Study on the mechanism of CHD4 regulating telomere function to promote cervical cancer HeLa cell proliferation

doi:10.11958/20222091

Abstract

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

CLC Number:

R737.33

Figures/Tables 11

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

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^＊＊

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^＊＊

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

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

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^＊＊

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

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

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

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

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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