Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: Comparative analysis of exosomes from the serum of patients with primary and metastatic colon cancer. (a) Serum exosomes were isolated from patients with primary tumor (P-exo) and distant metastasis (M-exo). Representative electromagnetic images of exosomes are shown. Scale bar: 200 nm. Western blots demonstrated increased CD9 and MCT1 (exosome markers) and cyclin D1 (tumor-specific marker) in the serum of patients with metastatic colon cancer (M, M-exo) compared with that of patients with primary tumor (P, P-exo). Serum exosomes of normal healthy people were included as controls (N, normal). (b) Comparative qPCR analysis showed that the levels of PVT1 and VEGFA were significantly higher in the M-exo than in the P-exo. (c) The sphere-forming assay showed that the addition of M-exo led to formation of an increased number of tumorspheres in both cell lines compared with controls and the group with P-exo. (d) Exosomes and colon cancer cell line coculture experiment. HCT116 and LoVo cells cocultured with M-exo demonstrated enhanced migratory and invasive abilities. (e) Western blot analysis indicated an elevation in metastatic markers, namely, Twist1, vimentin, and MMP2, and the stemness marker Sox2 in HCT116 and LoVo cells cocultured with M-exo compared with their counterparts cultured with P-exo. Coculture with exosomes derived from the serum of healthy individuals served as control. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Isolation, Western Blot, Marker, Cell Culture, Derivative Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: PVT1 silencing suppressed colon tumorigenic and metastatic potential. (a) The siRNA knockdown effect of PVT1 on two colon cancer cell lines. (b) The basal levels of PVT1 and VEGF (Western blot and gene expression) in cell lysates and exosomes. (c) Colony formation assay revealed that si-PVT1-transfected HCT116 and LoVo cells formed a significantly lower number of colonies compared with control parental cells. (d) Comparative tumorsphere-forming assay. HCT116 and LoVo cells transfected with si-PVT1 were significantly less potent in forming tumorspheres compared with their parental cells. (e) Comparison of expression between parental colon cancer cells and PVT1-silenced cells. Right panels: qPCR analysis demonstrated markedly decreased expression of metastatic markers, namely, VEGFA, Twist1, and MMP2, and the oncogenic marker EGFR in si-PVT1 colon cells. Left panels: Western blots of parental versus PVT1-silenced HCT116 and LoVo cells. Prominent reduction in the expression of VEGFA, Twist1, MMP2, and EGFR was observed after PVT1 silencing in both cell lines. Effect of PVT1 expression on cell (f) migration and (g) invasion of HCT116 and LoVo cells detected using Transwell assays. NC: negative control (scramble PVT1 oligonucleotides). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Western Blot, Gene Expression, Colony Assay, Transfection, Control, Comparison, Expressing, Marker, Migration, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: Target validation for miR-152-3p and its role in suppressing metastasis. (a) Target binding sequences of miR-152-3p in the 3′-UTR of PVT1 and VEGFA. These binding sites were predicted using both miRmap and MiRanda software. (b) qPCR analysis of PVT1, EGFR, and VEGFA levels in response to the sequential miR-152-3p mimic and inhibitor transfections (the control group did not add any reagents). A significant decrease in the mRNA levels of PVT1, EGFR, and VEGFA was observed after miR16-5p mimic transfection and subsequent restoration with the addition of the miR-152-3p inhibitor. Both HCT116 and LoVo cells showed a similar trend. (c) Tumorsphere formation assay. The tumorsphere-forming ability was considerably inhibited by the transfection of miR-152-3p in both HCT116 and LoVo cells; partial restoration of the tumorsphere-forming ability was noted when the miR-152-3p inhibitor was added. (d) Invasion assay revealed that an increase in miR-152-3p significantly reduced the invasive ability in both HCT116 and LoVo cells; however, the invasive ability was restored by the addition of the miR-152-3p inhibitor. (e) Western blot analysis. The addition of miR-152-3p mimics suppressed the expression of EGFR, vimentin, and VEGFA in both HCT116 and LoVo cells, and the inhibitor restored their expression. (f) A negative correlation was noted between miR-152-3p and PVT1 levels in colon cancer clinical samples from databases of TCGA ( n = 450). Control/NC: scramble hsa-miR-152-3p oligonucleotides. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Biomarker Discovery, Binding Assay, Software, Transfection, Control, Tube Formation Assay, Invasion Assay, Western Blot, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: Comparative analysis of exosomes from the serum of patients with primary and metastatic colon cancer. (a) Serum exosomes were isolated from patients with primary tumor (P-exo) and distant metastasis (M-exo). Representative electromagnetic images of exosomes are shown. Scale bar: 200 nm. Western blots demonstrated increased CD9 and MCT1 (exosome markers) and cyclin D1 (tumor-specific marker) in the serum of patients with metastatic colon cancer (M, M-exo) compared with that of patients with primary tumor (P, P-exo). Serum exosomes of normal healthy people were included as controls (N, normal). (b) Comparative qPCR analysis showed that the levels of PVT1 and VEGFA were significantly higher in the M-exo than in the P-exo. (c) The sphere-forming assay showed that the addition of M-exo led to formation of an increased number of tumorspheres in both cell lines compared with controls and the group with P-exo. (d) Exosomes and colon cancer cell line coculture experiment. HCT116 and LoVo cells cocultured with M-exo demonstrated enhanced migratory and invasive abilities. (e) Western blot analysis indicated an elevation in metastatic markers, namely, Twist1, vimentin, and MMP2, and the stemness marker Sox2 in HCT116 and LoVo cells cocultured with M-exo compared with their counterparts cultured with P-exo. Coculture with exosomes derived from the serum of healthy individuals served as control. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Isolation, Western Blot, Marker, Cell Culture, Derivative Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: PVT1 silencing suppressed colon tumorigenic and metastatic potential. (a) The siRNA knockdown effect of PVT1 on two colon cancer cell lines. (b) The basal levels of PVT1 and VEGF (Western blot and gene expression) in cell lysates and exosomes. (c) Colony formation assay revealed that si-PVT1-transfected HCT116 and LoVo cells formed a significantly lower number of colonies compared with control parental cells. (d) Comparative tumorsphere-forming assay. HCT116 and LoVo cells transfected with si-PVT1 were significantly less potent in forming tumorspheres compared with their parental cells. (e) Comparison of expression between parental colon cancer cells and PVT1-silenced cells. Right panels: qPCR analysis demonstrated markedly decreased expression of metastatic markers, namely, VEGFA, Twist1, and MMP2, and the oncogenic marker EGFR in si-PVT1 colon cells. Left panels: Western blots of parental versus PVT1-silenced HCT116 and LoVo cells. Prominent reduction in the expression of VEGFA, Twist1, MMP2, and EGFR was observed after PVT1 silencing in both cell lines. Effect of PVT1 expression on cell (f) migration and (g) invasion of HCT116 and LoVo cells detected using Transwell assays. NC: negative control (scramble PVT1 oligonucleotides). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Knockdown, Western Blot, Gene Expression, Colony Assay, Transfection, Control, Comparison, Expressing, Marker, Migration, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

