Effects Of Different IL-15 Gene Transfections Into NCI-H446 Cells On Proliferation And Cell Cycle-regulatory Molecule Expression

Objective The tumor threatens the health and life seriously. The anti-tumor cytokine gene transfection have become the important field on exploring tumor gene therapy. Interleukin 15 (IL-15) is a cytokine that was found in 1994 with similar biological activity to IL-2 enhancing CD8+CTL and NK cells to proliferate and to kill tumor. There was IL-15 mRNA expression but rarely IL-15 secretion in many tissues and cells of human body, suggesting that IL-15 carried out its biological functions mainly within cell. There had been the reports about that inoculating the tumor cells transfected by prototype IL-15 gene into nude mice had not achieved satisfying anti-tumor effect, doubting the inhibitory effect of its long signal peptide on IL-15 secretion. However, transfection of the modified IL-15 gene that could increase IL-15 secretion had not reached to result expected either.We found previously that the transfections of different IL-15 gene could obviously decrease the proliferation of NCI-H446 cells, but the decreased degree was inconsistent each other. The tumor cell originate from normal tissue cell mutation. Quickening proliferation was one of the malignant characters of tumor cells, and the cell cycle disorders were the important mechanism which could cause the tumor. By now discovered, the molecules affecting cell proliferation were the cell cycle-positively regulatory molecules, including cell cycle protein (cyclin) and cyclin-dependent kinase (CDK) , and the cell cycle-negatively regulatory molecules, including cyclin-dependent kinase inhibitor (CDKI) and some proteins encoded by anti-oncogene.In this study using three kinds of NCI-H446 cell models transfected respectively by different IL-15 genes(C_IL-15 transfected by the IL-15 prototype gene, C_IL-15mp transfected by the IL-15 mature peptide gene and C_{IL-2sp-IL-15mp} transfected by the IL-15 modified gene that the signal peptide of IL-15 was replaced with that of IL-2)successfully constructed in our laboratory as experimental groups and the wide NCI-H446 cells (C^w) as control group, the effects of different IL-15 gene transfections on the proliferations of NCI-H446 cells and the expressions of protein and mRNA of cell cycle-positively regulatory molecules ( cyclinD1, cyclinE, CDK2 and CDK4 ) and cell cycle-negatively regulatory molecules ( p21and Rb ) in NCI-H446 cells were studied, the molecular mechanism of these effects were revealed , and the clues and suggestions were provided for further exploring the anti-tumor roles of IL-15.Mothods1 Using the wide NCI-H446 cells (C^w) as control group and the three kinds of NCI-H446 cell models transfected respectively by different IL-15 genes(C_IL-15 transfected by the prototype IL-15 gene, C_IL-15mp transfected by the IL-15 mature peptide gene and C_{IL-2sp-IL-15mp} transfected by the IL-15 modified gene that the signal peptide of IL-15 was replaced with that of IL-2)successfully constructed in our laboratory as experimental groups, the alive cells were counted everyday in culture for 8 days by trypan blue dye exclusion and the growth curve was made, the proliferations were measured by MTT assay at 24h, 48h, 72h and 96h of culture, and the distributions of cell cycle were analyzed by FCM.2 Using the wide NCI-H446 cells (C^w) as control, the expression of protein and mRNA of the cell cycle-positively regulatory molecules cyclinD1,cyclinE ,CDK2 and CDk4 in the three kinds of NCI-H446 cell model cells(C_IL-15, C_IL-15mp, and C_{IL-2sp-IL-15mp}) transfected by three kinds of different IL-15 genes were detected by Western-blot for protein expression and by semi-quantitive RT-PCR for mRNA expression respectively.3 Using the wide NCI-H446 cells (C^w) as control, the expression of protein and mRNA of the cell cycle-negatively regulatory molecules p21 and Rb in the three kinds of NCI-H446 cell model cells (C_IL-15, C_IL-15mp, and C_{IL-2sp-IL-15mp}) transfected