Tumor angiogenesis regulatory protein TARBP2, and encoding gene and application thereof

Abstract

According to the invention, with tumor angiogenesis as a starting point, a protein TARBP2 and a coding gene thereof are identified to be novel tumor angiogenesis regulators and can specifically degrade mRNAs of anti-angiogenesis factor genes to induce tumor angiogenesis and promote tumor metastasis. The adenovirus vector expressing shRNA targeting TARBP2 is used for treating tumors and can significantly inhibit tumor angiogenesis, metastasis and growth. The tumor angiogenesis regulatory protein and the encoding gene thereof provided by the invention provide a novel molecular target for targeting tumor angiogenesis to treat tumors, and have important theoretical and practical significance for clinically resisting angiogenesis to treat the tumors in the future.

Description

technical field

[0001] The invention relates to the field of genetic engineering, in particular to a tumor angiogenesis regulatory protein TARBP2 and its coding gene and application. Background technique

[0002] Tumor angiogenesis is a key step in the further growth of solid tumor tissue and the distant metastasis of tumor cells (Fidler IJ, 2003; Weinberg RA, 2008). However, the molecular mechanisms that regulate tumor angiogenesis remain unclear, especially which molecules control tumor cell-vascular endothelial cell interactions. Tumor cells stimulate the activation of vascular endothelial cells by secreting and releasing some angiogenesis-related factors, and then form a rich vascular network inside the tumor tissue (Fidler IJ, 2002; Husemann Y, et al., 2008), and these new blood vessels will promote tumor growth. Further growth and distant organ metastasis (Viallard C et al., 2017). Therefore, a series of angiogenic factors secreted by tumor cells become the link betw...

Examples

Embodiment 1

[0029] Example 1: Identification of a novel function of TARBP2 in controlling tumor angiogenesis

[0030] The human TARBP2 gene (NM_134324.2) was synthesized by gene synthesis method and cloned into pEGFP-N1 eukaryotic expression vector after complete sequencing. A549 cell clones (A549 / TARBP2) and MDA-MB-468 cell clones (MDA-MB-468 / TARBP2) stably expressing TARBP2 were produced by G418 screening, and the protein expression results were as follows figure 1 A and 1B are shown. The control group and TARBP2-overexpressing tumor cells were cultured for 48 hours to collect the conditioned culture supernatant (CM), centrifuged at 2000g at 4°C for 10 minutes to remove cell debris, and aliquoted for use. After the HUVECs were cultured to confluence, they were replaced with 2% FBS growth factor-free medium. Use a 200uL pipette tip to scratch the endothelial cell monolayer and add tumor cell CM. Photographs were taken at the beginning of culture and after 24 hours. The percentage of ...

Embodiment 2

[0031] Example 2: Functional identification of TARBP2 specific degradation of anti-angiogenic factor mRNAs

[0032] 1) In order to further detect how TARBP2 regulates tumor angiogenesis, genes related to its regulation of tumor angiogenesis signaling pathways were detected. Pro-angiogenic factor mRNAs were upregulated after TARBP2 overexpression, while anti-angiogenic factors were downregulated. RNA-IP experiments further confirmed that TARBP2 can target and bind anti-angiogenic factors. Moreover, luciferase reporter experiments also confirmed that TARBP2 can target the 3'UTRs of anti-angiogenic factor mRNAs to degrade their mRNAs, but cannot target the 3'UTRs of pro-angiogenic factor gene mRNAs. In-depth examination of the half-life of anti-angiogenic factor mRNAs found that TARBP2 significantly reduced their half-life, but did not affect the half-life of pro-angiogenic factor gene mRNAs. This further confirms that TARBP2 specifically degrades mRNAs of anti-angiogenic facto...

