Construction of <i>CHD5</i> Gene Overexpressing Lentiviral Vector Based on CRISPR/Cas9-SAM System and the Effect of CHD5 on Proliferation, Migration and Invasion in T24 Cells

doi:10.13523/j.cb.1908028

China Biotechnology

2020, Vol. 40

Issue (3): 1-8 DOI: 10.13523/j.cb.1908028

Orginal Article

Construction of CHD5 Gene Overexpressing Lentiviral Vector Based on CRISPR/Cas9-SAM System and the Effect of CHD5 on Proliferation, Migration and Invasion in T24 Cells

HUANG Sheng^1,^**,YAN Qi-tao^2,^**,XIONG Shi-lin³,PENG Yi-qi¹,ZHAO Rui^3,^***(

)

1 Zhujiang Hospital of Southern Medical University,Guangzhou 510282,China
2 General Hospital of Southern Theatre Command Key Laboratory of Geriatric Infection and Organ Function Support,Guangzhou 510010,China
3 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University 510515,China

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Abstract

Objective: To construct a CHD5 gene overexpressing lentiviral vector using CRISPR/Cas9-SAM system and analyze its effect on the proliferation, migration and invasion in bladder cancer cell line T24.Methods: Three pairs of sgRNAs (sgRNA1, sgRNA-2, sgRNA-3) were designed targeting CHD5 gene. The sgRNA was inserted into the LV-sgRNA-MS2-P65-HSF1-Neo vector, and the lentivirus particles with high titer were obtained after 293T cell packaging. Subsequently, bladder cancer cell line T24 were infected with lentivirus at MOI=10. The expression of CHD5 mRNA and protein in T24 cells was examined by RT-qPCR and Western blot, respectively. CCK8 assay, flow cytometry, wound healing and transwell assay were performed to detect the effects of overexpression of CHD5 on the proliferation, apoptosis, migration and invasion of T24 cells.Results: The CHD5 overexpressing lentiviral vector was successfully constructed. The results of RT-qPCR and Western blot confirmed that the mRNA and protein expression of CHD5 in T24 cells were significantly higher than those in the blank and negative control groups after lentivirus infection(P<0.05), and the effect of sgRNA-3-MS2-P65-HSF1 was the most significant.CCK8 and flow cytometry analysis showed that overexpression of CHD5 inhibited the proliferation and promoted apoptosis of T24 cells, which had statistical significance compared with the control group (P<0.001). Wound healing and transwell assay showed that overexpression of CHD5 inhibited the migration and invasion of T24 cells (P<0.01).Conclusion: CHD5-SAM lentivirus has been successfully constructed. Overexpression of CHD5 could induce apoptosis, inhibit proliferation, migration and invasion of bladder cancer cell line T24.

Key words： CHD5 CRISPR/Cas9-SAM system Bladder cancer Lentivirus

Received: 15 August 2019 Published: 18 April 2020

ZTFLH:

Q819

Corresponding Authors: Rui ZHAO E-mail: zhaoruiruizhao@qq.com

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	HUANG Sheng
	YAN Qi-tao
	XIONG Shi-lin
	PENG Yi-qi
	ZHAO Rui

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HUANG Sheng, YAN Qi-tao, XIONG Shi-lin, PENG Yi-qi, ZHAO Rui. Construction of CHD5 Gene Overexpressing Lentiviral Vector Based on CRISPR/Cas9-SAM System and the Effect of CHD5 on Proliferation, Migration and Invasion in T24 Cells. China Biotechnology, 2020, 40(3): 1-8.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1908028 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/1

Table 1 sgRNA sequence

Table 2 Primer sequences of CHD5 and β-Actin

Fig.1 Sequencing results of CHD5-sgRNA expression vector

Fig.2 Expression of CHD5 in T24 cells infected with SAM dual-vector virus * P<0.05 vs NC or Blank;NC:Negative control;n.s.: No significance

Fig.3 CCK8 assay indicated overexpression of CHD5 inhibited growth of T24 cells *** P<0.001 vs NC or Blank

Fig.4 Effects of overexpression of CHD5 on cell cycle and apoptosis

Fig.5 The effect of CHD5 on migration and invision of T24 cell (a),(b) Wound-healing assay showed CHD5 overexpression could inhibit the migration of T24 cell (c),(d) Transwell assay demonstrated that overexpression of CHD5 significantly reduced the number of invasive cells ** P<0.01 vs NC or Blank; n.s.: No significance


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