by three kinds of different IL-15 gene were detected by Western-blot for protein expression and by semi-quantitive RT-PCR for mRNA expression respectively.Results1 The effects of different IL-15 gene transfections on NCI-H446 cell proliferation1.1 Alive cells counted by trypan blue dye exclusion.At day 5 of culture, the alive cell counts are [(71.18±5.16)×104 (C^w)],[(58.93±3.34)×104 (C_IL-15) ],[(35.82±2.45)×104 (C_IL-15mp) ] and [(25.4±2.84)×104 (C_{IL-2sp-IL-15mp}) ] respectively. In culture from day 5 to day 8, the cell proliferations of the three kinds of NCI-H446 cell models transfected by different IL-15 gene showed decreasing than that of C^w,but the decreasing degree was inconsistent each other (p<0.05 or p<0.01). The order of cell proliferation from quickness to slowness was C^w, C_IL-15, C_IL-15mp, and CIL-2sp-IL-15m in turn.1.2 Cell proliferation detected by MTT assay.The OD values were 1.124±0.091(C^w), 0.968±0.084(C_IL-15), 0.786±0.084(C_IL-15mp) and 0.627±0.156 (C_{IL-2sp-IL-15mp}) at 72h of culture and 1.564±0.163 (C^w), 1.262±0.135(C_IL-15), 0.928±0.070(C_IL-15mp) and 0.777±0.030(C_{IL-2sp-IL-15mp}) at 96h of culture respectively. At these two time points the OD values of the three kinds of NCI-H446 cell models transfected by different IL-15 gene were decreased than that of C^w, but the decreasing degree is inconsistent each other (P<0.05 or P<0.01). The order of OD values from highness to lowness was C^w, C_IL-15, C_IL-15mp, and CIL-2sp-IL-15m in turn.2 The effects of different IL-15 gene transfections on cell cycle of NCI-H446 cellsThe cell percentages at G0/G1 phase were [(69.44±3.26)%(C^w)], [(73.47±1.49)% (C_IL-15) ], [(76.81±1.41)% (C_IL-15mp) ] and [(81.26±2.31)% (C_{IL-2sp-IL-15mp}) ] respectively and had significant differences each other among the four kinds of cells(P<0.05 or P<0.01). The cell percentages at G0/G1 phase significantly increased in the three kinds of cell models transfected by different IL-15gene than that of C^w (P<0.05 or P<0.01). The order of cell percentages at G0/G1 phase from highness to lowness were CIL-2sp-IL-15m , C_IL-15mp, C_IL-15 and C^w in turn.The cell percentages at S phase were [(26.90±1.43)% (C^w) ], [(19.94±1.72)% (C_IL-15) ], [(16.11±1.80)% (C_IL-15mp) ] and [(12.80±0.59)% (C_{IL-2sp-IL-15mp}) ] respectively and had significant differences each other among the four kinds of cells (P<0.05 or P<0.01). The cell percentages at S phase significantly decreased in the three kinds of NCI-H446 cell models transfected by different IL-15 gene than that of C^w (P<0.01). The order of cell percentages at S phase from lowness to highness were CIL-2sp-IL-15m , C_IL-15mp, C_IL-15 and C^w in turn.3 The effects of different IL-15 gene transfections on expression of the cell cycle-positively regulatory molecules in NCI-H446 cells3.1 Expression of cyclin D1The protein expression of cyclin D1 was 0.893±0.182(C^w), 0.717±0.148(C_IL-15), 0.749±0.172 (C_IL-15mp)and 0.786±0.172 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the cyclin D1 protein expression of the three kinds of cell models transfected by different IL-15 gene was no significant difference (P>0.05), and there was no significant difference among the three kinds of NCI-H446 transfected by different IL-15 gene(P>0.05). It demonstrated that transfecting different IL-15 gene had no effect on the protein expression of cyclin D1 of NCI-H446.The mRNA expression of cyclin D1 was 0.52±0.046 (C^w), 0.47±0.125 (C_IL-15), 0.52±0.049 (C_IL-15mp) and 0.54±0.057 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the mRNA expression of cyclin D1of the three kinds of cell models transfected by different IL-15 gene was no significant difference (P>0.05), and there was no significant difference among the three kinds of NCI-H446 transfected by different IL-15 gene (P>0.05). It demonstrated that transfecting different IL-15 gene had no effect on the mRNA expression of cyclin D1 of NCI-H446.3.2 Expression of cyclin EThe protein expression of cyclin E was 1.154±0.153 (C^w), 0.911±0.196 (C_IL-15), 0.720±0.112 (C_IL-15mp) and 0.531±0.104 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w , the protein expression of cyclin E in the three kinds of cell models (C_IL-15,C_IL-15mp,C_{IL-2sp-IL-15mp} )transfected by different IL-15 gene significantly decreased (P<0.05, P<0.05, P<0.01), and there was significant difference among C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} (P<0.05). It was suggested that the transfection of three kinds of IL-15 gene could decrease the protein expression of cyclin E but decreasing degree was different. The order of protein expression of cyclin E from highness to lowness was C^w, C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} in turn. The decreasing of protein expression of cyclin E was negative correlation with the increasing of cell percentages at G0/G1 phase (r=-0.983,P<0.01) and positive correlation with the decreasing of cell percentages at S phase ( r=0.993,P<0.01) in the three kinds of cell models transfected by different IL-15 gene.The mRNA expression of cyclin E was 1.10±0.121 (C^w), 0.93±0.073 (C_IL-15), 0.76±0.059 (C_IL-15mp) and 0.48±0.056 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the mRNA expression of cyclin E in the three kinds of cell models (C_IL-15,C_IL-15mp,C_{IL-2sp-IL-15mp} )transfected by different IL-15 gene significantly decreased (P<0.05, P<0.05, P<0.01), and there was significant difference among C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp}(P<0.05). It was suggested that the transfection of three kinds of IL-15 gene could decrease the mRNA expression of cyclin E but decreasing degree was different. The order of mRNA expression of cyclin E from highness to lowness was C^w, C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} in turn. The decreasing of mRNA expression of cyclin E was negative correlation with the increasing of cell percentages at G0/G1 phase (r=-0.998,P<0.01) and positive correlation with the decreasing of cell percentages at S phase ( r=0.0.957,P<0.05) in the three kinds of cell models transfected by different IL-15 gene.3.3 Expression of CDK2The protein expression of CDK2 was 0.689±0.251 (C^w), 0.257±0.106 (C_IL-15), 0.254±0.120 (C_IL-15mp) and 0.385±0.046 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the protein expression of CDK2 in the three kinds of cell models (C_IL-15,C_IL-15mp,C_{IL-2sp-IL-15mp} )transfected by different IL-15 genes significantly decreased (P<0.01, P<0.01, P<0.05,), and there was no significant difference among C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} ( P>0.05). It was demonstrated that the transfection of the three kinds of different IL-15 genes could decrease the protein expression of CDK2 in NCI-H446 cells with equal degree.The mRNA expression of CDK2 was 0.964±0.139 (C^w),0.560±0.167 (C_IL-15), 0.683±0.049 (C_IL-15mp) and 0.622±0.023 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the mRNA expression of CDK2 in C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} was significantly decreased (P<0.01,P<0.05,P<0.01), and there was no significant difference among C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} ( P>0.05). It was demonstrated that the transfection of the three kinds of different IL-15 genes could decrease the mRNA expression of CDK2 in NCI-H446 cells with equal degree.3.4 Expression of CDK4The protein expression of CDK4 was 1.694±0.506 (C^w), 1.067±0.338 (C_IL-15), 1.038±0.311(C_IL-15mp) and 0.974±0.255 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the expression of CDK4 protein decreased in C_{IL-2sp-IL-15mp} (P<0.05) and had no significant changes in C_IL-15and C_IL-15mp (P>0.05). The expression of CDK4 protein had no significant differences among C_IL-15, C_IL-15mp and C_{IL-2sp-IL-15mp} (P>0.05). It was shown that the modified IL-15 gene transfenction could lightly decrease the expression of CDK4 protein in NCI-H446 cells, but either the prototype IL-15 gene transfection or the IL-15 mature peptide gene transfection could not