Hypoxia in the tumor microenvironment is a critical factor contributing to chemoresistance in glioblastoma. This study preliminarily investigates the role of hypoxia-inducible factor-2α (HIF-2α) and transforming growth factor-β2 (TGF-β2) in promoting chemotherapy resistance of glioblastoma cells under hypoxic conditions.

U87 and U118 cells (n=3) were cultured under normoxic and hypoxic conditions and treated with temozolomide (TMZ) for 24, 48, and 72 h. Cell proliferation was assessed by CCK-8 assay, and apoptosis was detected by flow cytometry after 48 h of culture. Using normoxic culture as control, U87 and U118 cells (n=3) were subjected to hypoxic culture for 4, 8, 12, and 24 h, followed by RT-qPCR to measure HIF-2α and TGF-β2 mRNA expression. After further hypoxic culture for 12, 24, and 48 h, the protein levels of HIF-2α and TGF-β2 were detected by Western blotting. After knockdown of HIF-2α and TGF-β2, the cells (n=3) were cultured under hypoxia and treated with TMZ for 24, 48, and 72 h. Then, the cell proliferation was detected by CCK-8 assay. After 24 h of culture, TGF-β2 (50 ng/mL) was added to the culture medium of some cells, and the cells were further cultured under hypoxia for another 24 h, followed by flow cytometry for cell apoptosis. Western blotting was applied to detect TGF-β2 expression in HIF-2α knockdown cells after 48 h of hypoxic culture.

CCK-8 and flow cytometry assays indicated that the hypoxia+TMZ group showed increased cell viability (P<0.05) and decreased apoptotic rate compared to the normoxia+TMZ group (P<0.05). RT-qPCR revealed that compared with the normoxia group, the expression levels of HIF-2α and TGF-β2 in hypoxia-cultured cells gradually increased with the prolongation of hypoxic time (P<0.05), and Western blotting also confirmed that the protein expression of HIF-2α and TGF-β2 increased in a time-dependent manner after hypoxic culture. Lower cell viability (P<0.05) and higher apoptotic rate (P<0.05) were observed in the HIF-2α-knockdown and TGF-β2-knockdown cells compared with the control non-knockdown group. Continued hypoxic culture of HIF-2α-knockdown cells indicated decreased TGF-β2 expression compared with the control non-knockdown HIF-2α group.

HIF-2α chemoresistance in glioblastoma cells via TGF-β2.