慢病毒介导的c-Myc启动子结合蛋白1过表达对结肠癌HCT116细胞增殖与凋亡的影响及其机制研究

doi:10.3877/cma.j.issn.2095-1221.2020.01.006

目的

探讨慢病毒介导的c-Myc启动子结合蛋白1 （MBP-1）过表达对结肠癌HCT116细胞增殖和凋亡的影响及其机制。

方法

将体外培养的HCT116细胞分为以下3组：空白对照组，细胞未做任何处理；空载感染组，空载体对照慢病毒（LV-GFP）感染HCT116细胞；MBP-1过表达慢病毒组（MBP-1组），MBP-1过表达的重组慢病毒（LV-MBP-1-GFP）感染HCT116细胞。RT-PCR检测各组细胞中MBP-1 mRNA的表达，CCK-8实验检测细胞的增殖能力，流式细胞仪检测细胞的周期变化，Western blot检测细胞中MBP-1、NF-κB p65、c-Myc、CyclinD1及细胞凋亡相关蛋白Bcl-2、Bax、cleaved caspase-3的表达。三组间比较采用单因素方差分析，方差齐性采用F检验，若方差齐则组间比较采用LSD检验，若方差不齐则组间比较采用Dunnett检验。

结果

与空载感染组比较，MBP-1组在MBP-1 mRNA （1.02±0.15比4.56±1.03）、蛋白表达水平（0.18±0.01比0.72±0.10）、S期百分比[（39.12±2.18）﹪比（45.64±3.21）﹪]、G2/M期的百分比[（14.81±1.02）﹪比（23.16±2.12）﹪]、细胞中Bax蛋白（0.55±0.10比0.76±0.11）、cleaved caspase-3蛋白（0.45±0.08比0.81±0.11）表达水平均升高，差异具有统计学意义（P均< 0.001），而细胞48 h OD值（0.58±0.08比0.43±0.03）、72 h OD值（1.10±0.13比0.52±0.09）、细胞G0/G1期的百分比[（46.06±1.89）﹪比（31.36±2.02）﹪]、细胞中Bcl-2蛋白（0.52±0.10比0.23±0.02）、NF-κB p65蛋白（0.61±0.13比0.16±0.03）、c-Myc蛋白（0.79±0.15比0.43±0.05）、CyclinD1蛋白（0.62±0.09比0.32±0.01）的表达水平均降低，差异具有统计学意义（P均< 0.001）；空白对照组和空载感染组之间各指标差异无统计学意义（P > 0.05）。

结论

MBP-1过表达可抑制HCT116细胞增殖及诱导细胞周期阻滞于S期，其作用机制可能与抑制NF-κB信号通路活化有关。

关键词: 慢病毒, 结肠癌, c-Myc基因, 细胞增殖, 凋亡, NF-κB信号通路

Objective

To investigate the role and mechanisms of lentivirus-mediated overexpression of c-Myc promoter binding protein-1 (MBP-1) gene in the regulation of proliferation and apoptosis of colon cancer HCT116 cells and its mechanism.

Method

HCT116 cells cultured in vitro were divided into the three groups: no-load infection group, cells were not treated; control lentivirus group, empty vector control lentivirus (LV-GFP) infected HCT116 cells; MBP-1 overexpression lentivirus group (MBP-1 group) : Lentivirus overexpressing MBP-1 (LV-MBP-1-GFP) infected HCT116 cells. The expression of MBP-1 mRNA in each group was detected by RT-PCR, the proliferation rate of cells was observedby CCK-8 test, cell cycle was determined by flow cytometry, and the expression of MBP-1, NF-κB p65, c-Myc, Cyclin D1, Bcl-2, Bax and cleaved caspase-3 proteins was measured by Western blot. SPSS 22.0 was applied to conduct statistical analysis. The data collected from the three groups were analyzed by one-way ANOVA, while F-test was conducted to determinethe homogeneity of variance. If the variances were shown to be homogeneous, LSD test would be carried out for comparisonto be performed between different groups, otherwise Dunnett test would be conducted for comparison to be drawn between different groups.

