The Influence Of Livin-ShRNA On The Subcutaneous Xenograft Tumor Model Of Glioma TJ905 Cells And Its Stem Cells, As Well As The Expressions Of Caspase3, 7 MRNA

[Objective] To explore the influence of Livin short hairpin RNA(sh RNA) on the subcutaneous xenograft tumor model induced by glioma TJ905 cells and its stem cells, as well as the expressions of Caspase3, 7 m RNA.[Methods] Glioma stem cells were isolated from TJ905 cells by immunomagnetic beads and serum-free medium. The sh RNA which expresses the specific sh RNA targeting Livin m RNA(Livin-sh RNA) or Negative-sh RNA were transfected into glioma TJ905 cells and its stem cells, RT-PCR was used to detect the expression of Livin after transfection. According to the transfected plasmids, all cells were divided into six groups, Group A(glioma TJ905 cells transfected with Livin-sh RNA plasmids), Group B(glioma TJ905 cells transfected with Negative-sh RNA plasmids), Group C(the monolayer cultured glioma TJ905 cells), Group D(glioma TJ905 stem cells transfected with Livin-sh RNA plasmids), Group E(glioma TJ905 stem cells transfected with Negative-sh RNA plasmids), Group F(the monolayer cultured glioma TJ905 stem cells). The cellular proliferation rate was evaluated by CCK-8 and cell cycle distribution was analyzed by flow cytometry. The nude mices subcutaneous xenograft tumor models were established accordingly. The m RNA expressions of Caspase-3, Caspase-7 were detected by RT-PCR both in vitro and vivo.[Results] 1. Little glioma stem cell was detected in TJ905 cells. The percentage was about 0.64% to 0.91%. 2. Compared to Group C and F, the relative expressions of Livin in Group A, Group B, Group D, Group E were 0.117±0.017, 0.996±0.025, 0.193±0.014, 1.003±0.033 corresponding, the differences between Group A and B, Group D and E were statistically significant(P<0.05). 3. After transfected with Livin-sh RNA for 48 h and 72 h, the absorbances detected by CCK-8 in Group A, Group B and Group C were 0.828±0.031, 1.377±0.026, 1.437±0.027 and 0.896±0.029, 1.533±0.035, 1.602±0.031, the differences between Group A and B, Group A and C were statistically significant( P<0.05) in both time points. 4. According to the results of flow cytometry, the percentage of G0/G1 phase cells in Group A, Group B and Group C were 68.68±2.01, 63.14±1.39, 62.97±1.79 corresponding, the differencesbetween Group A and B, Group A and Group C were statistically significant(P<0.05), the percentage of S phase cells in Group D, Group E and Group F were 35.42±2.18, 30.33±1.75, 30.57±1.49 corresponding, the differences between Group D and E, Group D and F were statistically significant( P<0.05). 5. Animal experiment showed that the tumor formation rate in Group F was 100%, while the tumor formation rate in Group C was 37.5%, the differences between Group F and C was statistical significance(P<0.05). Obviously,the tumorigenicity of glioma TJ905 stem cell was stronger than glioma TJ905 cell in vivo. The tumorigenicity of glioma TJ905 cell was not affected by Livin-sh RNA transfection, but the volume of tumor was significantly decreased compared to control and blank group, which means Livin-sh RNA may be slow down TJ905 glioma cell proliferation, but has no significant effect on tumorigenicity. Both the tumorigenicity and the volume of tumor induced by glioma TJ905 stem cells were not affected by Livin-sh RNA, which means Livin-sh RNA has no significant effect on the tumorigenicity and cell proliferation in glioma TJ905 stem cell. 6. Compared to Group C and F, the relative expressions of Caspase-3 in Group A, Group B, Group D, Group E were 5.163±0.364, 1.009±0.038, 4.076±0.043, 1.116±0.031 corresponding. The relative expressions of Caspase-7 in Group A, Group B, Group D, Group E were 3.067±0.157, 1.039±0.022, 2.993±0.109, 1.045±0.046 corresponding, the differences between Group A and B, Group D and E were statistically significant(P<0.05), which means Livin-sh RNA maybe induce the expressions of Caspase-3 m RNA and Caspase-7 m RNA.[Conclusion] A small amount of glioma stem cell was detected in glioma TJ905 cells. The tumorigenicity of glioma TJ905 stem cell is stronger than glioma cell in vivo. Livin-sh RNA maybe inhibit the expression of Livin and cellular proliferation, induce the expressions of apoptosis related genes, reduce the proliferation, but has no obvious influence on the tumorigenicity of glioma TJ905 cells. The apoptosis pathway of glioma stem cells may be different from glioma cell, so to find some genes related to Livin as the target for gene therapy of glioma maybe a good way to cure it.