| Purpose:To explore the role of Pim1 in the process of hypoxia regulating the cell proliferation and epithelial-mesenchymal transition of Triple Negative Breast Cancer(TNBC).Method:1.Use CCK-8,cell scratch and Transwell experiments to investigate the changes in the proliferation,migration and invasion of TNBC cells MDA-MB-231 under the stimulation of different concentrations of CoCl2.2.Through q RT-PCR and WB experiments to explore the changes in Pim1mRNA and protein expression levels in MDA-MB-231 cells stimulated by different concentrations of CoCl2.3.Establish Pim1 expression knockdown MDA-MB-231 cell line(Pim1-SH)and no-load transfection cell line(Pim1-NC)by lentiviral transfe-ction,and explore the effect of Pim1 expression level on MDA-MB-231 cells The influence of proliferation and apoptosis,cell cycle and migration and invasion ability.4.Use CoCl2 to stimulate Pim1-SH cells,detect the changes in HIF-1α mRNA and protein expression levels,and preliminarily explore the role of Pim1in the process of hypoxia regulating the HIF-1α pathway.5.Use CoCl2 to stimulate Pim1-SH cells,explore the role of Pim1 in the process of cell proliferation,migration and invasion promoted by hypoxia through CCK-8,cell scratch experiment and Transwell experiment,and detect cell proliferation and EMT correlation by WB Changes in protein expression.6.Use MDA-MB-231 and Pim1-SH cells to construct a nude mouse tumor-bearing model,compare the volume growth and final wet weight of the two groups of transplanted tumors,and detect the expression of proliferation and EMT-related factors in the tumor tissue by immunohistochemical method.Result:1.CCK8 experimental results:at 12h,the survival rate of the 100μM and200μM CoCl2 stimulation group was significantly higher than that of the 0μM CoCl2 stimulation group(P<0.001;P<0.05);at 24h,compared with the 0μM CoCl2 stimulation group,it was lower The survival rate of the cells stimulated by the concentration of CoCl2(25-100μM)increased;at 36h and 48h,the survival rate of the 200μM CoCl2 stimulated group began to decrease significantly,and compared with the 0μM group,there were significant statistical differences(P<0.01,P<0.05).Cell scratch test results:Compared with the scratch healing rate of the 0μM CoCl2 stimulation group(25.60%±0.30%),the scratch healing rate of the 25,50,and 100μM CoCl2 stimulation groups were significantly increased,respectively 31.03%±1.76%,35.58%±2.74%,38.01%±2.78%,all have statistical differences(P<0.05;P<0.001;P<0.001).Transwell migration experiment results:Compared with the number of cells migrating per unit field of view of the 0μM CoCl2 stimulation group(264.33±28.04),the number of cells migrating per unit field of view of the 25,50,and 100μM CoCl2 stimulation groups all increased significantly,respec-tively 354.00±39.51,518.33±48.13,597.33±49.33,all of which were statisti-cally different(P<0.05;P<0.001;P<0.001).Transwell invasion experiment results:Compared with the number of cells per unit field of view in the 0μM CoCl2 stimulation group(206.00±24.52),the number of cells per unit field of view in the 25,50,and 100μM CoCl2 stimulation groups all increased signifi-cantly,respectively 288.33±49.34,420.67±29.54,478.33±14.47,all have statistical differences(P<0.05;P<0.001;P<0.001).2.Compared with the 0μM CoCl2 stimulation group,the expression levels of Pim1 mRNA and protein in the 25,50,and 100μM CoCl2 stimulation groups increased,and the increase was the highest when the concentration was 50μM and there was a statistical difference(P<0.05;P<0.01).3.The cell line in which Pim1 expression was knocked down(Pim1-SH)and the no-load transfected cell line(Pim1-NC)were successfully established.CCK-8 experiment results:the OD values of the Ctrl group and the Pim1-NC group were significantly higher than those of the Pim1-SH group(P<0.01).Flow cytometry experiment results:the survival rate of cells in the Ctrl group and the Pim1-NC group was significantly higher than that of the Pim1-SH group(P<0.001),and the proportion of cells in the G2 phase of the Pim1-SH group was compared