture, cell proliferation was detected by CCK-8, and cell cycle was detected by flow cytometry. After 72 h of hypoxic culture with temozolomide, cell proliferation and apoptosis were assessed. Results: Immunohistochemical results showed that HIF1α and HIF2α were highly expressed in glioma tissues. After 72 h hypoxia culture, HIF1α and HIF2α were highly expressed which was indicated by immunofluorescence staining and Western blotting assay. CRISPR /Cas9 successfully knocked out the expression of HIF1α and HIF2α. After 72 h of hypoxia culture, CCK-8 showed that the proliferation rate of HIF1α+ HIF2α knockout group was higher than that of the vector control group, HIF1α knockout alone group or HIF2α knockout alone group (F =24.419, P <0.001). There was no significant difference between HIF1α or HIF2α knockout group and vector control group (P >0.05). Cell cycle analysis showed that the proportion of G0/G1 phase cells in HIF1α+ HIF2α knockout group was lower than that in the vector control group, HIF1α or HIF2α knockout group alone, and the proportion of G2/M+ S phase cells was higher than that in the vector control group, HIF1α or HIF2α knockout group alone (F =5.326, P =0.025). There was no significant difference in cell cycle between HIF1α or HIF2α knockout group and vector control group (both P >0.05). After adding temozolomide into the medium, the cell proliferation rate in HIF1α+ HIF2α knockout group was lower than that in vector control group, HIF1α or HIF2α knockout group (F =46.518, P <0.001), and the cell proliferation rate in HIF1α or HIF2α knockout alone group was lower than that in vector control group (both P <0.05). Conclusion: HIF1α and HIF2α play synergistic roles in regulating the proliferation and chemotherapy resistance of human glioma cells.
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