Results

Compared with control lentivirus group, the expression of MBP-1 mRNA and protein was significantly increased in MBP-1 group (MBP-1 mRNA:1.02±0.15 vs 4.56±1.03; MBP-1 protein: 0.18±0.01 vs 0.72±0.10; P< 0.001) . Relative to the control lentivirus group [S phase (39.12±2.18) ﹪, G2/M phase (14.81±1.02) ﹪], the MBP-1 group was observed to be in the S phase (45.64±3.21) ﹪ and the G2/M phase (23.16±2.12) ﹪, where a significantly increase of percentage was discovered (P< 0.001) . The expression level of Bcl-2 protein showed declining trend (0.23±0.02, P< 0.001) . In comparison with the control lentivirus group [ (0.55±0.10) , (0.45±0.08) ], the levels of Bax (0.76±0.11) and cleaved caspase-3 protein expression (0.81±0.11) were improved in MBP-1 group (P< 0.001) . Nevertheless, the proliferation of cells (OD value) at 48 h (0.43±0.03) and 72 h (0.52±0.09) , as well as the percentage (31.36±2.02) ﹪ of cells at the G0/G1 phase in the MBP-1 group were reduced when compared to the control lentivirus group [ (46.06±1.89) ﹪, (0.61±0.13) , (0.79±0.15) , (0.62±0.09) ] (P< 0.001) , as were the levels of NF-κB p65 (0.16±0.03) (P< 0.001) , c-Myc (0.43±0.05) (P< 0.001) and CyclinD1 protein expression (0.32±0.001) (P< 0.001) . There was no significant difference observed in the indicators between the control group and the NC group.

Conclusion

Overexpression of MBP-1 can be effective in suppressing the proliferation of HCT116 cells and inducing cell cycle arrest in S phase. Moreover, its mechanism is speculated to be associated with the inhibited activation of NF-κB signaling pathway.

Key words: Lentivirus, Colon cancer, c-Myc gene, Cell proliferation, Apoptosis, NF-κB signaling pathway

表1 引物序列表

基因	引物序列（5'→3'）
β-actin	上游GGACCTGACTGACTACCTC
β-actin	下游TACTCCTGCTTGCTGAT
MBP-1	上游CGTTCAATGTCATCAATGGCGGT
MBP-1	下游CTTCAGGTTGTGGTAAACCTCTG

表1 引物序列表

基因	引物序列（5'→3'）
β-actin	上游GGACCTGACTGACTACCTC
β-actin	下游TACTCCTGCTTGCTGAT
MBP-1	上游CGTTCAATGTCATCAATGGCGGT
MBP-1	下游CTTCAGGTTGTGGTAAACCTCTG

图1 Western blot检测结肠癌细胞中MBP-1蛋白表达

表2 三组细胞中MBP-1 mRNA和蛋白相对表达量比较（±s，n = 3）

分组	MBP-1 mRNA	MBP-1蛋白
空白对照组	1.00±0.13	0.16±0.02
空载感染组	1.02±0.15	0.18±0.01
MBP-1组	4.56±1.03^a	0.72±0.10^a
F值	103.086	259.543
P值	< 0.001	< 0.001

表2 三组细胞中MBP-1 mRNA和蛋白相对表达量比较（±s，n = 3）

分组	MBP-1 mRNA	MBP-1蛋白
空白对照组	1.00±0.13	0.16±0.02
空载感染组	1.02±0.15	0.18±0.01
MBP-1组	4.56±1.03^a	0.72±0.10^a
F值	103.086	259.543
P值	< 0.001	< 0.001

表3 两组结肠癌HCT-116细胞的OD值比较（±s，n = 3）

分组	24 h	48 h	72 h
空载感染组	0.45±0.02	0.60±0.02	1.13±0.05
MBP-1组	0.41±0.06	0.43±0.03	0.52±0.09
t值	1.551	8.276	15.895
P值	0.583	< 0.001	< 0.001

