Hypoxia inducible factor-1α induces tunneling nanotube formation and promotes the invasive ability of bladder tumor cells
Objective To explore the mechanism by which hypoxia inducible factor-1α(HIF-1α)promotes the invasive ability of bladder tumor cells and induces the formation of intercellular tunneling nanotubes(TNTs).Methods T24 cells and RT4 cells(purchased from ATCC)were cultured for 24 hours,mitochondria were labeled with Mito-Tracker Deep Red,and cytoskeletal was labeled with Actin-Tracker Green.Laser confocal microscopy was used to observe and count intercellular TNTs.HIF-1α was transfected into T24 cells,and the changes in the number of TNTs between T24 cells overexpressing HIF-1α were observed using laser confocal microscopy.The changes in the invasion and metastasis ability of T24 cells after transfection were observed by scratch test and Transwell test.The expression difference of mammalian target of rapamycin(mTOR)/4EBPl/P70S6K-eIF4e/S6RP signaling pathway proteins was de-tected by Western blotting.Results The number of TNTs between T24 cells overexpressing HIF-1α gene(HIF-1α-T24 cells)was greater than that between T24 cells transfected with empty vector(NC-T24 cells)(84.80±2.91 vs.60.40±0.91,t=8.01,P＜0.01).The results of scratch test showed that the healing speed of HIF-1α-T24 cells was faster than that of NC-T24 cells[(43.39±0.57)％vs.(25.76±0.53)％,t=22.93,P＜0.01].Transwell assay found that more HIF-1α-T24 cells migrated through the matrix than NC-T24 cells(55.00±1.53 vs.32.00±1.16,1=12.01,P＜0.01).Molecular studies showed that the phosphorylation level of mTOR in HIF-1α-T24 cells was higher than that in NC-T24 cells(0.58±0.12 vs.0.26±0.06,t=4.47,P＜0.01),and the phosphorylation levels of downstream mole-cules 4EBP1 and P70S6K were upregulated(0.51±0.06 vs.0.32±0.06,t=5.74,P＜0.01;0.44±0.02 vs.0.27±0.03,t=5.21,P＜0.01),thereby upregulating the phosphorylation levels of effector molecules eIF4E and S6RP(0.45±0.02 vs.0.22±0.03,t=11.61,P＜0.01;0.52±0.12 vs.0.29±0.06,t=3.32,P＜0.01).Conclusion HIF-lα induces the formation of TNTs between bladder tumor cells by activating the mTOR signaling pathway,thereby promoting the invasive ability of bladder tumor cells.
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