CHD4调控端粒功能促进宫颈癌HeLa细胞生长的机制研究

doi:10.11958/20222091

摘要/Abstract

摘要：

目的探讨染色质域解旋酶DNA结合蛋白4（CHD4）通过调控端粒功能对宫颈癌HeLa细胞增殖、迁移的影响。方法慢病毒感染构建CHD4稳定敲低的宫颈癌HeLa细胞系，实时荧光定量PCR（qPCR）和Western blot分别检测CHD4的mRNA和蛋白表达水平；将宫颈癌HeLa细胞分为对照组、shCDH4-1组、shCHD4-2组，CCK-8实验检测CHD4对HeLa细胞增殖能力的影响；集落形成实验检测细胞的集落数量；划痕愈合实验检测细胞迁移能力；裸鼠荷瘤实验观察移植瘤生长情况；免疫荧光-荧光原位杂交技术检测CHD4与HeLa细胞中端粒的共定位及各组细胞中端粒的损伤情况，端粒损伤以损伤因子γH2AX与端粒的共定位表示；中期染色体-荧光原位杂交技术检测各组细胞中端粒功能的变化，端粒信号缺失（SFE）或多端粒信号（MTS）的染色体比例升高代表端粒功能障碍。结果慢病毒感染成功构建CHD4稳定下调的HeLa细胞系（P<0.05）。与对照组相比，shCDH4-1组和shCHD4-2组的细胞增殖能力、集落形成能力、迁移能力、体内肿瘤生长能力均明显下降（P<0.05）。免疫荧光-荧光原位杂交实验表明，CHD4定位于HeLa细胞的端粒上。与对照组相比，CHD4的缺失不足以引起端粒的DNA损伤（P>0.05），但会导致HeLa细胞SFE的染色体比例明显增多（P<0.05）。结论 CHD4可通过调控HeLa细胞的端粒功能，促进宫颈癌细胞的增殖和迁移。

关键词: HeLa细胞, 端粒, 宫颈肿瘤, 细胞增殖, 染色质域解旋酶DNA结合蛋白4

Abstract:

Objective To investigate the effect of chromodomain helicase DNA-binding protein 4 (CHD4) on the proliferation and migration of cervical cancer HeLa cells by regulating telomeres function. Methods CHD4-depleted HeLa cell lines were constructed by lentivirus infection. The mRNA and protein expression levels of CHD4 were detected by real-time fluorescence quantitative PCR (qPCR) and Western blot assay. Cervical cancer HeLa cells were divided into the control group, the shCDH4-1 group and the shCHD4-2 group. The effect of CHD4 on cell proliferation of HeLa cells was detected by CCK-8 assay. The colony formation assay was performed to detect the number of cell colonies. Scratch-healing assay was performed to detect cell migration. In vivo, the tumor formation experiment was used to observe the growth changes of xenograft in nude mice. Immunofluorescence-fluorescence in situ hybridization was performed to detect the co-localization of telomeres and CHD4 in HeLa cells, and the level of damage at telomeres in each group. Telomere damage was indicated by the co-localization of damage factor γH2AX with telomeres. Metaphase-telomere fluorescence in situ hybridization was performed to detect changes in telomere function in each group, and the increased proportion of chromosomes with telomere signal deletion (SFE) or multiple telomere signals (MTS) represented telomere dysfunction. Results HeLa cell lines with stable down-regulated CHD4 were successfully constructed after lentiviral infection (P<0.05). Compared with the control group, the cell proliferation ability, colony formation ability, migration ability and tumor growth ability in vivo were significantly decreased in the shCHD4-1 group and the shCHD4-2 group (P<0.05). Immunofluorescence-fluorescence in situ hybridization assay showed that CHD4 localized to telomeres of HeLa cells. Compared with the control group, the deletion of CHD4 was insufficient to cause DNA damage at telomeres (P>0.05). However, the proportion of SFE chromosomes in HeLa cells increased significantly (P<0.05). Conclusion CHD4 can promote the proliferation and migration of HeLa cells by regulating telomere function.

