The Effect Of TRPS1 Knockdown On Vasculogenic Mimicry Of Human Triple-negative Breast Cancer Cell Line MDA-MB-231 And Its Related Mechanism

Vasculogenic mimicry(VM)tubules are lumen structures comprised of malignant tumor cells without the participation of endothelial cells.VM simulates blood vessel function in tumors to deliver a sufficient blood supply for proliferation,invasion,and metastasis of malignant tumors,thereby reducing the clinical effects of anti-angiogenic treatments.The elimination or prevention of malignant tumor VM development therefore represents an urgent research goal as a therapeutic strategy to and cut off nutrients required for tumor growth.The GATA transcription factor TRPS1 is abnormally upregulated in breast cancer,osteosarcoma,prostate cancer,and other tumor tissues,and is instrumental in regulating cell proliferation,differentiation,and tissue growth and development.Here,we investigated the effects of TRPS1 knockdown on VM and the proteins underlying its development in triple-negative breast cancer cell line MDA-MB-231.We found that TRPS1 knockdown resulted in obvious inhibition of VM development.Fluorescence microscopy of F-actin and tubulin revealed that loss of TRPS1 function resulted in disruption of cytoskeleton and microtubule formation,respectively.In addition,TRPS1-suppressed cells exhibited reduced accumulation of VM-associated proteins Eph A2,MMP-2,MMP-9,VEGF,and VE-cadherin.Moreover,we found that the capacity for migration and invasion were limited in MDA-MB-231 cells after TRPS1 knockdown and that the average number of VM tubules,their length,and number of intersections were also significantly decreased.Based on our results,and in light of previous studies,we thus proposed that TRPS1 suppression negatively affects vascular mimicry possibly through reduced TRPS1-mediated transcriptional regulation of VM-related protein VEGF-A.Methods Using Q-PCR technology,the best interference sequence(TRPS1 knockdown)cell lines were selected from normal MDA-MB-231 cell lines;hoechst33342 / propidium iodide(HO / PI)staining was used to detect the necrosis and apoptosis of sh-NC cells and MDA-MB-231 cells with TRPS1 knockdown;annexin After V-FITC / PI double staining,the apoptosis of sh-NC cells and MDA-MB-231 cells with TRPS1 gene knockdown were detected by flow cytometry;the migration and invasion ability of sh-NC cells and MDAMB-231 cells with TRPS1 gene knockdown were detected by cell scratch test and cell invasion test.The cytoskeleton microfilaments and microtubules of sh-NC cells and MDA-MB-231 cells with TRPS1 gene knockdown were observed by immunofluorescence staining under confocal microscope.Matrigel model was constructed in vitro to detect the ability of sh-NC cells and TRPS1 knockdown MDAMB-231 cells to form VM in vitro.Finally,Western blot(WB)was used to detect the expression of Caspase-3,cleaved caspase-3 and VE-Cadherin,Eph A2,MMP-2,MMP-9and VEGF in MDA-MB-231 cells and MDA-MB-231 cells with TRPS1 gene knockdown.Result1.In triple negative breast cancer cell line MDA-MB-231,knockdown of TRPS1 gene made MDA-MB-231 cells more prone to apoptosis;Western blot analysis showed that the level of cleaved caspase-3 in MDA-MB-231 cells with knockdown of TRPS1 gene increased significantly,which induced apoptosis.2.Through cell scratch test and cell invasion test,we found that knockdown of TRPS1 gene could inhibit the migration ability and invasion ability of MDA-MB-231 cells.3.By confocal microscopy,the F-actin cytoskeleton and tubulin structure of MDA-MB-231 cells were disrupted after knockdown of TRPS1,thus impairing some of the aggressive biological properties of breast cancer,such as migratory and invasive abilities,which may be one of the mechanisms by which vasculogenic mimicry is inhibited after TRPS1 knockdown.4.On Matrigel Matrigel,both sh-NC cells and MDA-MB-231 cells with knockdown of TRPS1 gene had the ability of vasculogenic mimicry formation.The ability of vasculogenic mimicry formation was significantly inhibited in MDA-MB-231 cells with knockdown of TRPS1,as indicated by the statistical results of the number of tubules formed,the number of intersections,and the average length of tubules.5.Western blot analysis showed that the expression levels of VE-cadherin,Eph A2,MMP-2,MMP-9 and VEGF were significantly down regulated in MDA-MB-231 cells with TRPS1 gene knockdown.Conclusion: Knockdown of TRPS1 gene inhibits some biological behaviors of human triple negative breast cancer MDA-MB-231 cells,such as cell migration,invasion and vasculogenic mimicry.The mechanism is related to the destruction of the cytoskeleton of MDA-MB-231 cells and the down-regulation of vasculogenic mimicry related marker proteins VE-cadherin,Eph A2,MMP-2,MMP-9 and VEGF.