N-terminal Polypeptide Derived From VMIP-? Exerts Its Antitumor Activity By Regulating Lncrna SPRY4-IT1 And GAS5 In Human Breast Cancer

Objective:To investigate the effect of NT21 MP on breast cancer by regulating lnc RNA SPRY4-IT1 and GAS5.Methods: 1.The expression of SPRY4-IT1 and GAS5 in human breast cancer cells was detected and the best si RNA of SPRY4-IT1 and GAS5 was screened by qRT-PCR.2.qRT-PCR was used to detect the influence of NT21 MP and CXCR4 on the expression of SPRY4-IT1 and GAS5 in breast cancer cells.3.After treated with down-regulated or up-regulated SPRY4-IT1 and GAS5,and combined with NT21 MP,the cells ability of proliferation was testified by SRB analysis;The cells ability of migration and invision was detected by wound healing analysis and Transwell analysis;The cell cycle and apoptosis analysis was performed by the museTM cell analyzer.4.qRT-PCR and Western blot was used to detect the effect of down-regulated or up-regulated SPRY4-IT1 and GAS5 on the m RNA and protein levels of cell cycle and apoptosis related genes.5.qRT-PCR was used to detect the expression of SKA2 in human breast cancer cells and the effect of down-regulated or up-regulated SPRY4-IT1 on the expression of SKA2.6.The best si RNA of SKA2 was screened by qRT-PCR and Western blot.7.After treated with down-regulated SKA2,combined with down-regulated or up-regulated SPRY4-IT1,or combined with NT21 MP,the cells ability of proliferation was testified by SRB analysis;The cells ability of migration and invision was detected by wound healing analysis and Transwell analysis;The cell cycle and apoptosis analysis was performed by the museTM cell analyzer.Results: 1.The expression of lnc RNA SPRY4-IT1 is the highest in pc DNA-CXCR4-MDA-MB-231 cells and is the lowest in MCF-7 cells.The expression of GAS5 is the highest in MCF-7 cells and is the lowest in SKBR-3 cells.The best si RNA of SPRY4-IT1 is si-SPRY4-IT1-3(p<0.05).2.NT21 MP antagonists the role of SDF-1?,down-regulated the expression of SPRY4-IT1(p<0.05)and up-regulated the expression of GAS5(p<0.05);Depletion of CXCR4 can down-regulated SPRY4-IT1(p<0.05)and up-regulated GAS5(p<0.01);The expression of SPRY4-IT1 and GAS5 is not change significantly in pc DNA-CXCR4-MDA-MB-231 cells.3.Depletion of SPRY4-IT1,the ability of cells proliferation,migration and invision was decreased,the cell cycle was arrested in G0/G1 phase(p<0.05)and induced cells apoptosis(p<0.05).At the same time,up-regulated the expression of apoptisis related genes(Bak1 and Caspase3),down-regulated the expression of Bcl-2/Bax and the cell cycle regulator(Cyclin D1)(p<0.05).Overexpression of SPRY4-IT1 received the opposite results.4.Depletion of GAS5,the ability of cells proliferation,migration and invision was increased(p<0.01),promoted the transition of cell cycle from G0/G1 phase to S phase and inhibited cells apoptosis(p<0.05).At the same time,down-regulated the expression of apoptisis related genes(Bak1 and Caspase3),up-regulated the expression of Bcl-2/Bax and the cell cycle regulator(Cyclin D1)(p<0.05).Overexpression of GAS5 received the opposite results.5.Compared to the NT21 MP group and the si-SPRY4-IT1 group,the ability of cells proliferation,migration and invision was significantly decreased,the ratio of G0/G1 phase cells and the apoptosis cells was significantiy raised compared with the NT21 MP group and the si-SPRY4-IT1 group;Treated with NT21 MP combined with pc DNA-SPRY4-IT1,the ability of cells proliferation,migration and invision was higher than the NT21 MP group,but was lower than the pc DNA-SPRY4-IT1 group,the ratio of G0/G1 phase cells and the apoptosis cells was lower than the NT21 MP group,but was higher than the pc DNA-SPRY4-IT1 group.6.Treated with NT21 MP combined with si-GAS5,the ability of cells proliferation,migration and invision was higher than the NT21 MP group,but was lower than the si-GAS5 group,the ratio of G0/G1 phase cells and the apoptosis cells was higher than the NT21 MP group,but was lower than si-GAS5 group;Compared to the NT21MP group and the pc DNA-GAS5 group,the ability of cells proliferation,migration and invision was significantly decreased,the ratio of G0/G1 phase cells and the apoptosis cells was significantiy raised;7.The expression of SKA2 is the highest in MCF-7 cells and is lowest in SKBR-3 cells;The best si RNA of SKA2 is si-SKA2-1(p<0.01);Depletion of SPRY4-IT1 can decrease the expression of SKA2(p<0.05)and overexpression of SPRY4-IT1 can increase the expression of SKA2(p<0.05);8.Depletion of SKA2,the ability of cells proliferation,migration and invision was decreased,arrested cell cycle in G0/G1 phase(p<0.05),induced cells apoptosis(p<0.01);Depletion of SKA2 combined with depletion of SPRY4-IT1,the ability of cells proliferation,migration and invision was significantly decreased,the ratio of G0/G1 phase cells and the apoptosis cells was increased significantly compared with si-SKA2 group and the si-SPRY4-IT1 group;9.Depletion of SKA2 combined with overexpression of SPRY4-IT1,the ability of cells proliferation,migration and invision was higher than si-SKA2 group but is lower than pc DNA-SPRY4-IT1 group,the ratio of G0/G1 phase cells and the apoptosis cells was lower than si-SKA2 group but is higher than pc DNA-SPRY4-IT1 group;10.Depletion of SKA2 combined with NT21 MP,the ability of cells proliferation,migration and invision was significantly decreased,the ratio of G0/G1 phase cells and the apoptosis cells was significantly increased compared with the si-SKA2 group and the NT21 MP group.Conclutions: 1.SPRY4-IT1 and GAS5 played the role of oncogene and tumor suppressor genes respectively in human breast cancer cells;2.NT21 MP can play the inhibiting effect on human breast cancer by regulating the expression of SPRY4-IT1 and GAS5;3.The inhibition role of NT21 MP on breast cancer is partially achieved through the downstream effects molecular of SKA2.