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. 2023 Mar 20;43(3):340-348.
doi: 10.12122/j.issn.1673-4254.2023.03.02.

[DTX2 overexpression promotes migration and invasion of colorectal cancer cells through the Notch2/Akt axis]

[Article in Chinese]
Z Ma  1 , X Zhao  1 , X Zhang  1 , G Xu  1 , F Liu  1
Affiliations

[DTX2 overexpression promotes migration and invasion of colorectal cancer cells through the Notch2/Akt axis]

[Article in Chinese]
Z Ma et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effect of changes in DTX2 expression level on migration and invasion of colorectal cancer (CRC) cells and explore the mechanism.

Methods: Two CRC cell lines SW620 and LoVo were transfected with a specific shRNA targeting DTX2 (DTX2-shRNA) or a DTX2-overexpressing plasmid (pcDNA-DTX2), and the transfection efficiency was evaluated with RT-qPCR and Western blotting. Scratch and Transwell assays were used to assess the changes in migration and invasion ability of the transfected cells, and the cellular expression levels of Notch2, NICD, AKT, p-Akt and MMP-2/9 proteins were detected with Western blotting. The CRC cells were co-transfected with pcDNA-DTX2 and Notch2 siRNA to assess the effect of Notch2 knockdown on DTX2 overexpression-induced enhancement of cell migration and invasion.

Results: The expression levels of DTX2 at both the mRNA and protein levels were significantly decreased in CRC cells transfected with DTX2- shRNA (P < 0.01) and increased in cells transfected with pcDNA-DTX2 (P < 0.01). Scratch and Transwell assays showed that the migration and invasion abilities of CRC cells were significantly lowered following DTX2 knockdown (P < 0.01) and were enhanced in cells with DTX2 overexpression (P < 0.01). The expression levels of Notch2, NICD, p-Akt and MMP-2 proteins decreased significantly in CRC cells with DTX2 knockdown (P < 0.05) and increased obviously in DTX2-overexpressing cells (P < 0.05). In both of the two CRC cell lines, transfection with Notch2 siRNA obviously reversed the effect of DTX2 overexpression in promoting cell migration and invasion (P < 0.01) and expressions of the related proteins.

Conclusion: DTX2 overexpression promotes migration and invasion of CRC cells through the Notch2/Akt axis, suggesting the potential of DTX2 as a new biological indicator of CRC.

目的: 探讨DTX2对结直肠癌(CRC)细胞迁移侵袭的影响及作用机制。

方法: 利用基因工具干预CRC细胞，分为敲低组(DTX2-shRNA)、敲低空载组(neg-shRNA)、未转染组(con)、过表达空载组(pcDNA)及过表达组(pcDNA-DTX2)，利用qRTPCR及Western blotting法检测转染效率。采用划痕和Transwell侵袭实验检测DTX2基因的表达改变对CRC细胞迁移侵袭能力的影响，并通过Western blotting检测各组中Notch2、NICD、Akt、p-AKT、MMP-2及MMP-9蛋白的表达水平。CRC细胞共转染pcDNA-DTX2和Notch2 siRNA，检测回复实验对CRC细胞迁移侵袭能力的影响。

结果: CRC细胞中DTX2 mRNA和蛋白表达量，敲低组中明显降低(P < 0.01)，过表达组中明显升高(P < 0.01)。划痕和Transwell侵袭实验提示，CRC细胞迁移侵袭能力，DTX2 shRNA显著低于con和neg-shRNA(P < 0.01)；pcDNA-DTX2显著高于con和pcDNA(P < 0.01)。Western blotting检测提示，Notch2、NICD、p-Akt及MMP-2蛋白平均表达水平，与con和neg-shRNA相比，DTX2 shRNA明显降低(P < 0.05)；与con和pcDNA相比，pcDNA-DTX2明显升高(P < 0.05)，而这些蛋白在con、neg-shRNA及pcDNA间的平均表达水平无明显差异(P>0.05)。回复实验提示，siRNA-Notch2可扭转pcDNA-DTX2增强的CRC细胞迁移侵袭的能力(P < 0.01)，相应蛋白水平的变化也具有统计学意义。

结论: DTX2通过Notch2/Akt轴促进CRC细胞迁移侵袭的能力，DTX2可能成为CRC的新型生物学指标。

Keywords: DTX2; Notch2/Akt axis; cell invasion; cell migration; colorectal cancer.