significantly affect the expression of CDK4 protein in NCI-H446 cells.The mRNA expression of CDK4 was 0.630±0.045 (C^w), 0.651±0.082 (C_IL-15), 0.561±0.061 (C_IL-15mp) and 0.666±0.027 (_{IL-2sp-IL-15mp}) respectively. The mRNA expression of CDK4 had no significant differences among the four kinds of cells (p>0.05), suggesting the three kinds of different IL-15 gene transfections could not significantly affect the mRNA expression of CDK4 in NCI-H446 cells.4 The effects of different IL-15 gene transfections on expression of the cell cycle-negatively regulatory molecules of NCI-H446 cells4.1 Expression of p21The protein expression of p21 was 0.907±0.042 (C^w), 1.052±0.086 (C_IL-15), 0.848±0.084 (C_IL-15mp) and 0.878±0.050 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the protein expression of p21 in C_IL-15 significantly increased (P<0.05), and had no significant differences in C_IL-15mp and C_{IL-2sp-IL-15mp} (P>0.05). It demonstrated that the prototype IL-15 gene transfection could increase the protein expression of p21 in NCI-H446 cells and that both the transfection of the mature peptide gene and the modified gene had no effect on the protein expression of p21 in NCI-H446 cells.The mRNA expression of p21 was 0.455±0.051 (C^w), 0.747±0.061 (C_IL-15), 0.516±0.093 (C_IL-15mp) and 0.512±0.117 (C_{IL-2sp-IL-15mp}) respectively. Compared with C^w, the mRNA expression of p21 in C_IL-15 significantly increased (P<0.01) and had no significant difference in C_IL-15mp and C_{IL-2sp-IL-15mp} (P>0.05); It demonstrated that the transfection of the prototype IL-15 gene could increase the mRNA expression of p21 in NCI-H446 cells and that both the trasfection of the IL-15 mature peptide gene and the IL-15 modified gene could not affect the mRNA expression of p21 in NCI-H446 cells.4.2 Expression of RbThe expression of Rb protein was 0.860±0.184 (C^w), 1.132±0.150 (C_IL-15), 0.959±0.221 (C_IL-15mp)and 0.938±0.091 (C_{IL-2sp-IL-15mp}) respectively, Compared with C^w, the protein expression of Rb in C_IL-15 significantly increased (P<0.05) and had no significant difference in C_IL-15mp and C_{IL-2sp-IL-15mp} (P>0.05). It demonstrated that the transfection of prototype IL-15 gene could increase the protein expression of Rb in NCI-H446 cells and that both the transfection of the IL-15 mature peptide gene and the IL-15 modified gene could not affect the protein expression of Rb in NCI-H446 cells.The mRNA expression of Rb was 0.787±0.075 (C^w), 1.176±0.080 (C_IL-15), 0.899±0.103 (C_IL-15mp) and 0.775±0.087 (C_{IL-2sp-IL-15mp}) respectively, Compared with C^w, the expressions of Rb mRNA in C_IL-15 and C_IL-15mp significantly increased (P<0.01) and had no statistical difference in C_{IL-2sp-IL-15mp} (P>0.05). It demonstrated that both the transfection of the prototype IL-15 gene and the IL-15 mature peptide gene could increase the mRNA expression of Rb in NCI-H446 cells and that the transfection of the modified IL-15 gene could not affect the mRNA expression of Rb in NCI-H446 cells.Conclusion1 Transfection of the three kinds of different IL-15 genes could decrease the proliferation of NCI-H446 cells and the decreasing degree from highness to lowness was C_{IL-2sp-IL-15mp}, C_IL-15mp and C_IL-15. Decreasing the proliferation of NCI-H446 cells was concerned with increasing the percentage at G0/G1 phase and decreasing the percentage at S phase in NCI-H446 cells.2 That the trnsfection of the three kinds of different IL-15 genes decreased the proliferation of NCI-H446 cells, increased the percentage at G0/G1 phase and decreased the percentage at S phase in NCI-H446 cells may be caused mainly by decreasing the expression of cell cycle-positively regulatory molecule cyclin E.3 That the transfection of the three kinds of different IL-15 genes decreased the proliferation of NCI-H446 cells,increased the percentage at G0/G1 phase and decreased the percentage at S phase in NCI-H446 cells did not be mainly caused by the effect on the expression of cell cycle-negatively regulatory molecules p21 and Rb.