Impact of perilipin 2 on the stemness of glioblastoma stem cells under hypoxic microenvironment
Objective To explore the expression of perilipin 2(PLIN2)in glioblastoma stem cells(GSC)under hypoxic microenvironment and its impact on the stemness and growth of GSC.Methods Construct a glioma stem cell line WF4 from glioblastoma samples collected at Tongji Hospital affiliated with Huazhong University of Science and Technology.Divide WF4 into two groups,culture under normoxic(21%O2)and hypoxic(1%O2)conditions for 24 hours,and use Western blotting to detect the relative expression of hypoxia-inducible factor-1α(HIF-1α)and PLIN2 proteins in the two groups of cells.Design and synthesize short hairpin RNA(shRNA)targeting HIF-1α and PLIN2,transfect WF4 cells,divided into control group(shNT group)and knockdown group(shHIF-1α,shPLIN2 group),and treat all cells under hypoxic conditions for 24 hours.Use Western blotting to detect the relative expression of PLIN2 protein in the control group and knockdown group after knocking down HIF-1α,and to detect the relative expression of glioma stem cell stemness indicators sex-determining region Y-box 2(SOX2)and oligodendrocyte tran-scription factor 2(OLIG2)in the shNT group and shPLIN2 group after knocking down PLIN2.Use the BODIPY 493/503 fluorescence probe at a concentration of 2 μmol/L to incubate cells for 30 mins,and de-tect cell fluorescence intensity by immunofluorescence(IF)and flow cytometry.compare differences be-tween two groups using independent sample t-tests,and compare differences among multiple groups using One-way ANOVA analysis.Results After hypoxia treatment of WF4 cells,the expression of HIF-1α(0.38±0.02 vs.0.07±0.01,t=17.313,P<0.01)and PLIN2(0.68±0.03 vs.0.46±0.04,t=6.566,P<0.01)in the hypoxia group was higher than that in the normoxia group,with statistically signif-icant differences.In the shHIF-1α group,the expression of PLIN2 was lower than that in the shNT group(0.87±0.01,0.82±0.01 vs.1.18±0.06,F=55.191,P<0.01).In the shPLIN2 group,the relative expression of SOX2(1.30±0.13,1.17±0.08 vs.2.74±0.10,F=138.535,P<0.01)and OLIG2(0.76±0.09,0.92±0.11 vs.1.65±0.10,F=44.497,P<0.01)was lower than that in the shNT group.Sphere formation assays showed that the sphere formation ability of cells in the shPLIN2 group was lower than that in the shNT group(0.34±0.01,0.40±0.01 vs.1.00±0.08,F=115.823,P<0.01).Immunofluorescence(IF)and flow cytometry results indicated that the fluorescence intensity of BODIPY 493/503 probe in the shPLIN2 group was lower than that in the shNT group(2.67±0.47,2.33±0.47 vs.6.00±0.82,F=22.200,P<0.01;27 440.20±117.80,25 908.73±375.21 vs.40 681.50±413.55,F=667.206,P<0.01).Conclusion In GSC,PLIN2 is upregulated under hypoxia and regula-ted by HIF-1α.Knockdown of PLIN2 affects the stemness,proliferation,and lipid droplet number of GSC.