Key words: HeLa cells, telomere, uterine cervical neoplasms, cell proliferation, chromodomain helicase DNA-binding protein 4

中图分类号:

R737.33

图/表 11

图1 慢病毒感染HeLa细胞后CHD4的蛋白表达水平 A：对照组；B：shCHD4-1组；C：shCHD4-2组。

Fig.1 Expression levels of CHD4 protein in HeLa cells after lentivirus infection

表1 各组HeLa细胞中CHD4的mRNA和蛋白表达水平比较（n=3，$\bar{x}\pm s$）

Tab.1 Comparisons of mRNA and protein expression levels of CHD4 between three groups of HeLa cells

组别	mRNA表达	蛋白表达
对照组	1.00±0.00	1.00±0.00
shCHD4-1组	0.37±0.04^a	0.44±0.15^a
shCHD4-2组	0.45±0.06^a	0.35±0.08^a
F	191.520^＊＊	39.067^＊＊

表2 各组HeLa细胞不同天数的OD450比较（n=3，$\bar{x}\pm s$）

Tab.2 Comparison of OD450 at different days between three groups of HeLa cells

组别	第1天	第3天	第5天
对照组	0.06±0.04	0.42±0.20	1.74±0.25
shCHD4-1组	0.05±0.02	0.27±0.09	1.00±0.12^a
shCHD4-2组	0.05±0.03	0.21±0.11	0.73±0.25^a
F	0.108	1.784	17.474^＊＊

图2 抑制CHD4表达对HeLa细胞集落形成的影响

Fig.2 The effect of inhibiting CHD4 on the colony formation of HeLa cells

图3 抑制CHD4表达对HeLa细胞迁移的影响（×40）

Fig.3 The effect of inhibiting CHD4 on the migration of HeLa cells (×40)

表3 各组HeLa细胞集落形成数量、细胞迁移率的比较（n=3，$\bar{x}\pm s$）

Tab.3 Comparison of colony formation and migration between the three cell groups

组别	集落形成数量/（个/视野）	细胞迁移率/%
对照组	230.00±26.66	44.13±2.99
shCHD4-1组	19.33±12.50^a	32.37±3.56^a
shCHD4-2组	36.67±10.07^a	28.21±2.41^a
F	127.070^＊＊	22.411^＊＊

图4 抑制CHD4表达对移植瘤生长的影响

Fig.4 The effect of inhibiting CHD4 on the growth of xenograft

图5 HeLa细胞中CHD4与端粒共定位的免疫荧光图（免疫荧光染色， ×1 000）

Fig.5 Representative images showing the colocalization of CHD4 and telomere in HeLa cells (immunofluorescence staining, ×1 000)

图6 IF-FISH检测HeLa细胞中端粒与γH2AX的共定位（免疫荧光染色，×1 000）

Fig.6 The co-localization of telomeres and γH2AX in HeLa cells detected by IF-FISH (immunofluorescence staining, ×1 000)

图7 Metaphase-FISH检测CHD4对HeLa细胞中染色体末端端粒信号的影响（免疫荧光染色，×1 000）

Fig.7 Effect of CHD4 on chromosome terminal telomere signals in HeLa cells by Metaphase-FISH (immunofluorescence staining，×1 000)

表4 各组HeLa细胞中含SFE、MTS的染色体占比的比较[n=60，%，M（P25，P75）]

Tab.4 Comparison of the proportion of chromosomes with SFE and MTS between three groups of HeLa cells

组别	含SFE的染色体占比	含MTS的染色体占比
对照组	3.15（1.34，5.56）	1.29（0，2.48）
shCHD4-1组	9.23（4.50，12.12）^a	0.64（0，1.87）
shCHD4-2组	8.32（5.30，12.09）^a	1.28（0，1.42）
H	46.554^＊＊	1.460

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