Embodiment 3

[0035] Example 3: Treatment of Tumor Metastasis by Targeted Knockdown of TARBP2 Gene and Protein

[0036] In order to detect the role of targeted knockdown of TARBP2 gene in inhibiting tumor angiogenesis and tumor metastasis, firstly, lentiviruses expressing shRNAs targeting TARBP2 gene were used to infect tumor cells in vitro, and the effect of anti-angiogenesis after knockdown of TARBP2 protein expression was detected. Factor mRNAs expression and its half-life. Knockdown of TARBP2 was found to significantly increase the expression of anti-angiogenic factors and increase the half-life of their mRNAs. In vivo, using adenoviral vectors expressing shRNAs targeting the TARBP2 gene to simulate clinical treatment of tumor-bearing nude mice, it was found that the number of blood vessels in the treated tumor tissue was significantly reduced, and the lung tumor metastasis of the mice was also significantly reduced. In summary, the experimental results showed that targeted knockdown o...

technical field

[0001] The invention relates to the field of genetic engineering, in particular to a tumor angiogenesis regulatory protein TARBP2 and its coding gene and application. Background technique

[0002] Tumor angiogenesis is a key step in the further growth of solid tumor tissue and the distant metastasis of tumor cells (Fidler IJ, 2003; Weinberg RA, 2008). However, the molecular mechanisms that regulate tumor angiogenesis remain unclear, especially which molecules control tumor cell-vascular endothelial cell interactions. Tumor cells stimulate the activation of vascular endothelial cells by secreting and releasing some angiogenesis-related factors, and then form a rich vascular network inside the tumor tissue (Fidler IJ, 2002; Husemann Y, et al., 2008), and these new blood vessels will promote tumor growth. Further growth and distant organ metastasis (Viallard C et al., 2017). Therefore, a series of angiogenic factors secreted by tumor cells become the link betw...

Examples

Embodiment 1

[0029] Example 1: Identification of a novel function of TARBP2 in controlling tumor angiogenesis

[0030] The human TARBP2 gene (NM_134324.2) was synthesized by gene synthesis method and cloned into pEGFP-N1 eukaryotic expression vector after complete sequencing. A549 cell clones (A549 / TARBP2) and MDA-MB-468 cell clones (MDA-MB-468 / TARBP2) stably expressing TARBP2 were produced by G418 screening, and the protein expression results were as follows figure 1 A and 1B are shown. The control group and TARBP2-overexpressing tumor cells were cultured for 48 hours to collect the conditioned culture supernatant (CM), centrifuged at 2000g at 4°C for 10 minutes to remove cell debris, and aliquoted for use. After the HUVECs were cultured to confluence, they were replaced with 2% FBS growth factor-free medium. Use a 200uL pipette tip to scratch the endothelial cell monolayer and add tumor cell CM. Photographs were taken at the beginning of culture and after 24 hours. The percentage of ...

Embodiment 2

[0031] Example 2: Functional identification of TARBP2 specific degradation of anti-angiogenic factor mRNAs

[0032] 1) In order to further detect how TARBP2 regulates tumor angiogenesis, genes related to its regulation of tumor angiogenesis signaling pathways were detected. Pro-angiogenic factor mRNAs were upregulated after TARBP2 overexpression, while anti-angiogenic factors were downregulated. RNA-IP experiments further confirmed that TARBP2 can target and bind anti-angiogenic factors. Moreover, luciferase reporter experiments also confirmed that TARBP2 can target the 3'UTRs of anti-angiogenic factor mRNAs to degrade their mRNAs, but cannot target the 3'UTRs of pro-angiogenic factor gene mRNAs. In-depth examination of the half-life of anti-angiogenic factor mRNAs found that TARBP2 significantly reduced their half-life, but did not affect the half-life of pro-angiogenic factor gene mRNAs. This further confirms that TARBP2 specifically degrades mRNAs of anti-angiogenic facto...

Embodiment 3

[0035] Example 3: Treatment of Tumor Metastasis by Targeted Knockdown of TARBP2 Gene and Protein

[0036] In order to detect the role of targeted knockdown of TARBP2 gene in inhibiting tumor angiogenesis and tumor metastasis, firstly, lentiviruses expressing shRNAs targeting TARBP2 gene were used to infect tumor cells in vitro, and the effect of anti-angiogenesis after knockdown of TARBP2 protein expression was detected. Factor mRNAs expression and its half-life. Knockdown of TARBP2 was found to significantly increase the expression of anti-angiogenic factors and increase the half-life of their mRNAs. In vivo, using adenoviral vectors expressing shRNAs targeting the TARBP2 gene to simulate clinical treatment of tumor-bearing nude mice, it was found that the number of blood vessels in the treated tumor tissue was significantly reduced, and the lung tumor metastasis of the mice was also significantly reduced. In summary, the experimental results showed that targeted knockdown o...

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