doi: 10.1155/2021/9959807

Figure Lengend Snippet: Target validation for miR-152-3p and its role in suppressing metastasis. (a) Target binding sequences of miR-152-3p in the 3′-UTR of PVT1 and VEGFA. These binding sites were predicted using both miRmap and MiRanda software. (b) qPCR analysis of PVT1, EGFR, and VEGFA levels in response to the sequential miR-152-3p mimic and inhibitor transfections (the control group did not add any reagents). A significant decrease in the mRNA levels of PVT1, EGFR, and VEGFA was observed after miR16-5p mimic transfection and subsequent restoration with the addition of the miR-152-3p inhibitor. Both HCT116 and LoVo cells showed a similar trend. (c) Tumorsphere formation assay. The tumorsphere-forming ability was considerably inhibited by the transfection of miR-152-3p in both HCT116 and LoVo cells; partial restoration of the tumorsphere-forming ability was noted when the miR-152-3p inhibitor was added. (d) Invasion assay revealed that an increase in miR-152-3p significantly reduced the invasive ability in both HCT116 and LoVo cells; however, the invasive ability was restored by the addition of the miR-152-3p inhibitor. (e) Western blot analysis. The addition of miR-152-3p mimics suppressed the expression of EGFR, vimentin, and VEGFA in both HCT116 and LoVo cells, and the inhibitor restored their expression. (f) A negative correlation was noted between miR-152-3p and PVT1 levels in colon cancer clinical samples from databases of TCGA ( n = 450). Control/NC: scramble hsa-miR-152-3p oligonucleotides. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Scale bar: 100 μ m.

Article Snippet: The human colon cancer cell lines HCT116 (characteristics: derived from the primary colon ascendens tumor, TGF β 1+/TGF β 2+, suitable transfection host, and tumorigenic in nude/immunodeficient mice) and LoVo (characteristics: derived from metastatic colon cancer, Dukes' type C, grade IV, colorectal adenocarcinoma, MYC+/KRAS+/HRAS+/NRAS+, suitable transfection host, and tumorigenic in immunodeficient mice) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Biomarker Discovery, Binding Assay, Software, Transfection, Control, Tube Formation Assay, Invasion Assay, Western Blot, Expressing