with that of the Ctrl group and Pim1-NC group were significantly reduced(2.87%±0.60%.vs.12.02%±2.58%.vs.11.36%±3.37%,P<0.01),while the proportion of cells in the G1 phase was significantly increased(75.37%±0.74%.vs.62.57%±7.81%.vs.61.9%±5.21%,P<0.05).Cell scratch test results:The scratch healing rate of cells in the Pim1-SH group(14.07%±1.16%)was significantly lower than that of the Ctrl group(25.36%±2.27%)and the Pim1-NC group(25.37%±1.84%)(P<0.001;P<0.001).Transwell migration experiment results:the number of migrating cells in the Pim1-SH group was144.00±32.08/per field,which was significantly lower than the Ctrl group(259.33±53.13/per field)and Pim1-NC(236.00±49.15/field)Per field of view)(P<0.01;P<0.01).Transwell invasion experiment results:The number of invasion cells in the Pim1-SH group was 103.33±14.47 cells/field,which was significantly lower than that of the Ctrl group(206.00±24.52 cells/per field)and Pim1-NC(214.00±18.68 cells/field)Per field of view)(P<0.01;P<0.001).WB experiment results:Compared with the Ctrl group and the Pim1-NC group,the expressions of the proliferation-related proteins PCNA and BCL2 in the Pim1-SH group were significantly reduced(P<0.01),while the expression of BAX increased(P<0.05).The expression of EMT-related protein E-Cadherin was significantly increased(P<0.01),while the expression of N-Cadherin and Vimentin were significantly decreased(P<0.05).4.Compared with the CoCl2 stimulation group,the HIF-1α mRNA and protein expression levels in the CoCl2+Pim1-SH group were lower(P<0.05).5.CCK8 experiment results:the cell survival rate of the CoCl2+Pim1-SH group was significantly lower than that of the CoCl2+Pim1-NC group(P<0.001).Cell scratch test results:The healing rate of the CoCl2+Pim1-SH group was20.31%±0.44%,which was significantly lower than that of the CoCl2+Pim1-NC group(35.84%±0.64%)(P<0.001).Transwell migration experiment results:The number of migrating cells in the CoCl2+Pim1-SH group was 265.00±91.76/per field,which was significantly lower than the number of migrating cells in the CoCl2+Pim1-NC group(580.00±70.34/per field,)P=0.001.Trans-well invasion experiment results:The number of invaded cells in the CoCl2+Pim1-SH group was 203.33±29.05/per field,which was significantly less than the number of invaded cells in the CoCl2+Pim1-NC group(403.33±23.76/per field),P<0.001.WB results:Compared with the CoCl2+Pim1-NC group,the expression levels of PCNA,BCL2,N-Cadherin and Vimentin in the CoCl2+Pim1-SH group were lower(P<0.01;P<0.05;P<0.001;P<0.01),while the expression levels of BAX(P<0.001)and E-Cadherin(P<0.05)were higher.6.Animal tumor-bearing experiment results:the two groups of tumors showed statistical differences on the 15th day after implantation(243.85mm~3±90.70mm~3.vs.90.07mm~3±16.85mm~3,P=0.045),the final on the 27th day The tumor volume was also significantly different(1252.04mm~3±322.53mm~3.vs.560.88mm~3±276.63mm~3,P=0.048),and the final wet-to-weight ratio was 1.42g±0.26g.vs.0.66g±0.27g,which was statistically different(P=0.025).Immun-ohistochemical results:Compared with the Ctrl group,the expression of Pim1 in the tumor tissue of the Pim1-SH group was significantly decreased(P=0.005),and the proliferation and apoptosis related factors PCNA(P=0.004)and Ki-67(P<0.001)The expression of E-Cadherin was also significantly reduced;the expression of EMT-related factor E-Cadherin was significantly increased(P=0.012),and the expression of Vimentin was significantly reduced(P<0.001).Conclusion:1.The hypoxic environment simulated by CoCl2 can improve the prolifera-tion,migration and invasion ability of MDA-MB-231 cells,and at the same time can up-regulate the expression levels of Pim1 mRNA and protein in the cells.2.Knocking down the expression level of Pim1 can significantly inhibit the proliferation,cycle progression,migration and invasion of MDA-MB-231 cells.3.Pim1 plays an important role in the process of hypoxia promoting the proliferation,migration and invasion of MDA-MB-231 cells and activating the HIF-1α pathway. |
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