表3 两组结肠癌HCT-116细胞的OD值比较（±s，n = 3）

分组	24 h	48 h	72 h
空载感染组	0.45±0.02	0.60±0.02	1.13±0.05
MBP-1组	0.41±0.06	0.43±0.03	0.52±0.09
t值	1.551	8.276	15.895
P值	0.583	< 0.001	< 0.001

表4 MBP-1过表达对结肠癌HCT-116细胞周期的影响（±s，n = 3）

分组	G0/G1期	S期	G2/M期
空白对照组	47.58±2.25	38.45±2.06	13.95±1.15
空载感染组	46.06±1.89	39.12±2.18	14.81±1.02
MBP-1组	31.36±2.02^a	45.64±3.21^a	23.16±2.12^a
F值	170.405	22.070	101.904
P值	< 0.001	< 0.001	< 0.001

表4 MBP-1过表达对结肠癌HCT-116细胞周期的影响（±s，n = 3）

分组	G0/G1期	S期	G2/M期
空白对照组	47.58±2.25	38.45±2.06	13.95±1.15
空载感染组	46.06±1.89	39.12±2.18	14.81±1.02
MBP-1组	31.36±2.02^a	45.64±3.21^a	23.16±2.12^a
F值	170.405	22.070	101.904
P值	< 0.001	< 0.001	< 0.001

图2 MBP-1过表达对结肠癌HCT-116细胞凋亡蛋白表达的影响

表5 MBP-1过表达对结肠癌HCT-116细胞凋亡蛋白表达的影响（±s，n = 3）

分组	Bcl-2	Bax	cleaved caspase-3
空白对照组	0.50 ± 0.03	0.56 ± 0.08	0.43 ± 0.06
空载感染组	0.52 ± 0.10	0.55 ± 0.10	0.45 ± 0.08
MBP-1组	0.23 ± 0.02^a	0.76 ± 0.11^a	0.81 ± 0.11^a
F值	62.681	13.295	55.873
P值	< 0.001	< 0.001	< 0.001

表5 MBP-1过表达对结肠癌HCT-116细胞凋亡蛋白表达的影响（±s，n = 3）

分组	Bcl-2	Bax	cleaved caspase-3
空白对照组	0.50 ± 0.03	0.56 ± 0.08	0.43 ± 0.06
空载感染组	0.52 ± 0.10	0.55 ± 0.10	0.45 ± 0.08
MBP-1组	0.23 ± 0.02^a	0.76 ± 0.11^a	0.81 ± 0.11^a
F值	62.681	13.295	55.873
P值	< 0.001	< 0.001	< 0.001

图3 Western blot检测NF-κB p65、c-Myc和CyclinD1蛋白的表达

表6 MBP-1过表达对NF-κB信号通路相关蛋白表达的影响（±s，n = 3）

分组	NF-κB p65	c-Myc	CyclinD1
空白对照组	0.56±0.10	0.82±0.13	0.60±0.06
空载感染组	0.61±0.13	0.79±0.15	0.62±0.09
MBP-1组	0.16±0.03^a	0.43±0.05^a	0.32±0.01^a
F值	59.083	30.351	64.373
P值	< 0.001	< 0.001	< 0.001

表6 MBP-1过表达对NF-κB信号通路相关蛋白表达的影响（±s，n = 3）

分组	NF-κB p65	c-Myc	CyclinD1
空白对照组	0.56±0.10	0.82±0.13	0.60±0.06
空载感染组	0.61±0.13	0.79±0.15	0.62±0.09
MBP-1组	0.16±0.03^a	0.43±0.05^a	0.32±0.01^a
F值	59.083	30.351	64.373
P值	< 0.001	< 0.001	< 0.001

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Role and mechanisms of lentivirus mediated MBP-1 overexpression in the regulation of proliferation and apoptosis of colon cancer HCT116 cells

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Role and mechanisms of lentivirus mediated MBP-1 overexpression in the regulation of proliferation and apoptosis of colon cancer HCT116 cells