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Figures

图 1
图 1
CRC细胞中DTX2 mRNA和蛋白的表达水平 Expression levels of DTX2 mRNA and protein in CRC cells in different groups. A: Expression levels of DTX2 mRNA and protein in SW620 and LoVo cells. B, C: Relative expression levels of DTX2 mRNA and protein in cells with DTX2 knockdown or overexpression. con: non-transfected group; neg-shRNA: Negative control shRNA group; DTX2 shRNA: DTX2 knockdown group; pcDNA: empty plasmid group; pcDNA-DTX2: DTX2 overexpression group. *P < 0.01.
图 2
图 2
改变CRC细胞中DTX2基因表达后对CRC细胞迁移能力的影响 Changes in migration ability of CRC cells after DTX2 knockdown or overexpression. A, B: Scratch assay of SW620 and LoVo cells with DTX2 knockdown (Original magnification: ×100). C: Cell migration rates of the CRC cells. D, E: Scratch assay of the cells with DTX2 overexpression (×100). F: Cell migration rate. *P < 0.01.
图 3
图 3
改变CRC细胞中DTX2基因表达后对CRC细胞侵袭能力的影响 Changes in invasion ability of the CRC cells with DTX2 knockdown or overexpression (×200). A, B: Transwell assay of SW602 and LoVo cells after DTX2 knockdown. C, D: Transwell assay of the CRC cells overexpressing DTX2 (×200). *P < 0.01.
图 4
图 4
CRC细胞共转染DTX2过表达基因和Notch2 siRNA后对CRC细胞迁移能力的影响 Scratch test for assessing changes of migration ability of SW620 (A, C) and LoVo (B, D) cells after co-transfection with DTX2-overexpressing plasmid and Notch2 siRNA (×100). *P < 0.01.
图 5
图 5
CRC细胞共转染pcDNA-DTX2基因和Notch2 siRNA后对CRC细胞侵袭能力的影响 Transwell invasion assay for assessing changes of migration ability of SW620 and LoVo cells after co-transfection with DTX2-overexpressing plasmid and NOTCH2 siRNA. A: Microscopic observation of the cells (×200). B: Average number of invasive cells in different groups. *P < 0.01.
图 6
图 6
CRC细胞敲减DTX2基因后Notch2/Akt轴中相关蛋白的变化 Changes of protein expressions in the Notch2/Akt axis in SW620 and LoVo cells with DTX2 knockdown. A, C: Western blotting of the proteins in SW620 and LoVo cells. B, D: Quantitative analysis of the protein expressions. GAPDH was used as the internal control. *P < 0.05.
图 7
图 7
CRC细胞过表达DTX2基因后Notch2/Akt轴中相关蛋白的变化 Changes of protein expressions in the Notch2/Akt axis in SW620 and LoVo cells with DTX2 overexpression. A, C: Western blotting of the proteins in SW620 and LoVo cells. B, D: Quantitative analysis of the protein expressions. GAPDH was used as the internal control. *P < 0.05.
图 8
图 8
CRC细胞共转染pcDNA-DTX2+Notch2 siRNA后Notch2/Akt轴中相关蛋白的变化 Changes of protein expressions in the Notch2/Akt axis in SW620 and LoVo cells co-transfected with DTX2-overexpressing plasmid and DTX2-shRNA. A, C: Western blotting of the proteins in SW620 and LoVo cells. B, D: Quantitative analysis of the protein expressions. GAPDH was used as the internal control. *P < 0.05.
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